明 里 宏 文

Research on Retroviruses：Implication in the Effects
of the Viral and Host−Cellular Factors on Retroviral
Infection．
（レトロウイルスに関する研究：ウイルス側及び宿主細胞側
因子のレトロウイルス感染における影響）
明 里 宏 文
Re se ar ch on re tr ov ir us es ; lmpl ic at io n in th e ef fe ct s of
the vi ral and ho st -c el lu la r fact ors on re tr ov ir al
infection.
Contents
I Review of retroviruses
I -1 Hist ory of re tr ov ir us es
I-2 Morphological properties of retroviruses
I -3 Ge ne tics
I -4 Re pl ic at ion cycle
I-5 Taxonomy of retroviruses
I--6 Pathogenesis of retroviral infection
I -6 -1 Ac qu ir ed im mu no de fici enc y sy nd ro me (A ID S)
I -6 --2 Onco ge ne sis
'
I -6 -3 Ot her dise as es
A. Anemza
B. Arthritis and disorders in CNS
ll General introduction -Aim of this research-
M Effects of viral and cellular factors on early stages
of primate leukemia virus infection
i
Ifi -1 Es tabl is tment of a simp le as say sy st em for de te ct ing
HT LV -: -b in di ng ce Zls and bi nd ing in hi bi to ry anti bo di es in
sera.
M-1-1 rntroduction
M -1-2 Ma te ri als and me th ods
A. Cells
B. Vlrus preparatlon
C. Virus binding assay
D. Sera
E. Binding inhibitory assay
F. In di re ct im mu no fl uo re sce nce (I F) as say
G. Sy nc yt ium in hibiti on as say
I[{ -1-3 Re su lts
A. HTLV-I binding assay
B. In hi biti on of the HT LV -Z bi nd ing by the pr et re at me nt
of HTLV-r with sera from HTLV-I-infected individuals.
C. HT LV -Z bi nd ing on va ri ous hu man ce lls
III -- 1-4 Di scus si on
M -- 1-5 Co nc lu si ons
M-2 Detection of cellular mernbrane proteins on HTLV-Z
envelope.
M-•2-1 Zntroduction
I[I-2--2 Materials and methods
A. Cells
ii
B. Detection of cellular membrane proteins on
HTLV"-I
C. Tnhibition of HT]"V-I binding by sera
I[[ -2 -3 Re su lts
llI--2-4 Discussion
M-2-5 Conclusion
llI-3 Alteration of the envelope and exposure of
gag p19
matrix protein of HTLV-Z on the target cells in
the post-
binding (fusion) steps.
M-3-1 !ntroduction
I[I -3 -1 Ma te ri als and me th ods
A. Cells
B. :nhibitors
C. Detection of gp46 and MA on HTLV-:-binding
ce lls
D. Detection of intracellular gp46 and MA
llI-3-3 Results
M -3 --4 Di sc us sion
M-3-5 Conclusion
IV Effects of viral factors on the infection of
im mu no de fi cienc y vi ru se s.
W-1 Introduction
IV-2 Materials and methods
A. Cells
B. DNA construct
iii
primate
C. DNA transfection
D. Infection
E. Re ve rse tr an sc ri pt ase as sa ys
F. Syncytium formation
G. Ch lo ra mp he nicol ac et yl -t ra ns fer ase as says
IV -3 Re su lts
IV-4 Discussion
rv -5 Co nc lusi on
V Ge ne ral co nc lu sion
VI Acknowledgments
N'V Re fe re nc es
iv
I Review of retrovirus
Z-1 History of retrovirus
The history of retroviruses is mainly as that of RNA
tum or v ir us es. Rous et al. fou nd an inf il tr ab le age nt from
avian leukosis in 1911, which was later designated as a
Rous sarcoma virus (1). In 1914 Fujinami et al. also
rep or ted s im il ar tum ors whi ch cou ld be t ra ns mi tted by
ultrafiltrates to healthy chickens and ducks (2). However,
t he s ig ni fica nce of t he obs er va ti on was not rec og ni ze d
until much iater. Thereafter, a variety of viruses shown
in Table 1 were found between 1960 - 1980. During this
period, there were two important discoveries for
ret ro vi ral res ea rc h; one was the ide nt if ic at ion of the
reverse transcriptase (RT, RNA-dependent DNA polymerase),
which was found from RNA tumor viruses by Temin et al. (3)
and Baltimore et al. (4), independently, and another was
the detection of oncogenes in RNA tumor viruses, which was
found in RSV by Vogt and Duesberg in the early 197Os.
Afterward, retroviral research progressed with the help of
the immunological and molecular biological techniques.
The n, ret ro vi ral res ea rc he rs ' int er es ts bec ame cen te red on
an enigma as to whether human retroviruses exists or not.
:n spite of the labors of many researchers, the works had
been in vain until 1980. The first human retrovirus found
was human T-cell leukemia virus type I (HTLV-Z) isolated
1
from adult T-cell leukemia (ATL) by Poiesz et al. and
Hinuma et al., independently (5-7). This finding and
further studies contributed to the understanding of the
c au se o f t he acq ui re d immun odef ic ie nc y s yn dr ome ( AI DS ),
pan de mi ca lly pre va il ing all over the wor ld from 198 0s; jn
1982r Montagnue et al. isolated a retrovirus from
lymphocytes of the AIDS patients and proved the virus to
be the causative agent of ATDS (8), then it was named as
human immunodeficiency virus type 1 (H:V-1).
T-2 Mor ph ol og ic al p ]ro pe rt ies of ret ro vi ru ses
The retrovirus characteristically has a reverse
transcriptase (RT) within the virion. The spherical
virions, 80 to 100 nm in diarneter, have glycoprotein
surface projections approximately 8 nm in diameter on the
envelope derived from the plasma membrane of the host
cell. The glycoprotein is narned env protein and the
pre cu rs or is c le av ed by host-- ce ll enz ymes int o the
transmembrane portion (TM) and the surface external
glycoprotein (SU). Within the envelope of the virion, the
group-specific antigens (gag protein) form two distinct
constituents, the membrane-associated matrix protein (MA)
layer and the viral core, which is made up of the inner
shell or capsid protein (CA). The CA contains two
identical genomic viral RNAs. The RNAs, together with the
nuc le ic -acid-- as soci at ed bas ic pro te ins (NC ), make up the
2
ribonucleoprotein (RNP). The genomes form the inverted
dimer of linear, single-stranded, positive RNAs. Fig. 2
shows two-dimensional diagram of HIV--lr as a
r ep re se nt at ive o f ret ro vi ru se s. T he p ro ce ss ed gag a nd p oZ
proteins form proper mature virions, and the virions bud
from cells, coated by the cellular ripid-bilayer membrane.
This life cycle of the retrovirus is illustrated in Fig.
2.
Z-3 Genetics
The genome of the retrovirus consists of positivestranded RNA molecules. The prototype genomic RNA contains
three genes: gag, poZ (protease, RT, and integrase)r and
env. These replicative genes are flanked at both ends by
regulatory sequences (R and U5 at 5'; U3 and R at 3').
During the reverse transcription, these sequences are
duplicated in a way resulting in the presence of identical
units of U3-R--U5, now called long terminal repeats (LTR),
at both ends of the genome (Fig. 3). Some of the
oncoviruses contain a variety of oncogenes (v-onc) in the
genome. Other oncoviruses, like HTLV-: and simian T-cell
leu ke mia vir us es (ST LV ), pos se ss a tra ns -a ct iv at or gene
narned tax and a regulatory gene called rex instead of v-
onc. The tax product transactivates not only 3 copies of
21 base pair repeats which serve as a transcriptional
enhancer in the U3 region of the LTR (9,10) but also genes
3
for both ZL-2 (11,12), the :L-2 receptor chain (12,13),
oncogenes (14,15), and others (16-20), which may be the
rea son for the ind uc ti on of mal ig na nt leu ke mi a. The r ex
pro te in enh an ces tra ns po rt of uns pl ic ed or p ar ti al ly
spliced transcripts from the nucleus to the cytoplasm
(21). The net effect of high levels of rex expression is
to induce the accumulation of gag and env mRNA expression
of vir al str uc tu ral genes whi le d im in is hi ng lev els of
dou bly spl ic ed sma ll mRNA enc od ing tax and zr ex (22 ). This
neg at ive fee db ac k mec ha ni sm all ows r ex- re gu la ted v ir al
gene expression to be divided into early and late phases.
During the early phase, low levels of tax and rex are
pro du ce d. The tax pro du ct inc re as es t he t ot al rat e of
transcription. In the late phase, increasing levels of rex
pro du cts acc umul ate the cyt op la sm ic uns pl ic ed or s in gly
spl ic ed mRNA lea di ng to the exp re ss ion of vir al str uc tu ral
genes, and to the diminution of tax and rex genes'
e xp re ss io n. This reg ul at ion sys te m of gene e xp re ss ion
could be critical for governing viral latency. Also,
transient bursts of viral expression resulting from tax
and r ex-me di at ed reg ul at ion c ou ld eff ec ti ve ly min im ize
immune surveillance by not allowing sufficient time for
immune mediated clearance of infected cells (23). The
genomic organization of HTLV-! is shown in Fig. 3. As for
lentiviruses, similar regulatory genes are also found as
tat and rev genes, furtherrnore, they have several other
4
regulatory genes, which might be important for the
pathogenicity of the viruses.
T-4 Rep lica ti on cyc le
Replication cycle of the retrovirus -s umque among
RNA viruses, because of the presence of the RT. Firstr
mature virions attach to the target cell surface in the
interaction of the viral env protein and cellular receptor
like CD4 molecule for HTV-1. Then fusion between viral and
cel lu lar mem br ane occ urs and int er na li za ti on of v ir al core
follows. The core may be internalized by pinocytosis. The
core is uncoated in the cytoplasm where the viral genome
is exp os ed . Then gen orn ic RNA is rev er se --t ra ns cr ib ed by
viral RT and the product DNA is integrated into
chromosomal DNA. The integrated viral DNA is called
proviral DNA. Transcription from proviral DNA depends on
cel lu lar RNA pol ym er as e II. Tra ns cr ib ed p ro du cts are
genomic RNA, full length mRNA coding gag-poZ frame and
spl ic ed sub ge no mic mRNA cod ing en v. In s ome of the
ret ro vi ru se s, sma ll mRN As spl ic ed two or thr ee tim es are
transcribed. They code various regulatory genes such as
revr tatr nef and others, which may regulate viral
replication and pathogenicity, as mentioned above. Viral
particles are made of gag, poZ and env products translated
from mRNAs described above. rn this step, the genomic RNA
interacts with gag-poZ precursor and is packaged into
5
viral particle and simultaneously budding of virion occurs
from the surface of the infected cells. :n the postbudding step, gag-poZ precursor is processed by viral
protease and the processed gag and poZ proteins form
proper mature virion. The life cycle of the retrovirus is
illustrated in Fig. 2.
:-5 Taxonomy of retrovirus
Ret ro vi ri dae f am ily is c la ss if ied int o t hr ee
sub fami li es, Oncovir in ae, L en ti vi ri na e, and Spuina vi ri na e.
The subfamily Oncovirinae is divided into types Ar Br
C, and D, according to the morphologic, antigenic, and
enzymatic differences. For example, HTLV-! is classified
=nto type C oncovlrus.
L en ti vi ri nae was not cla ss if ied cle ar ly befor e, but
kno wn as slow vir us es. L en ti vi ri nae is mor ph ol og ic al ly and
c he mi ca lly l ike other mem be rs of the Ret ro vi ri da e f am il y.
Zt does not show any oncogenicity but induce
immun od ef ic ie nc y (HI V- 1, H IV -2 , s im ia n i mmun od ef ic ie nc y
viruses (SXVs), feline immunodeficiency (F:V), and bovine
immunodeficiency virus (BIV)), anemia (equine infectious
anemia virus (EIAV)), and disorders of central nervous
sys tem (vi sna vir us and c ap ri ne art hr it is e nc ep ha litis
vlrus (CAEV)).
Spumavirinae has a characteristic of forming syncytia
in vitro so that it is called the foamy virus. Howeverr
6
the virus does not induce tumor or other lentiviruscausing diseases but shows persistent asymptomatic
infections in vivo. To date the pathogenesis of foamy
viruses is not found. Table 1 summarizes the taxonomy of
retrovlruses.
r-6 Pat ho ge ne sis of ret ro vi ral i nf ec ti on
T-6 --1 Acqui red immun odef ic ie ncy syn dr ome (A: DS)
AIDS is the disease that the patients become highly
sus ce pt ib le to many kin ds of inf ec ti ous dis ea se sr
including opportunistic infections, tumorsr or sometimes
disorders of the central neivous system. HTV-1, HTV-2,
SIVs, F:V and BIV are the causative agents of AIDS in
humans and animals. Here, the AIDS caused by HZVs is
explained below. The course of AIDS virus is characterized
by a long phase of asymptomatic periods and by the
terminal development of immunodeficiency. :n acute phase,
transient antigenemia occurs for weeks and soon
dis ap pe ar s. At the s ame t ime, ant i-- vi ral ant ib od ies app ear
aft er abo ut 3 to 12 wee ks, and v ir us -s pe ci fic cyt ot ox ic
lym ph oc yt es also ari se. In t he c ou rse of inf ec ti on, t he
titer of the anti-core antibodies and the number of CD4+
T-lymphocytes gradually decline, which are known as the
marker of the beginning of the symptoms. During the
periods of ArDS-related complex (ARC) or ATDS, the number
of CD4+ T cells decreases usually below 300 cells per pcl
7
and the viral load in the blood is much higher than that
in the asymptomatic state.
T-6-2 Oncogenesis
As descr ib ed a bo ve , o nc ov irus es c au se neo pl as ia r
including leukemias, lymphomas, and sarcomas. Oncoviruses
conta-n-ng oncogenes generate sarcomas, or somet-mes
leukemias in cat, mouse, rat, chicken, and others. The
period of transfonnation by these viruses is generally
acute. An enhanced production of transformation proteins
rnight also be the mechanism by which chronic leukemia
viruses cause leukernia. Oncoviruses which do not contain
oncogenes (H[PLV-T, HTLV-!I, STLVs and BLVÅr usualiy possess
t ra ns --act iv at or genes and t he pro du ct s act iv ate many
cellular genes as mentioned above. For exampler HTLV-:
cau ses mai ig na nt and chr on ic leu ke mi a, ATL. :t has been
est im at ed that the i nc id en ce of T cell mal ig na nc ies in
H[PL V- r- in fe ct ed perso ns is abo ut 1 to 2 in 100 0.
I-6-3 Other diseases
A. Anemia
Equine infectious anemia (EIA) caused by E!AV is a
chronic relapsing anemia with'
fever. Periodic viral
rep li ca ti on lea ds to an immun ol og ic al ly med ia ted dis ea se
with fever and severe anemia. The halimark of EIA is
periodic remissions. E:AV persists for life in the
8
infected animals.
B. Arthritis and disorders in CNS
C AE V p ri ma ri ly c au se s i nf lamm at io n o f t he s yn ov ia a nd
arthritis in goats. The most common and severe
loc al iz at io ns are in t he car pal j oi nt s. :n y ou ng ani ma ls
neurologic disease may be dominant. The disease develops
insidiously, and the course is slowly progressive as in
other lentiviral infections.
:n case of HIV-1, disorders in CNS sometimes occur on
ARC or A!DS stages. HTZV-I develops not only ATL but also
HTLV-r-associated myelopathy (HAM). So far the cause of
these neural diseases is unclear.
9
ll
General introduction -Aim of this research-
:n the early to middle of 20th century the retrovirus
was found as the tumor--generating RNA virusr howeverr
definition of retrovirus was not established until the
discovery of RT in 197O. :n those days immunology and
molecular biology rapidly developed, which influenced the
development of retrovirology very much. The informations
for animal retroviruses had been accumulated, but the
existence of the human retroviruses had been suspicious
even in 1970s. In the early 1980s, HTLV-I was found in
Japan and USA independently as the causative agent of ATL
(5-7). Pandemic of AIDS concurrently occurred, howeverr
HIV -1 c ou ld be iso la ted and ide nt if ied as an AIDS vir us in
1982 owing to the previous accumulation of the knowledges
on retroviruses, especially those on HTLV--I. Nowadays much
has been understood about A:DS and HIV--1, or other
retroviruses-related diseases, whereas they have been
incurable so far. In this regard, much more studies should
be required for the eZucidation of them. Especially viral
and the host or the host cell interaction still remains
unc le ar. As to HIV -:, for exa mp le, t he cel lu lar rec ep tor
(ie, CD4 molecule) and viral interaction has been studied
well, however, the function of the regulatory genes'
products in vivo remains to be elucidated. Moreoverr the
fact is that the cellular receptor(s) for HTLV-r or many
10
other retroviruses have not identified yet. Therefore, the
interaction between primate retroviruses and the host
cells was brought into focus in this research.
First, the effects of viral and cellular factors on
early stages of HTLV-I infection, especially on the
binding and fusion steps, were studied as a representative
of primate leukemia viruses because HTLV-Z has already
been studied much so that virus-producer cell lines and
mon oc lo nal ant ib od ies aga in st v ir al ant ig ens are e as ily
ava il ab le. (i ) To det ect the spe ci fic bin di ng of HTLV-- :
to the cel ls, a s imple ass ay sys tem was est ab li sh ed by
usi ng mon oc lo nal ant ib od ies (mA bs ) and f lowc yt omet ry, and
was applied to see if the antibodies in sera could inhibit
the HTLV--I binding. (ii) In the application of the system,
envelope of HTLV-: was examined to know the presence of
the cell-membrane proteins on the virions. (iii) For
further application of the system, it was investigated
that envelope of HTLV-! was altered or degenerated on the
target cells in the binding and post-binding steps.
Second, the effects of viral factors were examined on
the infectivity of H:V-1 and HIV-2 as primate
imm un odetic ie ncy v ir us es bec au se of t he ava il ab il ity of
materials, infectious clones and cell lines. (i) Several
mutants in the regulatory genes of the infectious
molecular clones of HIV-1 and HrV-2, were infected to the
per ip he ral blo od mon on uc le ar cel ls (PB MC ) of hum ans to see
11
the phenotypes of the mutants, in comparison to those in
the est ab lished cell lin es. ( ii ) To eva lu ate the funct ion
of nef gene as a negative factor, the potential role of
nef gene expression in transcription directed by the LTRs
of various primate immunodeticiency viruses was examined.
(iii) Tt was found from these experiments that a variety of
interaction among viral and cellular factors may be
specific and selective for sufficient infection of
vlruses .
12
Ig Effects of viral and cellular factors on early steps
of the primate leukemia virus infection.
III-1 Establishment of a simple assay system for detecting
H[VLV- I- bi nd ing cel ls and bin di ng inh ib it ory ant ib od ie s .
M-i--1 :ntroduction
HTLV-Z is a causative agent of ATL and HAI![/tropical
spastic paraparesis (TSP) as mentioned above. rn order to
explain the diverse clinical manifestations of HTIV-:
infectionr much information about events or consequences
of interaction of the viral infection with the host immune
res po ns e is nee ded. Unl ike other ret ro vi ru ses, H[PLV- I
seems to have difficulty in infecting target cells in its
cell-free form, although cocultivation of various target
cel ls w ith HTL V- I- pr od uc ing cel ls i nd uc es syn cy tia (24-
26). To analyze HTLV--I infection, especially the virus
binding to the target cells, the use of labeled HTLV-:
(27-29), or vesicular stomatitis virus (VSV) pseudotype
with HTLV-I envelope (30) have been reported. However,
the se met ho ds inv ol ve lab or io us tec hn iq ues and the re fo re
are not widely used.
In this study, a simple, rapid, and quantitative assay
sys tem is dem on st rated to det ect HTLV-- T s pe ci fic bindi ng
to human cells, using HTLV-I concentrate, mAbs and
flo wc yt omet ry. In app licati on of this sys te m, t he
13
ant ib od ies in sera from HTL V- Z- in fe ct ed i nd iv id ua ls
inc lu di ng ATL and HAM pat ie nts were sho wn to inh ib it the
binding of HTLV-I.
III-1-2 Materials and methods
A. Cells
The following established human cell lines were used:
MT- 2, a T cell l ine from umb il ic al c ord blo od lym ph oc yt es
exogenously infected with HTLV-I (31); ILT-8M2, an HTLV--:
positive T cell line (32); MOLT-4#8 (33), A3.01 (34), and
H9 (35), CD4+ lymphoma cell lines; BJAB (36) and Raji
(37), Burkitt lymphoma lines; K562, a chronic myelogenous
leukemia cell line (38); U937, a promyelocytic cell line
(39), UI05MG (40) and U251MG (41), glioma cell line; HeLa,
an epitheloid carcinoma line (42); and FL, an amnion cell
line (43). They were maintained in RPM:1640 medium
supplemented with antibiotics and 10% heat--inactivated
fet al b ov ine ser um (FB S) . Hum an per ip he ral b lo od
lym ph oc yt es were sep ar at ed from hea lt hy don or blo od on
F ic ol --Hyp aq ue gra di ents , sti mu lated wit h
phytohemaglutinin-M for 3 days before use.
B. Vlrus preparatlon
The culture supernatant of MT--2 cells was spun at 300
Å~ g for 5 minutes to remove the cells, filtered with O.8
ptm filter and centrifuged at 30,OOO Å~ g for 2 hours at
14
4Åé. The viral pellets were resuspended in RPMI 1640
medium to give a 100 times concentrated virus and stored
in aliquots at -800C until use.
C. Virus binding assay
Cells (5 Å~ 105) were washed with RPMI 1640 medium
containing 20-o FBS (washing medium), resuspended in a 100
pt l of vir us pre pa rati on, and inc ub at ed at 37 0C for 1 hour
with occasional agitation. Then the cells were washed
twi ce with was hi ng med ium and rea ct ed with 1 00 pt l of
1:250 diluted REY-7, rat anti-HTIJV-T gp46 mAb (44), on ice
for 30 minutes. They were again washed twice with washing
medium and resuspended in 100 ptl of 1:100 diluted goat
fluorescence isothiocyanate (FITC)-conjugated anti-rat Ig
(Ta go, rnc ., Cal if or ni a, USA ). They were inc ub at ed on ice
for 30 minutes, washed once with washing medium and
res us pe nd ed in O.5 ml of lg for ma ld eh yde in p ho sp ha te --
buffered saline (PBS). The stained samples were analyzed
for flu or es ce nce int en si ty on FAC Sc an (Be ct on & D ic ki ns on,
C al if or ni a, U SA ).
D. Sera
Sera were obtained from normal healthy adults and
HTLV-: seropositive individuals including asymptomatic
carriers, ATL, and HAM patients. They were inactivated at
560C for 30 minutes and stored in aliquots at -200C untU
15
use. Dilutions of sera were made with RPM: 1640 medium
c on ta in ing 10 rg FBS .
E. Bin di ng inh ib it ion ass ay
Bin di ng inh ib it ion ass ay was c ar ried out by
pretreatment of the virus with serially diiuted sera. The
diluted sera (IOO pt 1) were mixed with the same volume of
the virus and incubated on ice for 1 hour. The mixture
(200 ILI) was added to 5 Å~ 105 of MOLT-4#8 cells and
inc ub at ed at 37 0C for 1 hou r. The n, the vir al bin di ng
assay was carried out as described above.
F. :ndirect immunofluorescence (TF) assay
Tndirect :F assay was carried out as previously
described (45). Smeared and fixed MT-2 cells were reacted
with 2-fold diluted sera at 370C for 30 minutes. After
washing with PBS, the cells were incubated with F:TCconjugated rabbit anti-human TgG (Daco, Inc., Glostrup,
Denmark) at 370C for 30 minutes. The stained cells were
exa mi ne d by flu ores ce nce mic ro sc op e. Ant ib ody t it ers were
determined by the final dilution of sera which showed
pos it ive flu or es ce nc e.
G. Syncytium inhibition assay
For the titration of antibodies that inhibit the
syn cy ti um forma ti on by HTL V- Z, 1 OO /Lc l of ser ia lly dil ut ed
16
sera were added to 100 ptl of a mixture containing an
equal number (5 Å~ 104) of MOLT-4#8 and TLT-8M2 cells in a
96 flat well tissue culture plate. Experiments were
performed in duplicate. They were cultured at 370C for 48
hours and the syncytium formation was evaluated by
microscope examination. Titers of syncytium inhibition
were det er mi ned by fin al dil ut io ns of sera that inh ib it ed
syncytium formation.
I[I-1-3 Results
A. HTLV-I binding assay
Crude HTLV-Z preparations (about 100 times
concentrated) were obtained from the concentrated
supernatant of MT-2 cell cultures. Protein concentration
of the viral preparation was about 1 mg/ml. The virustreated cells were then stained with REY-7 mAb (anti-HTLVI gp46) and subsequently with F:TC-labeled anti--xat :g. A
flow cytometer was used for the detection of HTLV-I
binding MOLT-4#8 cells (Fig. 4AÅr. Approximately 80g of the
vir us -t re at ed MOL T- 4#8 cel ls were det ec ted to be pos it ive
for fluorescence. The staining was specific as evidenced
by the background level of fluorescence when RPMI 1640
medium was used instead of the virus preparation or REY-7
mAb. Treatrnent of the virus-adsorbed MOLT-4#8 cells with
O.025% of trypsin for 3 minutes at 370C decreased the
fluorescence intensity of the positive cells to the
17
b ac kg ro un d l ev el . T hus, t he v ir al -b in di ng was res tr ic te d
on the cell membrane.
To det er mi ne whe th er flu or es ce nc e- po si ti ve cel ls are
specific to the viral binding, the relationship between
the viral dose and the fluorescence-positive cells
detected by flowcytometry was studied. Figure 5
dem on st ra tes the res ul ts of a 1-h our inc ub at ion at 37 0C
after adding the serially diluted virus. The frequency of
the positive celis decreased with increasing viral
/
dilution. Viral binding to MOLT-4#8 cells yields a linear
inc re ase in the perce nt age of pos it ive cel ls and
fluorescence intensity between 1/16 and 1/2 dilution (50
pt l) of the virus, reaching a rnaximum (80&) at 1/1
dilution of the virus.
Figure 6 shows the kinetics of fluorescence-positive
cells at 37Åé (triangle) and 40C (circle) on the viralbin di ng. Aft er 5 min ut es pos ti nf ec ti on at 37 0C , alm ost 50 06
of the cells became positive for fluorescence. After 30
minutes postinfection, 80% positive cells were recognized.
Thereafter, the positivity plateaued until 8 hours.
Incubation of the virus with the MOLT-4#8 cells at 40C
red uc ed the fre quency of the pos it ive cel ls. The max im um
of positive cells at 40C was about 20g. Thus,
approximately 60g of the inhibition of fluorescence
increase was observed by the incubation at 40C.
The difference in viral binding at 37 and 40C was
18
further studied by a temperature-shift experiment. MOLT4#8 cells were treated with the virus at 40C for 30
minutes, washed twice to remove the excess virus, and
transfered to 370C. Incubation of the cells at 370C was
terminated at 5, 15, and 30 minutes by the addition of
ice-cold washing medium. The viral-binding cells incubated
for 30 minutes at 370C showed nearly 3.5-fold increase in
the percentage of positivity (open) and mean fluorescence
intensity (closed) ÅqFig. 7).
Pretreatment of the cells with puromycin, a translational inhibitor, did not affect the viral binding or
-
the increase in fluorescence positivity (See Table 6)r
suggesting that the de novo synthesis of gp46 after viral
binding is not likely. However, cytochalasin B which
blocks membr ane mot il ity by i nh ib it ing t he pol ymer iz at ion
of act in fil amen ts mar ke dly red uc ed vir al bin di ng at 37 0C
(see Fig. 15). Thus, membrane fluidity might affect the
affinity of the viral binding or fusion process of the
viral envelope glycoprotein.
B. Inhibition of the HTLV-I binding by the pretreatment of
the virus with sera from HTLV-I-infected individuals.
Figure 8 illustrates the procedure used to determine
the tit er of bin di ng inh ib it ory ant ib od ies ÅqBr A) aga inst
HTL V- I. MOLT-- 4#8 cel ls were t re at ed with HTL V-r a ft er a 1hou r pre in cu bati on at 4 0C with 1:2 O, 1:2 00, or 1:2 OOO
19
dil ut io ns of fou r pre se le ct ed sera from a hea lt hy (HT LV -r
noninfected) donor, an asymptomatic carrier (AC) and
patients with ATL or HAM. Fluorescence-positive cells were
t he n ass es sed by flo wc yt om et ry. Sera from HTL V- I- in fe ct ed
individuals inhibited viral binding at various levels.
Titers of BIA were tentatively calculated by 50g
inh ib it ion of the vir al bin di ng (ab out 40g p os it ive cel ls
ind ic at ed by hor iz on tal das hed line in Fig. 8). Tit ers of
BTA were less than 1:2O, 1:8O, 1:7O, and 1:240 in cases of
a seronegative donor (closed circle}, an AC (closed
square) , and patients with ATL (open circle) or HAM (open
square), respectively. Thus, Fluorescence-positivity
detected with this method was exclusively inhibited by the
HTLV-I-positive sera, suggesting that HTLV-r was
spe ci ti ca lly ads or bed to the MOL T- 4#8 cel ls.
To rule out the possibility that the epitope of REY-7
mAb on the HTLV-: virion was occupied by the patient's
antibodies during the pretreatment of the virus with the
serum, the virus-adsorbed cells were reacted with or
without the serum and then stained with REY-7 mAb. No
difference in number of positive cells was observed. Thusr
occupancy of the mAb epitope by human serum in our
experiments was unlikely.
Usi ng the same met ho d, an add it io nal 8 sera inc lu di ng
2 healthy HTLV-I noninfected donors, 2 AC, 1 patient with
ATL and 3 patients with HAM were titrated. All sera were
20
also examined by IF and syncytium inhibition in order to
compare with BIA titers. Table 2 summarizes the data. All
sera frorn 3 healthy donors showed negative in all three
assays (!F, syncytium inhibition, and BIA). The range of
the titers of BrA by the treatment of the virus with 9
HTLV-: infected individuals was from 1:50 to 1:800. No
correlation between clinical category and titers of B:A
was observed albeit the cases examined were limited.
However, titers of BIA were roughly correlated with those
of IF and syncytium inhibition.
C. HTLV--: binding on various human cells
To assess the relationship between HTLV-: and host
cel ls, the vir al bin di ng ass ay was per fo irm ed to 7 flo at ing
cell lines, 4 adherent cell lines, and peripheral blood
lym ph oc yt es from a non in fe ct ed don or (Ta ble 3 and Fig. 4).
Among floating cell lines, T cell lines (MOLT-4#8, H9, and
A3.01 cells), K562 and U937 cells had a high percentage
(61 to 838) of positive cells. However, relatively low
binding level (40 and 5g) of HTLV-I was observed in B cell
1ines (Raji and BJAB, respectively). Figure 4B shows the
case of Raji cells. Adherent cell lines had a lower (2 to
33%) percentage of positive cells. UIO5MG cells showed
only 1.89o of positive cells (Table 3 and Fig. 4C). About
half of phytohemaglutinin--activated peripheral blood
lymphocytes were also able to adsorb the virus. Thusr
21
HTL V-I is see mi ng ly ads or bed to a wide var ie ty of cel ls.
III - 1-4 Dis cu ss ion
The results of these studies demonstrate the HTLV-I
specific binding to the target cells by the simple and
qua nt it at ive ass ay sys tem usi ng flo wc yt om et ry . Sec on dl yr
inh ib it ion of the vir al bin di ng to t he MOL T- 4#8 cel ls was
observed by the pretreatment of the virus with sera from
HTL V- : -- in fe ct ed ind iv idua ls , s ug ge st ing t hat sera
neu tr al iz ed the HTL V- I. [P hird ly, a rat he r bro ad ran ge of
hos t cell pop ul at io ns was sho wn to be sus ce pt ib le to HTL V-
Z binding.
HTL V-I ads or pt ion was c on fi rm ed by dos e- re sp on se
rel at io ns hip (Fi g. 5 ), kin et ics of t he flu or es ce nc e-
pos it iv ity and its t em pe ra tu re dep en de nc y (Fi gs. 6 and 7),
try ps in tre at me nt, and block ing by ser op os it ive sera (Fi g.
8 and Table 2). The mechanism of viral adsorption may be
dif fe re nt from t he ant ig en -a nt ib ody rea ct io n, bec au se the
inc re as e in flu or es ce nc e- po si ti vi ty was i nh ib it ed by 4 0C
(Fi gs. 6 and 7). [V he s ame inh ib it ory eff ect of 40C
inc ub at ion was rep or ted in the case of mur ine leu ke mi a
virus binding (46). The parameters of the HTLV-! membrane
binding and the penetration process have been difficult to
establish because of the inability of the productive
inf ec ti on by cel l- fr ee vir us es, and l ac k of a rel ia bZe
biological assay. The approach described here should be
22
readily adaptable for elucidating the characteristics of
HTLV-I membrane events.
The viral binding detected by the present method may
also be useful for predicting the infectibility of cells
by dif fe re nt str ai ns of H[VLV- !. !V he vir al bin di ng on
various cells was also reported by using F:TC or
rho da mi ne -1 ab el ed HTL V-I (27 -2 9) or VSV -H TL V- I pse ud ot ype
virus (30). However, these assays need a large amount of
highly purified HTLV-T particles and special techniques to
prepare the labeled virus or pseudotype virus. The present
met hod is simple and can be qua nt it at iv ely ava il ab le for
rapid and mass screening of HTLV-: receptor and
neutralizing antibody.
Pretreatment of the virus with sera from HTLV--Iinf ec te d ind iv id ua ls inh ib it ed t he vir al ads or pt ion (Fi g.
8 and Table 2). This finding further confirmed that H[PLV-:
par ticl es were res po ns ib le for t he ind uc ti on of
fluorescence-positivity in this system. :t is reported
that sera from ATL patients and healthy carriers contain
antibodies against cells-specific glycopolypeptides, gp68
and gp46, using radioimmuno-precipitation assay (47). Sera
from patients with ATL or HAM, and healthy carriers
inh ib it ed the H[PLV- I-bi nd ing to the MOL T- 4#8 cel ls by the
pretreatment of the virus, however, neither sera from
HTLV-:-uninfected controls (Fig. 8 and Table 2) nor G:N-14
mAb (an ti-gag p19 mat rix pro te in) inh ib it ed t he vir al
23
bin di ng. The inh ib it ion phe no me non is pro ba bly due to
antibodies directed against the viral envelope
glycoprotein. Although further studies are needed to
c on fi rm t hat t he ant ib od ies det ec ted by this ass ay are
specific to the epitope on viral envelope glycoprotein,
this assay system using flow cytometry is useful for rapid
screenlng.
:n order to delineate the relationship between HTLV-I
a nd t ar ge t c el ls , t he v ir al -b in di ng c el ls a ft er t re at me nt
with the cell--free virus has been studied. When F!TC-
labeled, highly purified HTLV-I was used to detect the
binding to various cell lines, most of the cell lines from
various species had HTLV-I binding activity (27). These
findings are more or less compatible with ours. Howeverr
BJAB and U1O5MG cells showed low binding activity,
suggesting that the cell lines may express less amount of
cellular receptor for HTLV-T. [Po date, the receptor has
not been identified. Zt should be ascertained whether true
cellular receptors are present on these cells, and further
the mechanism of viral infection should be studied.
M-1-5 Conclusions
A simple and rapid assay system for the detection of
HTLV-I-binding cells was developed to assess the virus
specific receptor and to titrate the antibodies that block
the viral binding. The specificity of the viral-binding
24
was shown by dose--response relationship, kinetics of the
b in di ng, a nd tem pe ra tu re dep en de nc y. HTL V-I was ads or be d
on a wide range of human cell lines and peripheral blood
lymphocytes at various levels. Antibodies to inhibit the
viral-binding were also quantitatively detected in sera
from HTLV-I infected individuals, including AC and
patients with ATL or HAM, but not from healthy
seronegatives. This assay system is useful in screening
the virus specific receptor and neutralizing antibodies to
H[VLV-r and in the analysis of virus-cell interaction.
25
gl-2 Detection of cellular membrane proteins on HTLV--:
envelope.
M--2-1 Introduction
In the budding stages of viral replication, enveioped
viruses obtain the envelope consisting of viral glycopro te ins and lip id -b il ay er fro rn t he cel lu lar mem br ane.
Simultaneously, the cellular membrane proteins rnay also be
taken up onto viral envelope. The host-derived membrane
proteins might function as the cofactor(s) of viral
inf ec ti on, esp ec ia ll y, in the ini ti al att ac hm ent or
bin di ng ste ps. Act ua ll y, it was rep or ted that ant is era to
B2 microglobulin, MHC-I, and MHC-::DR neutralized the
HIV -T inf ec ti on (48 ), and als o that P 2 mic ro gl ob ul in
enhances the infectivity of cytomegalovirus (49). However,
the pre se nce of the hos t- de ri ved mem br ane pro te ins on
HTLV-Z is unknown.
As described above, a simple assay system was
established for the detection of HTLV-: binding to the
target cells. It is shown here that not only human T cells
but also mouse T cells were able to adsorb HTLV-Tr and
that in application of this phenomenon the presence of the
host cell--derived membrane proteins were demonstrated on
HTLV-! virions, by using mAbs which react to cellular
proteins of human but not to those of mouser and
flowc yt omet ry.
26
llI-2-2 Materials and methods
A. Cells
BW5147 (50) and EL--4 (51) cells, mouse T cell lines,
and MOLT-4#8 cells (33), a human T lymphoma cell line,
were used for the target cells of HTLV-Z binding. MT-2
cells (31), an HTLV-Z producer cell line, was also used
for the determination of the cell-surface molecules.
B. Detection of the cellular membrane proteins on HTLV-Z
envelope.
The detection of HTLV-: binding to the ceils is
described above. Tn the application of this system, the
HTLV-I-binding cells were treated with mAbs against the
cellular membrane molecules of human, Leu4 (CD3), Leu3a
(CD4), and MHC-rZDR (Becton & Dickinson, California, USA);
LFA-1, ICAM-1 (Biodesign, Kennebunkport, ME); LFA-3, MHC-r
(Co sm ob io ); and CD2 (Nich irei ), res pe ct iv el y, at 40C for
40 minutes. The cells treated with HTLV-Z and each mAbs
were then stained with FTTC-labeled anti--mouse or rat Ig
at 40C for 40 min ut es, and ana ly zed by flo wc yt om et ry•
C. Inhibition of HTLV-! binding by sera
For the inhibition of H[VLV-! binding to the cells, 50
IL 1 of HTLV--I concentrates were pretreated with the same
volume of the serially dUuted sera from HTLV--I seropositives at 40C for 1 hour, thereafter the mixture was
27
treated with BW5147 cells.
llI-2-3 Results
Preliminarily, the expression levels of the cellular
mem br ane mol ec ul es on MOL T- 4#8 cel ls were c om pa re d with
those on MT-2 cells (Table 4). Both MOL[P-4#8 and MT-2
cells expressed high levels of CD4, LFA-1, LFA-3, and MHC1 molecules, as shown by mean channel fluorescence,
however, the expression of CD2 and CD3 molecules was
almost undetectable on both cells. Furtherrnore, :CAM-1 and
MHC -I rDR mol ec ul es were str on gly det ec ta ble on MT-2 cel ls
but not on MOLT-4#8 cells. These results prompted that if
:CAM-1 or MHC-ITDR molecules are present on the HTLV-r
envelope, they could be detected on MOLT-4#8 cells
ads or bi ng the HTL V- I. The re fo re, HTL V-I was added to
MOLT-4#8 cells at 370C for 1 hour, washed, and the cells
were treated with mAbs to :CAM-1, MHC--rTDR, and as a
control, LFA-1. Figure 9 shows the histograms of the
stained cells; the expression levels of !CAM-1 and MHCIrDR on MOLT-4#8 cells were very low (line b) compared
with the background (line a) as shown in Table 4, however,
the fluorescence of the HTLV--T-treated MOLT-4#8 cells to
t he se mol ec ules bec ame obv io us ly pos it ive (li ne c ). As for
.
LFA-lr MOLT--4#8 cells originally expressed the molecule so
str on gly (li ne b to a) that the inc re ase in flu or es ce nce
intensity was little even after the virus treatment
28
(line c).
:t is reported that strong induction of rCAM-1 was seen
in HTL V- I- po si ti ve [V --c el l lin es (52 ). The re fo re, it is
still possible that the stimulation of HTLV-Z binding
might induce the increase in the expression levels of
:CAM-1 and MHC-I!DR molecules. To exclude this
possibility, MOLT-4#8 cells were pretreated with the
t ra ns la ti on al inh ib it or cyc lo he xi mi de at a c on ce nt rati on
of 1 ptglml for 1 hour at 370C, and then HTLV-r was added
to the cells. The detectable levels of !CAM-1 or MHC--I:DR
mol ec ul es on HTL V- !- tr ea ted MOL T- 4#8 cel ls were n ot
affected with or without cycloheximide treatment,
suggesting that the stimulation of HTLV-Z binding did not
affect the expression levels of the molecules.
From these results, TCAM-1 and MHC-IIDR were seemingly
present on HTLV-I envelope, however, the presence of other
molecules was still undetectable by the method above. To
investigate the presence of the other cellular proteins,
mouse T cell lines were applied for the target cells of
HTLV-I because possibly the mAbs to the cellular membrane
proteins of human do not react to those of mouse so that
any human proteins may be detectable specifically.
Therefore, the efficiency of HTLV-Z binding to mouse T
cell lines, BW5147 and EL-4 cells, was investigated.
Figure 10 demonstrates that HTLV-! gp46 was detectable on
both HTLV-Z--treated mouse T cell lines, indicates that
29
HTLV-r was able to bind to the cells. Thus, BW5147 cells
were selected for further experiments.
Preliminarily the cross-reactivity of the mAbs, which
react to the membrane proteins of humanr to the membrane
molecules on BW5147 cells was examined. No cross-reaction
of the mAbs to the molecules on BW5147 cells was observed.
Thenr the detectable levels of cellular membrane molecules
of human on HTLV-:-binding BW5147 cells were tested.
Fig. 11 and Table 5 describes the results on HTLV-Ibinding BW5147 cells. Table 5 also compared the results
with the expression levels on MT-2 cells, shown as mean
channel fluorescence. All the molecules expressed on MT-2
cells were also detectable on viral-binding BW5147 cells.
Furthermore, the levels of the mean channel fluorescence
of these molecules on HTLV-I-treated BW5147 cells were
correlated with those on MT-2 ce:ls. These results suggest
that the hos t cel l- me mb ra ne pro te ins may be tra ns fe rr ed to
t he vir al env el ope acc or di ng to t he exp re ss io n lev els of
them on MT-2 cells, although the variety of molecules
screened was 1imited.
There remains the possibility that the soluble
cellular proteins in HTLV-I concentrate might attach to
the cells so that they became detectable. To exclude this
possibility, it was determined if pretreatment of HTLV-I
with sera from HTLV-r-seropositive individuals, which is
sho wn to inh ib it the HTL V-I bin di ng to the t ar get cel ls,
30
affect the detectable levels of cellular membrane-derived
molecules of human on BW5147 cells. The HTLV-Z concentrate
was pretreated with the same volume of the serially
diluted sera from HTLV-I-seropositive individuais or
ser on eg at ive con tr ols at 40C for 1 hou r, then the mix tu re
was added to BW5147 cells and stained as mentioned above.
Figure 12 demonstrates the representative result of the
exp er irn ents . The dat a were pre se nt ed as per ce nt age
c om pa re d wit h the pos it ive c ontr ols (pr et re at me nt with
serum-free medium). As expected, HTLV-I pretreatment with
t he ser um from a s er op os it iv e i nd iv id ual ( as ympt om at ic
carrier) reduced the detectable levels of !CAM--1 and
MHC-I:DR molecules on BW5147 cells (open and closed
triangle, respectively). The rate of inhibition was
dependent on the concentration of the serum. On the other
hand, the serum from a seronegative individual did not
affect the detectable levels of the molecules on the cells
even at a concentration of 1:40 dilution (open and closed
circle). Similar results were obtained in the sera frorn
different individuals. These results indicated the
pre se nc e of HTLV- :-tr ansf er re d hos t cel{ l-- me rnb ra ne pro te ms
on BW5147 cells.
I[I-2--4 Discussion
In this study, first, the expression levels of the
membrane molecules on MOLT-4#8 cells were compared with
31
those on MT-2 cells (Table 4). Secondly, rCAM-1 and MHCIZDR, which were merely expressed on MOLT-4#8 cells,
bec ame det ec table on HTL V- 1- bi nd ing MOL [V- 4#8 cel ls ( Fig.
9). Thirdly, HTLV-r binding to mouse T lymphoma cell lines
was demonstrated (Fig. 10), thus, a batch of moleculesr
inc lu di ng adh es ion mol ec ul es (LF A- 1, ICA M- 1, and LFA -3 ),
MHC -Z, MHC -I ID R, and CD4 mol ec ul es, was sho wn to bec ome
detectable on viral--binding BW5147 cells (Fig. 11 and
Table 5). It was also demonstrated that the detectable
levels of the molecules on viral-binding BW5147 cells were
correlated with those expressed on MT-2 cells (Table 5).
Finally, the pretreatment of HTLV-T with the serum from an
HTL V-I ser op os it ive ind iv id ua l, whi ch was sho wn to inh ib it
the viral binding (Fig. 5 and Table 2), reduced the
detectable levels of the cellular proteins of human on
BW5147 cells (Fig. 12). From these results, it is
concluded that the HTLV-I envelope carries cellular
membrane proteins derived from the host cells.
Previously, many groups have reported the presence of
hos t cel l-- me mb ra ne pro te ins on e nvel op ed vir us es ,
inc lu di ng H:V -1 (48, 53- 55 ), SIV from mac aq ue mon key (56 ),
CMV (57 ), Friend leu ke mi a vir us (58 ), avi an leu ko sis vir us
(59), VSV (60), murine leukemia virus (60), Sindbis virus
(61), and influenza virus (62). However, it is contro-
versial so far whether the uptake of cellular membrane
proteins on virions is selective or not. The results in
32
this sec ti on sug ge st non -s el ec ti ve u pt ake of cel lu lar
proteins to the viral envelope. The discrepancy might be
the differences in budding system of these viruses,
int er ac ti on of t he mol ec ul es wit h vir al p ro te in s, or in
the assay system used for the detection of the membrane
proteins.
rt is possible that adhesion molecules or MHC molecul es on vir io ns may fun ct ion as sec on da ry mol ec ul es for
viral attachment or binding. Tn fact, Auther et al. (48)
reported that antisera to P2 microglobulin, MHC-: and
MHC-IrDR neutralized the H:V-1 infection. In this regard,
it should be elucidated whether mAbs against some of the
cellular proteins observed could affect the early steps of
H[VLV- T inf ec ti on .
M-2-4 Conclusion
A bat ch of cel lu lar membr ane pro te ins of hum an bec ame
det ec table on HTLV-- 1- bi nd ing mou se T cel ls by mAb s and
flo wc yt om et ry, sug ge st ing the pre se nc e of hos t cei l-
derived membrane proteins on HTLV-I virions.
33
III-3 Alteration of the envelope and exposure of gag p19
mat rix pro te in of HTL V-I on t he tar get cel ls in t he pos t-
binding (fusion) steps
M -3-1 Znt ro du ct ion
HTLV-I is the causative agent of ATL/HAM. In vivo, A[VL
shows a preferential incidence in CD4+ T lymphocytes (63).
H ow ev er, t he cel lu lar rec ep to r( s) for HTL V-Z is see mi ng ly
distinct from CD4 molecule (32,64). HTLV-I is usually
transmitted to uninfected cells by co-culture of the
vir us -c ar ry ing cel ls wit h per ip he ral blo od lym ph oc yt es
from healthy donors (7,65). Because of the inability of
the cell-free HTLV-: to infect cells, little is known
about the viral receptor and the early steps of viral
inf ec ti on suc h as ads or pt io n, pen et ra ti on and u nc oa ti ng.
In the previous section, the assay system for the
detection of HTLV-I binding to the target cells was
described, in which MOZT-4#8 and U937 cells were shown to
specifically adsorb high levels of H[PLV-T ([Pable 3).
Kuroda et al. reported that MOLT-4#8 cells showed marked
cell-fusion after co-cultivation with MT-2 cells but U937
cells did not, and also that fluorescence to envelope
glycoprotein gp46 and gag p19 matrix protein (MA) was
detected on HTLV-r-treated MOLT-4#8 cells, whereas virus-
treated U937 ceUs were only gp46-positive, using the same
ass ay sys tem (66 ). Bas ed on the se obs er va ti on s, it was
34
hyp ot he si zed that MA of HTL V-Z rn ay be exp os ed dur ing the
pos t- bi nd ing ste ps on the pla sma mem br ane of MOL T- 4#8
cells but not on U937 cells. The present results showed
that MA was exposed on MOLT-4#8 cells in post-binding
steps but not on U937 cells.
Ifi -3-2 Mat er ia ls and in et ho ds
A. Cells
MOIT-4#8 (33), U937 (39) and MT-2 (31) cells were used
for the target cells of HTLV-I as mentioned above.
B. :nhibitors
As t ra ns la ti on al i nh ib it ors, c yc lo he xi mi de a nd
puromycin were used at the final concentration of O.1 or 1
pt g lm l, and 5 or 50 pt g /m l, res pe ct iv el y. Cyt oc ha la sin B,
an inh ib it or of pol yrneriz at ion of cyt os ke le tal act in
filaments, was used at the concentration of 10 ptglml.
C. Det ec ti on of gp46 and MA on HTL V- I- bi nd ing cel ls
Detection of gp46 and MA on viral-binding cells is
described above, except for the use of Gin-14 mAb (antiMA) (66) for the detection of MA.
D. Detection of intracellular gp46 and MA.
For the detection of intracellular gp46 and MAr MT-2
cells or HTLV-I-binding MOLT-4#8 and U937 cells were
35
tre at ed with 70ig met ha nol at 40C for 3 min ut es, was he d,
then stained by mAbs as described above.
ilI -3- 3 Res ul ts
For detection of envelope gp46 and MA on the surface
of pla sma membr ane aft er ads or pt ion of cel 1- fr ee HTLV-- :,
flowcytometric analyses using mAbs to the antigens were
app li ed as des cr ib ed . Pre limi na ri iy , MOL T-- 4#8 and U93 7
cells were treated with HTLV-r, then stained as mentioned
above, except for the use of REY-7 (anti--gp46) or GIN-14
(anti-MA) mAbs for the first labeling, followed by
flowcytometric analysis. Figure 13 showed that both cell
lines treated with virus and REY-7 mAb became positive for
fluorescence. On the other hand, MOLT--4#8 cells treated
with virus and REY-7 mAb became positive for fluorescence
but U937 cells did not (Fig. 13), as reported previously
(67).
Then, the kinetics of Os positivity for fluorescence of
MOLT-4#8 and U937 cells treated with HTLV--I at 370C (Fig.
14). As for the fluorescence to gp46 on MOLT-4#8 cells,
nea rly 40 rg pos it iv ity bec ame det ec table 5 min ut es aft er
inc ub at io n, then 80g pos it iv ity was obs er ved aft er 30
minutes (panel A, open circle). The kinetics of
flu or es ce nce pos it iv ity of v ir us -t re at ed U937 cel ls was
similar to that of MOLT-4#8 cells, although maximum
positivity was about 602 (panel B, open circle). When MA
36
on vir us -t re at ed MOL T- 4#8 cel ls was sta in ed, pos it iv ity
for flu or es ce nce inc re as ed to 50g aft er 60 min ut es
inc ub at io nr tho ugh the i nc re as e in pos it iv ity was s lo we r
than that for gp46 (panel A, closed circle). However,
vir us -t re at ed U937 cel ls were alm os t neg at ive for MA even
after 60 minutes (panel B, closed circle). These results
ind ic ate the ina bi li ty of U937 cel ls to pre se nt MA on
the ir s ur face in the pos t-- ad so rp ti on ste ps , s ug ge st ing
that MA mig ht be exp os ed by deg en er at ion or alt er at ion of
the intact viral envelope or that de novo synthesis of MA
might occur in MOLT-4#8 cells but not U937 cells. To
assess the possibiiity that MA was expressed by de novo
protein synthesis after the viral inoculation, MOLT-4#8
cel ls were pre tr ea te d with cyc lo he xi mi de (O. 1 or 1 pt glml)
or puromycin (5 or 50 ptg/ml) as translational inhibitors
at 370C for 1 hour. [Phen HTLV--: was added to the
pretreated cells and stained as described above. Compared
with the negative control (without pretreatment), the rate
of fluorescence positivity for gp46 and MA was not
affected by the cycloheximide or puromycin-pretreatment
(Table 6), indicates that de novo synthesis after the
viral inoculation is not likely.
Tn the section III-1, increase in fluorescence
pos it iv ity for gp46 was shown to be t em pe ra tu re -d ep en de nt
(Figs. 6 and 7). It was also demonstrated that viraZ
binding occurred even at 40C, however, increase in
37
f lu ores ce nce pos it iv ity f or gp4 6 on H[PLV- :- bi nd ing cel ls
req ui re d 37 0C inc ub at ion (Fi g. 7).
Here, it was studied whether MA was exposed in the
post-binding steps at 370C (possibly fusion step) or
already MA was exposed in the binding step. MOLT-4#8 cells
were treated with HTLV-I at 40C for 30 minutes, washed
twice, and resuspended in warm medium at 370C (Fig. 15).
: nc ub at ion of t he vir us -t re at ed MOL T- 4#8 cel ls at 4 0C for
30 minutes induced less than 20% positivity for both gp46
and MA. The incubation of the cells at 370C was terminated
at 5, 15, 30 and 60 minutes by adding cold medium. The
tem pe ra tu re shi ft fro rn 4 0C to 37 0C res ul ted in t he gra du al
inc re ase in pos it iv it y, and the rate of pos it iv ity
increased from 18 to 65g for gp46 and from 16 to 40g for
MA after 60 minutes (Fig. 15). Alternatively, HTLV-:binding MOLT-4#8 cells at 40C for 30 minutes were treated
with 10 ptg/ml of cytochalasin B and incubated at 370C for
60 minutes. Cytochalasin B treatment completely inhibited
the increase in fluorescence positivity for both
molecules, similar to that of virus-treated cells without
inc ub at ion at 37 0C (Fi g. 15, open and hat ched bar ). The se
data demonstrate that the increase in fluorescence
pos it iv ity for gp46 and MA on HTL V- r- bi nd ing MOL T- 4#8
cells required 370C incubation and cytochalasin Bd ep en de nt p he no me no n, i.e . p ol ym er iz at io n o f c yt os ke le ta 1
actin filaments. It may be possible that the envelope of
38
HTLV-I was degenerated or broken on MOLT-4#8 cells and the
inner matrix proteins resultantly were exposed.
If this is the case, HTLV--! virion or at least
envelope and MA structure may stay intact on U937 cell
membraner so that MA might not become detectable on HTLV:-t re at ed U937 cel ls. Thu s, to c on fi rin whe th er U937 cel ls
treated with HTLV--Z were able to adsorb intact viral
particles sufficiently and yet exposure of MA did not
occur, intracellular gp46 and MA in HTLV-I-treated MOLT4#8 and U937 cells were monitored. For the detection of
intracellular antigens, the cells were treated with 70g
met ha nol at 4 0C for 3 min ut es, was he d, then sta in ed with
mAbs as described. As for MT-2 cells without methanol
treatment, gp46 was strongly detectable but MA was merely
detectabler whereas more than 90Z of fluorescence-pos it iv ity for both gp46 and MA was obs er ved in met ha no l-
treated MT--2 cells (Fig. 16). The result indicates that
rn et ha nol fix at ion of t he cel ls all ow ant ib od ies to
penetrate the membrane into the cells. Figure 17A displays
that methanol fixation of the HTLV-I-adsorbed MOLT-4#8
cells decreased the gp46-positivity from 85g to 5% but
increased MA-positivity from 5506 to 959o. :n case of the
vir us -t re at ed U937 cel ls, flu or es ce nc e for MA of the f ix ed
cells was clearly detectable, reached to 93g positivity,
however, fluorescence of the unfixed cells was merely
detectable (Fig. 17B). These data indicated that the
39
intact viruses truly bound to U937 cells but MA was not
exp os ed on the sur fa ce of the unf ix ed cel ls.
Alternatively, fluorescence-positivity for gp46 on viralbinding MOLT-4#8 and U937 cells decreased to the
background level by methanol treatment (Figs. 17A and B)r
which was probably due to the degeneration of the epitope
of gp46 on the cell surface.
I[I -3-4 Dis cu ss ion
Previously, Kuroda et al. demonstrated that coculturing MT-2 cells with MOLT--4#8 cells led to syncytiumformation but not with U937 cells (67). By celld-free HTLV-
: adsorption, fluorescence for gp46 became detectable on
the surface of both celis, however, that for MA became
positive on MOLT--4#8 cells only (67, Table 3 and Fig. 13).
These data necessitated studies to compare these two types
of cells in terms of the properties of the plasma membrane
aft er t he vir al ads or pt io n. T he d at a pre se nt ed sug ge st
that MA may be exposed on the cell membrane in consequence
to the lysis of the envelope and that the increased
fluorescence-intensity and positivity after 370C
incubation may be due to the spread of the envelope on the
cell sur fa ce, as sho wn by cyt oc ha la sin B tre at me nt (Fi g.
15). Lysis of viral envelope might be necessary for the
next steps of H[VLV-I infection such as fusion and
penetration. !n fact, U937 cells induce no fusion when co40
cultured with MT--2 cells (67). This finding is similar to
the case of H!V-1, in which V3 region of envelope gp120 is
cleaved on the cellular membrane by proteolysis (68). :n
cas es of ort ho rny xo vi ru se s, par am yx ov ir us es and
cor on av ir us es, act iv at in g pro te ol yt ic c le av ag es c at al yz ed
by cellular proteases late in the secretory pathway are
critical for the fusion activity (69). In this regardr
several available anti-protease agents (amastatinr
phosphoramidon, E-64, arphamenine A, leupeptin, antipain
and pepstatin A) were tested to block the exposure of MA
on the virus-adsorbed MOLT-4#8 cells. However, no
effective agent has been found so far. It still remains to
be elucidated whether the exposure of MA is followed by
successive uncoating of the virus. Further studies would
be needed to identify the mechanism.
M -- 3- 5 Concl us ion
This study demonstrated that MA was exposed on HTLV--rbinding MOLT--4#8 cells in the post-binding steps, probabiy
in fusion step, but not on viral-binding U937 cells,
suggesting that the phenomenon may affect the ability of
syncytium formation or infectivity of HTLV-Z.
41
IV Effects of viral factors on the infection of primate
immunodeficiency viruses
rv -1 Int ro du ct ion
Major disease of lentiviruses is acquired immunodeficiency syndrome (ATDS), shown by F:V, BIV, SrVrnacr HIV-
1 and HIV-2. ATDS is the disease that the patients become
hig hly sus ce pt ib le to a var ie ty of opp or tu ni st ic
infections, tumors, and sometimes disorders of central
nervous system as rnentioned above.
The primate immunodeticiency viruses are classified
into four groups; HIV-1 and STV from chimpanzee (STVcpz)
group, STV from african green monkey (SrVAGM) and from
afr ic an whi te --c rown ed mon ga bey mon key ( SI Vw cM ) gro up , H :V-
2, STVMAc and SZV from sooty mongabey monkey (SrVsM) group,
and SIV from mandrill (SIVMND) group. [Vhese fouir groups are
gen et ic al ly equ id is ta nt from eac h oth er, hav ing 55g to 60g
homology, although more than 80g homology within the
group.
Both H!V-1 and HIV-2 cause A:DS in humans, but
pathogenicity of SIVs to humans is unknown. Howeverr some
of the S:Vs (SIVMAc and STVsM) induce AIDS in the macaque
monkeys (70,71), and others (SIVAGM and SIVMND) do not
ind uce AZDS to mon ke ys (72 -7 4). Alt ho ugh dif fe re nce of the
gen es bet we en pat ho ge nic and non -p at ho ge nic v ir us es is not
so obvious as the grouping by the homology of the nucleic42
acid sequences (74), the determinant(s) of the
pathogenicity is still unknown. On the other hand, HTV-1
can inf ect t he c hi mp an zee but sho ws no AID S- li ke dis ea se,
furthermore, HTV-1 can not establish infections in other
mon ke ys. Sim il ar ly, S:VMAc c au ses A:DS in mac aq ue mon ke ys
but not in other monkeys like the african green monkey.
These tindings suggest that virus-host interaction is
exactly regulated for establishing the infection or
dev el op ing t he A:D S. Esp ec ia ll y, t he se v ir us es have
several small regulatory genes other than structural
genesr gagr poZ and env, different from other
retroviruses, suggesting that these regulatory genes may
play important roles for the infection and pathogenicity
of viruses. From this point of view, data from functional
analyses of the regulatory genes have been accumulating.
The fun ct io ns of two tra ns -a ct iv at or gen es, tat and revr
are known well and are indispensable for viral replication
(75-78), but the other regulatory genes, named vif, vpr,
vpur vpx and nef, are not always essential for infectivity
(79-94). The tat and rev genes are present in the genomes
of all these viruses. H!V-1 genome contains vif, vprr vpu
and nef genes. HIV-2/STVMAc/SIVMND have a vpx gene instead
of the vpu gene in HrV-1. SZVAGM genome is similar to HIV2ISrVMAc/SIVMND except for the deficient of the vpr gene.
Fig. 18 shows the gen om ic org an iz at io ns of t he se four
groups of primate immunodeficiency viruses.
43
To analyze the function of these regulatory genesr
infectious molecular cloned viruses introducing genespe ci fic mut at io ns have been used for in vit ro inf ec ti on
experiments. A recent report showed that vpx mutants of
H:V-2 was able to propagate normally in T cell and
mon oc yt ic l ines , b ut d id n ot e st ab li sh p ro du ct ive
infection in primary 1ymphocytes (95). The behavior of the
mut ants in pri ma ry c ell cul tu re s, the re fo re, may ref le ct
the functions of the genes in vivo more precisely than
those in established cell lines.
Her e, phe no ty pes of t he gen e- sp ec if ic inf ec ti ou s
mutants of HIV-1 (vif, vpr, vpu and nef mutants) (79) and
HIV -2 ( vif, vpx, vp2r and nef mut an ts) as rep re se nt at iv es
of the primate irmunodeficiency viruses (89) in human
primary blood mononuclear cells (PBMC) were compared with
those in established cell lines. The results demonstrate
that some of the mut an ts dis pl ay dif fe re nt gro wth
potentials in PBMC from those in established cell lines.
IV--2 Materials and methods
A. Cells
Human PBMC were separated from blood samples obtained
from hea lt hy ser on eg at ive ind iv id ua ls on Fic ol -Hyp aq ue
gradients, stimulated with 5 ptglml of Concanavalin A and
cultured in RPM! 1640 supplemented with 10% heatinactivated FBS for one day. The culture medium was then
44
replaced by the same medium containing 38 recombinant
human !L-2. The CD4+ human T cell lines, A3.01 (34) and
MOLT-3 (33) cells were maintained in RPMX 1640
supplemented with 10g heat-inactivated FBS. A human colon
carcinoma cell line, SW480 (96), was maintained in
Dulbecco's modified Eagle medium supplernented with 10g
heat-- in ac tiva ted FBS .
B. DNA construct
The infectious molecular clones pNL-432 (96) and pGH123 (89) were wild type (wt) proviral DNAs of HIV-1 and
HIV-2, respectively. The HZV-1 mutants designated as pNLNd (vif mutant), pNL-Af2 (vpr mutant), pNL--Ss (vpu mutant)
and pNL-Xh (nef mutant) were derived from pNL--432. The
HrV-2 mutants named pGH--Xb (vilf mutant), pGH-St (vpx
mutant), pGH-Ec (vpr mutant) and pGH-Ml (nef mutant) were
der iv ed from pGH -123. Tab le 7 sho ws the mut at io ns in the se
DNA clones.
Some biological properties of the constructs under the
control of viral LTRs were made by placing LTR fragments
derived from HTVslSIVs in front of the CAT gene of pHd-CAT
(89,97). The nef expression plasmid, pcD-SRanef460, and
the control plasmid, pcD-SRanefAKpn:473 were used in this
experiment. Briefly, the Hinf: (nucleotides 8623)-HindlU
(nucleotides 9606) fragrnent, containing the nef gene was
excised from the pNL-432 DNA and was repaired with Klenow
45
fragment. After adding BamHT linkers to both ends, the
fra gment was ins er ted into dep ho sp ho ry la ted Bgl ll s ite of
the pcDL-SRaBglE456 (98), generating the pcD-SRanef473
plasmid, in which the nef gene was expressed under the
control of the SRa promoter (98). Tn the pcD-SRanefA
Kpnl473, the Kpnl site (nucleotides 9605) in the nef gene
was del et ed by T4 DNA pol ymer as e, cre at ing a fra me -s hi ft
mutation in the nef gene.
C. DNA transfection
Twenty ptg of plasmid DNA were mixed with 50 pt1 of
2.5M CaC12 for 30 minutes. The mixture was added to SW480
cel ls, fol lo wed by the inc ub at ion at room tem pe ra tu re for
5 to 7 hours. Then the cells were treated with 30rg
gly ce rol for 3 min ut es, was he d, and cul tu re med ium was
added to the cells.
D. Xnfection
The growth kinetics of the mutants was determined in
PBMC and CD4+ T cell lines. Culture supernatants of
transfected SW480 cells was filtered with O.45 ptm pore
size and appropriate volume of filtrates which contains
same amount of virions adjusted by RT activity were
infected to 1 Å~ 106 cells. The culture supernatants were
harvested every 3 or 4 days to check the rate of viral
replication and then new medium was supplemented to the
46
culture.
E. Reverse transcriptase (RT) assays
Virion-associated RT activity was assayed for
scr ee ni ng the amo unt of pro ge ny vir io ns in cul tu re
sup er na ta nt. Fif ty pt 1 of the RT c oc kt ail conta in ing 50 mM
Tris-HC1, 75 mM KCI, 1O mM dithiothreitol, 4.95 mM MgCl2,
10 ptg/ml poly A, 5 ptg/ml Oligo dT, O.05g NP-40, and O.37
MBq of 32P-dTTP were incubated with 10 ptl of the culture
supernatant at 370C for 3 hours. Ten pt1 of the mixture
were spotted onto DE81 filter, followed by several
was hi ng. The rad io ac ti vi ty of the fil ter was measu red by
scintillation counter.
F. Syncytium formation
Syncytium formation was monitored at intervals by
m ic ro sc op ic exa mi nati on.
G. C hl or amph en ic ol a ce ty l- tr ansf eras e ass ay s
Chloramphenicol acetyl--transferase (CAT) assays were
car ried out as pre vi ou sly des cr ib ed (99 ). 20 pt 1 of the
cell lysates from transfected SW480 cells were mixed with
170 lz1 of the reaction buffer containing 3.7 KBq of i4c-
chloramphenicol, O.25 M Tris-HCI (PH 7.8), and 4 mM of
acetyl-coenzyme A, at 37Åé for 1 hour. One ml of ethyl
acetate was added to the mixture, shaked vigorously, then
47
spinned down. The supernatant was dried, resuspended in 15
pt 1 of ethyl acetate. It was spotted onto the thin-layer
chr om at og ra phy (TL C) fil te r, then all ow t he sol ve nt,
chl or of or m/ me th an ol (95 :5 ), fro nt to move to the top of
the tilter. The filter was dried, exposed to X-ray tilm.
IV--3 Results
A. Biological characterization of mutants
For ass es sment of the bio lo gi cal act iv it ies of HIV -1
and HIV-2 mutants in PBMC, equivalent amounts of cell-free
viruses (5 Å~ 104 cpm of RT activity) prepared from
transfected SW480 cells were inoculated into PBMC and CD4+
T cell lines. Virus replication and syncytium formation
were monitored by measuring RT production and by
microscopic examination, respectively. Both of the wt
viruses replicated well in PBMC, A3.01 (HIV-1) or MOLT-3
(H!V-2) cells.
Figure 19 shows the growth kinetics of HZV-1 mutants
in A3.01 cells (panel A) and PBMC (panel B). The mutants
of the vpu and n ef gen es exh ib it ed dif fe re nt growth
patterns in the two cell types. Virus replication of the
vpu mutant was clearly less efficient than that of wt
virus in A3.01 cells but replicated equally to wt virus in
PBMC. rn contrast, growth of the nef gene mutant was
similar to that of the wt virus in A3.01 cells, but was
slightly slower than that of the wt virus in PBMC. The vif
48
mutant did not grow in either type of the cells. The vpr
mutant and the wt virus replicated similarly in both types
of cells when infected at a relatively high input dose
(Fig. 19), but 10-fold decrease in the input dose of the
former delayed the virus growth markedly in A3.01 cells
(Fig. 20A) and slightly in PBMC (Fig. 20B).
Figure 21 shows the gronth kinetics of H!V-2 mutants
in MOLT-3 cells (panel A) and PBMC (panel B). The growth
characteristics of the vif and vpx mutants in PBMC were
different from those in MOLT-3 cells. The vpx mutant
replicated well in MOLT-3 cells but very poorly in PBMC.
To examine whether the retarded growth of the vpx mutant
in PBMC was a general feature, a similar infection
experiment was carried out by using PBMC samples prepared
from another individual. As shown in Fig. 22, the vpx
mut ant pro pa ga ted poo rly but the red uc ti on in its gro wt h
rate to one-third of that of the wt virus was not so
remarkable as that shown in Fig. 19. The vif mutant grew
poorly in MOLT-3 cells, and showed no productive infection
in PBMC. The kinetics of growth and the levels of virus
production of the vpr, nef mutants and wt virus were
similar in MOLT-3 cells and PBMC. A 10-fold reduction in
the input dose had no significant effect on the relative
growth kinetics of the wt virus and the vpr mutant in
MOLT-3 cells and PBMC (Figs. 20C and D).
Results of the syncytium formation by the wt virus and
49
the mutants are summarized in Table 8. The wt virus and
all mutants of HIV-1 except the vif mutant induced
syncytium formation in both of the cell types. On the
other hand, neither HrV-2 wt virus nor any of its mutants
induced syncytium formation in PBMC, which was in sharp
'
contrast to their effects on MOLT-3 cells.
B. Effects of expression of HIV-1 nef on HTVIS!V LTRs
Earlier studies showed that mutations of the nef gene
lead to more eff ic ie nt v ir al rep licati on (80 ,8 6, 94) or
transcription directed by the HIV-LTR (80,94). Howeverr
the possible function of the nef gene is still
controversial (85,87,100,101). As described above, the nef
gene product of HIV-1 showed no negative effect on virus
replication (Fig. 19). For further examination of the
potential role of H:V-1 nef gene expression in
transcription directed by the LTRs of various primate
immunodeficiency viruses, cotransfection experiments were
car ri ed out; SW4 80 cel ls were tra ns fe ct ed with a ser ies of
LTR -C AT con st ru cts and a pla sm id exp re ss ing e it he r the
HTV -1 n ef gene (pc D- SR a nef4 60) or the mut at ed neif gene
(pc D- SR a n ef A Kpn 47 3), and the CAT exp re ss ion was
monitored. As shown in Fig. 23, pcD-SRanef460 did not
rep re ss CAT exp re ss ion of any of t he HIV /S IV LTR -C AT
constructs compared with that by pcD-SRanefAKpn473.
Essentially the same results were obtained in repeated
50
experlments.
IV-4 Discussion
The regulatory genes of H:Vs (vif, vpr, vpu and nef of
HIV-1, and vif, vpx, vpr and nef of HIV-2) are known to be
dis pe ns ab le for vir al rep lica ti on in est ab li sh ed T cell
lines (79-94). Recently, Guyader et al. reported that vpx
mutants of HTV--2 replicated normally in T and promonocytic
cell lin es, but not in per ip he ral blo od lymph oc yt es (95 )r
suggesting that the vpx gene product may have an important
role in vivo. Therefore, to see if the mutants of these
genes might exhibit distinct characteristics in PBMC, the
virus replication and syncytium formation in PBMC were
compared with those in established T cell lines. The
results are summarized in Table 8.
The vpu gene is unique to the H:V-1 genome. The
results of the vpu mutant (Fig. 19A) and previous data
(79,92,93) showed that the vpu gene is necessary for
efficient viral replication in established T cell lines.
Unexpectedly, the vpu mutant showed similar growth
kinetics to that of wt virus in PBMC (Fig. 19B). This
obs er va ti on sug ge sts that the req ui re me nt of the vpu
product for viral replication may differ depending on the
origins or character of the cells. Further investigations
should be carried out for the understanding of the
function of the vpu gene in vivo.
51
The vpx gene is conserved in all primate lentiviruses
except HIV-1. This gene of HIV-2 is reported to be
necessary for viral replication in human PBL but not in
established cell lines (95). The results demonstrated here
(Fig. 21) is consistent with the reported findings.
However, the phenotype of the vpx mutant in PBMC was found
to vary individually (Figs. 21 and 22). [Vhese results also
ind ic ate the imp or ta nce of t he cha ra ct er of tar get ceZ ls
for infectivity of mutant viruses.
The vif genes of HZV-1 and H!V-2 have been
dem on st ra ted to be req ui re d for e ff ic ie nt rep lica ti on in
established cell lines, but the effect of mutation in the
vif gene varies depending on the cells used (79,84,89-91).
:n this experiment, mutants of the vif genes of neither
HTV-1 nor H:V-2 replicated in PBMC (Figs. 19 and 21),
sug ge st ing t hat vif gene pro du ct may be ind is pe ns ab le for
virus replication in vivo.
[Vhere are reports that the vpr product of HZV-1
increases the rate of replication and accelerates the
cytopathic effect of the virus in established T cell lines
(82r88). In case of HIV-2, however, vpr mutants exerted no
effect on the growth kinetics (83,89). The results
pre se nt ed on PBMC (Fi gs. 19- 21) a re ess en ti al ly c on si st ent
with these previous reports, therefore, the function of
vpr genes is seemingly not obvious, at least in vitro.
Earlier studies showed that nef mutants multiply
52
faster than the wt virus in established cell lines
(80r86r94). However, the present investigation
demonstrated that the nef genes' products did not affect
the rate of viral replication (Figs. 19 and 21). The nef
product has also been reported to reduce HTV-1 LTRmediated transcription (80,87), but more recent data
indicated the absence of the negative influence of the
gene (85,100,101). Cheng-Mayer et al. found that the nef
products from several isolates had different effects on
HIV replication in vitro (80). Moreover, the nef protein
of H:V--1 NL-432 did not repress CAT activity directed by
LTRs derived from HIV-1, HrV-2r S:VMAc, SIVMND and S:VAGMr
which are representatives of major primate immunodeficiency viruses, in the present experiments (Fig. 22).
This finding is consistent with the results of the
infection experiments showed above. Lately, Kestler I[I et
al. showed the importance of the nef gene of SIVMAc for
maintenance of high virus loads and for development of
ATDS (102).
So far, the real function of these regulatory genes in
vivo has been unknown, however, biological functional
analyses using animal models for AIDS such as the report
of Kestler Ifi et al. (102) are required to evaluate the
fun ct ion and imp lica ti on of t he gen es for the p at ho ge ne sis
of immunodeficiency viruses.
53
rv-5 Conclusion
The phe no ty pes of the mut an ts of sev er al regul at ory
genes in HIV-1 and H:V-2 were studied in PBMC and
established cell lines. The results showed that some of
the mutants displayed different growth potentials in PBMC
from those in established cell lines, suggesting that the
functions of some genes could be detected only in PBMC.
Studies on the roles of these genes in PBMC may provide a
bet ter u nd er st an di ng of the ir fun ct io ns in vivo.
Furthermorer infection experiments of these mutants in
vivo should be required for analyzing the pathogenesis of
HIV and AIDS.
54
V Final conclusion
The main object of the present study was to determine
the significance of the interaction among viral and
cellular factors in the retroviral infection.
First, the effects of viral and cellular factors on
ear ly s ta ge s of HTLV-- ! inf ec ti on , esp ec ia lly on the
binding and fusion steps, were studied as a representative
of primate leukemia viruses. (i) A simple assay system
for detecting HTLV-Z-binding cells was established by
using mAbs and flowcytometry. (ii) The system was
applicable for the determination of the antibodies in sera
inhibiting the H[VLV-I binding, (iii) and for the detection
of the host cell-derived membrane proteins on the envelope
of HTLV-I virions. (iv) As for further application of the
system, it was found that the envelope of the virions
altered or degenerated on the surface of the cells, and in
this process MA was exposed on the cells, dependent on the
tem pe ra tu re and pro bably cyt os ke le tal act in
p ol ymer iz at io n.
Secondly, the effects of viral factors on the
infectivity of H:V-1 and HIV-2 as prirnate immunodeficiency
viruses were examined, and it was found that; (i) several
mut ant virus es of the inf ec ti ous mol ec ul ar c lo ne s of H:V -- 1
and HTV-2, directed to the portions of the several
regulatory genes, showed distznct replication patterns -n
55
PBMC from those in established cell lines, (ii) and the
potential role of the nef gene expression in transcription
directed by the LTRs of various primate immunodeficiency
vir us es was exa mi ne d.
These findings suggest that the infection of primate
retroviruses is established by the interaction among viral
and cellular factors at each step of the life cycle of the
viruses. The interaction may restrict not only the target
cells, organs and the host but also pathogenesis of the
viruses. The understanding of these phenomena would be
meaningful for the elucidation of the diseases caused by
ret ro vi ru ses and also of the mec ha ni sm of t he fun ct io ns of
the cells.
56
VI Acknowledgments
The author is sincerely grateful to professors
Mas an ori Hay am i, Shi nj i Har ad a, Mas am it su Kan oe, Yos hi ta ka
Goto and Toshiharu Shinjo, for the supervision and
mea ni ng ful adv ic e, and to the col le ag ues in the Dep ar tment
of Vet er in ary Mic ro bi ol og y, F ac ul ty of Agr ic ul tu rer
Miy az aki Uni ve rs it y, Ins titu te for Vir us Res ea rc h of Kyoto
University, and Department of Biodefence and Medical
Virology, Kumamoto University, School of Medicine, for
helpful discussions and suggestions.
57
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Hayamir M.r and Adachi, A. (1991) Functional analysis of
long terminal repeats derived from four strains of simian
irmunodeficiency virus STVAGM in relation to other primate
77
lentiviruses. Virology 185, 455-459.
98. Takebe, Y., Seiki, M., Fujisawa, J., Hoy, P., Yokotar
K., Arai, K., Yoshida, M., and Arai, N. (1988) SRa
promoter: an efficient and versatile mammalian cDNA
expression system composed of the simian virus 40 early
promoter and the R--U5 segment of human T-cell leukemia
virus type 1 long terminal repeat. Mol. Cell. Biol. 8r
466-472.
99. Gorman, C.M., Moffat, L.F., and Howard, B.H. (1982)
Rec om bi na nt gen omes which exp re ss chl or am ph en ic ol
acetyltransferase in mammalian cells. Mol. Cell. Biol. 2r
1044-1051.
1OO. Bachelerie, F., Alcarni, J., Hazan, U.r rsraelr N.,
Goud, B., Arenzana-Seisdedos, F., and Virelizierr J.L.
( 19 90 ) C on st it ut ive e xp re ss io n o f h um an i rmun od ef ic ie nc y
virus (HrV) nef protein in human astrocytes does not
influence basal or induced H:V long terminal repeat
activity. J. Virol. 64, 3059-3062.
1 01 . H amme s, S .R ., D ix on , E .P ., M al im r M .H ., C ul le nr B .R .r
and Greene, W.C. (1989) Nef protein of human
imm un od ef ic ie ncy vir us t ype 1: e vi de nce aga in st its role
as a transcriptional inhibitor. Proc. Natl. Acad. Sci. USA
78
86, 9549-9553.
102. Kestler rll, H.W., Ringler, D.J., Morir K., Panicalir
D.L.r Sehgalr P.K.r Daniel, M.D., and Desrosiers, R.C.
(19 91) Imp or ta nc e of the nef gene for mai nt en an ce of high
virus loads and for development of AZDS. Cell 65, 651-662.
r
103. Gallo, R. C., and Montagnier, R (1988) ArDS in 1988.
Scientific American 18, 9-19.
104. Dulbecco, R.: In Brown, F., and Wilson, G.[ed] (1990)
Principles of bacteriology, virology and immunity, vol. 4,
Virology, 355-382.
79
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Antibody titers
Table 2.
syncyt1um inhibition and
infected individua1s and
detected
H[l]LV-I-bin
by indirect immunofluorescence (IF),
ding inhibition in sera from HTLV-I
uninfected controls.
Titers
detected by:
Serum*
IF
Syncytium inhibition
Binding inhi bition
Nor-1
Åq1:10
Åq1:10
Åq1:20
Nor-2
Åq1:10
Åq1:10
Åq1:20
Nor-3
Åq1:10
Åq1:10
Åq1:20
AC-1
1:80
1:40
1:80
AC-2
1:640
1:320
1:700
AC-3
1:80
1:80
1:50
ATL-1
1:80
1:40
1:70
ATL-2
1:160
1:40
1:400
HAM-1
1:160
1:160
1:240
HAM-2
1:320
1:320
1:600
HAI!l-3
1:80
1:320
1:80
HA)I-4
1:1280
1:640
1:800
*Nor,
normal uninfected control; AC, asympto]natic
T-cel1 leukemia; HAI!t, HTLV-I-associated myelopathy.
carrier; ATL,
adu1t
Table 3.
Binding of HTLV-I to
peripheral
blood lymphocytes.
var1ous huHun cel1 1ines and
I FIoating cell lines
MOLT-4#8
H9
A3.01
Rajj
BJAB
K562
U937
% positive
Origin
Cells
cells*
T-cell leukemia
82. 7
Å}
5.4
T-cell leukemia
70.9
Å}
2.1
T-cell leukemia
61. 4
Å}
9e1
Burkitt' s lymphoma
39. 7
Å}
1.0
Burkitt' s lymphoma
4.6
Å}
3.1
Myelogenous leukemia
64. 2
Å}
1.9
Promyelocytic ce11 1ine
63.6
Å}
5.2
Amnion ce11
29.8
Å}
4.3
Glioma cel1 1ine
1.8
Å}
Oe2
Glioma ce11 1ine
33.3
Å}
9e2
Epithelioid
12. 7
Å}
5.0
47. 5
Å}
O.4
ll Adherent cell lines
FL
UI05MG
U251MG
Hela
M Peripheral blood lymphocytes
*
Percentage represents the average
Å} S• de
l ine
carclnoma
from three independent experlments.
Tab1e 4. The expression levels
of the molecules
on the surfaee of MOLT-4#8 and MT-2 eel1s.
Mean channel f1uoreseenee
mAbs to:
MOLT-4#8
CD3
CD4
LFA-1
ICAM-1
MT-2
5. 6
4. 0
62. 4
80. 7
75. 5
46. 9
8e 8
12 6. 0
LFA-3
33. 7
101. 6
CD2
MHC-1
6. 2
5. 7
74. 5
13 6. 8
L9
71. 0
MHC-IIDR
Mean ehannel fluoreseenee on eaeh eellular
moleeules was averaged from the results of
several experiments. The values are shown as
the mean ehannel fluoreseenee of stained
eells minus that of the baekground control
(MOLT-4#8; about 33, MT-2; about 35, in average).
Table 5. Deteetion of eellular
membrane moleeules of
human on HTLV-I-binding BW5147eells and its
MT-2 eel1s.
eorrelation with the produeer
Mean ehanne1 fluoreseenee
mAbs to:
HTLV-I-treated BW5147
CD3
CD4
LFA-1
ICAM-1
LFA-3
CD2
MHC-I
MHC-IIDR
MT-2
3. 8
4. 0
35. 8
80. 7
15. 5
46. 9
58. 3
12 6. 0
38. 3
101. 6
5. 2
5. 7
87. 3
13 6. 8
33. 0
71. 0
Mean ehannel fluoreseenee on each eellular membrane
moleeules was averaged from the results of several
experiments. The values are shown as the mean ehannel
fluoreseenee of stained eells minus that of baekground
eontrol (BW5147; about 30, MT-2; about 35, in average).
Table 6. Inhibition of de novo synthesis by
translational inhibitors.
% positive eells
Pretreatment of
MOLT-4#8 eells
REY-7
untreated
84. 0
61. 0
O.1 pg/ml
1 pg/ml
88. 9
67. 0
85. 0
67. 3
5 pg/ml
84. 8
56. 2
50 pt g/ml
86. 8
5 8. 1
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(Referred from ref. 103)
HIV-1.
,
r
.
e Retrovirus
Membrane
.
s'
g p e 3t
NL-;-Lx"A"x(A)n RNA
ADSORPTION
orlltltllÅ}=ll=uen3' DNA
REVERSE
TRANSCRIPTION
Cytoplasm
s'
,
pipr e
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cell
g
INTEGRATION
2 o!tÅ} t=Å} #zr x me
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ASSEMBLY
MATURATION
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.
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Fig. 2.
.
r
o
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The genom-c organization of
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e
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Flg.
4. HTLV-I binding
and u1osrvfG (c)
cells.
to MOLT--4#8 (A)r
Raji
(B),
1OO
80
m
=o
o
o
År
60
.-
-
.
e-
m
o
40
a
oxo
20
o
tl
1OO
1: 10
1: 1
Ditution of virus
Fig. 5.
r"!OLT -- 4#8
Concentration-dependent H[VLV-I
cells.
binding to
1OO
80
co
=o
o
o
År
60
.-
i
.
e-
m
o
40
a
ÅrosÅqo
20
o
o
lncubation time (Minutes)
The kinetics of HTLV-: binding to
Fig. 6
cells at 370C (closed circle) or 40C (open
.
MOLT-4#8
circle).
1OO
10
80
8
m
=o
o
o
År
6
o
b6
=
o
=
-o
o
=
o
o
ut
o
M
o
=
,-,,-
60
6
•.-.
'
i
40
a
oXo
4
20
=
=
as
o
=
o
2
15
o
30
lncubation time (Minutes)
Fig. 7. Fluorescence positivity (open circle) and
mean fluorescence intensity (closed circle) were
increased by the 370C incubation of MOLT-4#8 cells
treated with HTLV-I at 40C.
1OO
80
m
o
o
År
=o
60
.-.
e
'
6
o
a
40
)o$Åqo
20
o
1: 20
1: 200
1: 2000
Dilution of sera
Fig. 8. Titration of binding inhibitory antibodies
in sera from HTLV-I-infected individuals. The virus
was pretreated with 1:20, 1:200, or 1:2000 diluted
sera from a seronegative donor (closed circle), an
asymptomatic carrier (closed square),an ATL patient
(open circle), and a HAM patient (open square).
LFA-1
ICAM-1
MHC-11 DR
e
=e
o
-o
b
o
E
=
.n
Z
Relative fluorescence intensity
Fig. 9. Detection of the host cell-membrane
proteins on HTLV--I-binding MOLT-4#8 cells. MOLT-4#8
cells were treated with medium (lines a and b) or
HTLV-: (line c) at 370C for 1 hour. The cells were
washed and reacted with medium (line a) or each mAb
(lines b and c) or medium (thin line) at 4Åé for 40
min. The washed cells were stained with goat antimouse rgG-FITC and the fluorescence intensity was
measured by FACScan.
BW5147
EL'-4
Je
-oo
"."'
5
A
E
=
=
Relative fluorescence intensity
Fig. 10.
cell
linesr
Binding of
HTLV-I to mouse T-lymphoma
BW5147 or EL-4 cells.
'
ICAMe' 1
MHC'"'11 DR
oo
=o
o
o
b
o
A
E
=
-em
=
Relative fluorescence intensity
F-g. 11
Detection of
( ZCAM -- 1 and
ceUs.
cellular molecules of human
MHC-IIDR) on H[PLV-I-binding BW5147
1OO
80
o
o
es
,e.,
=
o
o
M
o
60
a
40
20
1: 40
1:160
1: 640
Dilution of sera
Fig. 12. The detectable levels of cellular
membrane molecules on BW5147 cells were reduced by
pretreatment of the serum from an HTLV-Z
seropositive individual (circle) but not from a
seronegative control (triangle) with HTLV-! at 40C
for 1 hour. The rate of reduction by serum
pretreatment is shown for ICAM-1 (open) and MHC-ZZ
DR (closed) molecules as percentage of mean channei
fluorescence against positive controls
(pretreatment of serum-free medium).
-
REY-"7 GIN-"14
11
MOLT--4#8
m
=o
o
-o
8b
I• i"
U937
,
Relative fluorescence intensity
Fig. 13 Detection of HTLV-I gp46 (REY-7
mAb) and
MA (GrN-14 mAb) on viral--binding MOLT-4#8
cells .
.
and U937
r
1OO
80
.se
"o"'
o
o
.2
60
•---
en
o
a
40
;oso
20
o
20
40
60
o
20
40
Time after adding the virus (Min)
Fig. 14.
Kinetics of fluorescence positivity
gp46 (open circle)
and MA (closed circle) on
I-binding lvlOLT-4#8 (A) and U937 (B) cells.
for
HTLV •-
1OO
80
m
o
o
År
=o
60
.-
e .t
p-
m
o
a
40
;os(o
20
o
60
o
lncubation time (Minutes)
Fig. 15. Fluorescence positivity for gp46 (open
circle) and MA (closed circle) were increased by
the 370C incubation of MOLT-4#8 cells treated with
HTLV-: at 40C. However, cytochalasin B treatment
completely inhibited the increase in fluorescence
positivity for both gp46 (open bar) and MA (hatched
bar) after 60 minutes incubation at 37Åé.
1OO
80
m
=o
o
o
År
60
m unfixed
% methanol-fixed
.-.
e
'
6
o
40
g
yoo
20
o
REY-7
G1N"14
Fig. 16. Effects of methanol fixation on
fluorescence positivity for gp46 (REY-7 mAb)
(G!N-14 mAb) of MT-2 cells.
and MA
A
1OO
80
9
5o
60
o
.2
m untixed
z methanol-fixed
•.-.
o
o
a
40
)OikO
20
o
REY-7
GIN-14
B
1OO
80
9
5o
60
o
.2
m unfixed
z methanot-fixed
•.-.
e
o
a
40
oxo
20
o
REY-7
GIN-14
on
Fig. 17. Effects of methanol fixation
mAb) and MA
fluorescence positivity for gp46 (REY-7
(GIN-14 mAb) on HTLV-I-binding MOLT-4#8 and U937
cells.
1'
klkbH
HIV-1
-.rev..
LTR
vpr vpu
env
rev
HIV-2
vpx vpr
pol
S1VAGM
LTR
{
v; 'f
gag
I
1
J
l
pol
LTR
SIVMND
11111111111t
vif
gag
l
1
po1
LTR
-- -- -- tat
vt
llIl
,iiiil
------- ---p-
---"
l
tlvpx
[iiiil
II
nef
env
l
tat rev -
LTR
il::ll
..-.---i
1
---------
lneI
L
env
--....-
----•
vpr
.
Fig.
18.
Genomic organization of the
of primate immunodeficiency viruses.
tat-''"'-"rev-----•--
l
env
LTR
ilNi
I
nef
four groups
1
5 8 12 16 19 22 25
A
NL
Nd
Af2
Ss
eeee
Xh
Cr
5 8 12 16 19 22 25
B
NL
Nd
Af2
ss
Xh
Cr
Fig. 19. Growth kinetics of various HIV-1 mutant
clones in A3.01 cells (A) and pBMC (B). 1 x 106
cells were infeeted with 5 x 106 cpm of RT activity
of cell-free virus from transfected SW480 cells or
medium (mock infected; Cr), and progeny virus
production was monitored by RT production at every
3 to 4 days. NL virus is derived from pNL-432 wild
type clone of HIV-1. Nd (vif mutant), Af2 (vpr
mutant), Ss (vpu mutant), and Xh (nef mutant) are
mutants of wild type virus.
".N
5 8 12 16 19 22 25
4 7 " 15 18 22 25
c
GH•
Ec 1/
Crl
Cr
5 8 12 16 19 22 25
D GH[
E.C,
4 7 11 15 18 22 25
de ee'ee
eeee
Fig. 20. Growth kinetics of wild type viruses and
vpr mutants of H!V-1 (A, B) and HIV-2 (C, D) in
A3•Ol (A), MOLT-3 cells (C) and PBMC (B, D). 1 x 106
cells were infected with 5 x 105 cpm of RT activity
of cell-free virus or medium (mock infected; Cr).
NL and Af2 viruses from the pNL-432 wild type and
vpr mutant clones of HIV-1; GH and Ec from pGH-123
wild type and vpr mutant clones of HIV-2r
respective ly
A
4
7 11 15 18 22 25
GH
Xb
ee ipt e
st
Ec
Ml
Cr
B
4 7 11 15 18 22 25
GH e
Xb e
ew
st e
e dv ma•
tw ee
Ec e
Ml e
Cr
Fig. 21. Growth kinetics of various HIV-2 mutant
clones in MOLT-3 cells (A) and pBMC (B). 1 x 106
cells were infected with 5 x 106 cprn of RT activity
of cell-free virus from transfected SW480 cells or
medium (mock infected; Cr), and progeny virus
production was inonitored by RT production at every
3 to 4 days. GH virus is derived from pGH-123 wild
type clone of HIV--2. Xb (vif mutant), St (vpx
mutant), Ec (vpr mutant), and MJ (nef mutant) are
mutants of wild type virus.
4 7 11 15 18 22 25
GH
Xb
st
Ec
Ml
Cr
Fig. 22. Growth kinetics of various HIV-2 mutant
clones in another PBMC preparation. 1 x 106 of PBMC
separated from a different individual were infected
with 5 x 106 cpm of RT activity of cell-free virus
from transfected SW480 cells or inedium (mock
infected; Cr), and progeny virus production was
monitored by RT production at every 3 to 4 days.
O/o conversion
NY5-CAT 460
473
GHI-CAT 460
473
3.3
1.6
3.7
3.4
GH2-CAT 460
473
•
MAC-CAT 460
473
TYOI-CAT 460
473
TY02-CAT 460
473
TY05-CAT 460
473
TY07-CAT 460
473
e
MND-CAT 460
e
473
.
8.7
s
5.9
"
6.7
3.0
.e
•
e
28.3
pt e
21.9
ee
32.1
1 7.0
•
r
•
e-
•
•
e
e
7.1
ee
26.2
8.4
16.3
e
5.7
5.1
e
nef gene expresslon
F-g. 23. Effect of
]]TRs. 10 pcg of
expression vector of
'
on various
the nef gene
(460) or its
clone pcD-SRa ne f4 60
from pNL-432 HIV-1
mutant pcD-SRanefAKpnl473 (473) werecotransfected
with 10 ptg of each
-nto SW480 cells
of HIVISIV LTRCAT constructs,
NY5-CAT (HIV--1), GHI-CAT (HzV-2)r
GH2-CAT (HIV-2),
MAC-CAT (SIVMAc) TYO1--CATto TY07CAT (SIVAGM)
and
MND--CAT
(SIVMND). CAT actzvlty ln
the cell lysates was
determined at 48
hours posttransfection. The
percent conversion
of chloramphenicol to itsacetylated forms is
indicate on the
right.