Abstracts Vol. 1 [International Conference on AIDS (11th: 1996: Vancouver, Canada)]

Tuesday, July 9, 1996

Tu.A.495 -Tu.A.512

Tu.A.495
THE MEMBRANE-PROXIMAL INTRACYTOPLASMIC TYROSINE RESIDUE OF
HIV- I ENVELOPE GLYCOPROTEIN IS CRITICAL FOR BASOLATERAL TARGETING
OF VIRAL BUDDING IN EPITHELIAL CELLS
Cohen, Lric A, L odge R, L al nde J. I emay G. i)Departm ent de ni i, a t nmunologie,
Universite de Montreal, Montreal, Otuebec, Canada
Budding of retrovi uses from infected cells takes place specifically,t iir iso!ateral membrane surace of polarized epile lial adin -Darty canine kidney,i (Mil)( K). I his sorting
event is suspected to require a specific signal hirbored by the viral!ycp o tro!n envelope
and we previously showed tht, ats for most basolateral proteins, thp. o inti.rc top'isnic
dormain plays a crucial role in this ren phenomenon. It is well, th,, tyrosinebased motifs are a central ler 'nt in asolate l t argeting signals. ii ti, r:sernit study we
used site-directed mutagenesis to generate conservative or non one vattv substitution of
each four envelope glycopiotein intracytopiasmic tyrosines of human ev ' nodeficiency
virus (HIV I). The membrane proxiinal tyrosine is essn tial to e re solaeral targeting
of HIV I vinions. Substitutions of th e membrane proxirnal tyrosine did not appear to affect
incorporation of envelope glycoprotein to the virions nor its capaiilit, ti en-r1 its role in
viral infection. The effect of tihe four accessory HIV -I proteins nef, vi(f, and v,, was
also examined and it was clearly shown that they are not involved in basolateral targeting.
EA Cohen, CP6128, Succursale Centret ville, Monrtreal, Quebec, H 3Y C ada
Tel: 51t-343-5967 Fax: [phone]
Tu.A.50 I
SUPPRESSION OF HIV-I REPLICATION BY RANTES OCCURS AT AN EARLY
PRE-INTEGRATION LEVEL
SPoi, Guido, Ghezzi S, Alfano M,\icenzi E San R affa ele Scientif stic,t,,. M ',i, Italy
Objective: to identify the mchanisin of actionr of RANTES as acr antiHIV ctemrrokine.
Methods: 1 cell blasts from runinfected individuals were infected willt, igh primary HIV isolates in the presence or abse n e of RAN IfES (100Ing/ml), and th e, itii 5-(,e monitored
for the production of RT ac tivity of p24 Ag production. MT-2 calls were used for determining the fusogenic abilities of different primary isolates. For HIV DNA P(R t. experimrents,
aliquots of infected blasts were tharvested at different hours after irnfection.. r sets of
primer pairs, detecting very eily (minu s strong stop DNA; prime rpair I, U and nearly
complete (US -gag) HItV DNA were used.
Results: RANTES consistently inhibited the replication of several primary HIV isolates in T
cell blasts, although with different effiiency (range of inhibition: I4- 9/). No correlation
was observed between RAN1 F5 suppressive capacity and the i,,ia viral pihenotype (SI vs
NSI). The suppressive effect of RANT ES was observed in expe rinental conditions were
cells were first infected for I Ii and then exposed to the chermoiire, m aking it unlikely that
RANTES inhibited the initial phases of receptor binding and entry into target cells. A
more sustained effect of the chemokine was, however frequently obse red when blasts
were pre incubated with RANiFS, and this experimental condition was used to, investigate
the step of reverse transcription.i lwo primary isolates that had previously shown complete
and partial susceptibility, respectively, to the inhibitory effect of RANI fES were selected.
Complete and partial inhibition of reverse transcription was observed in T cells blasts after
a single incubation with the chen okine. Inhibition or suppression of rioviral DINA formation by RANTES involved also the synthesis of minus strong stop DNA.
Conclusions: RANTES is a ipotent suppressor of HIV replication which acts at an early
step of the retrovirus life cycle, likely following entry into target,ells, d p roeeding or
directly interfeng with the reverse transcriplion of the viral gerroe,.
G Poli,Via Stamira d Ancona N.l 20, Centr o San Luigi 20 I 27, Milano, Italy Tel: [phone]
Fax [phone]
Tu.A.503

the entire env gene or a fragment spanning the V2 to V5 region of env. Nucleotide,equences of clones that exhibited unusual mobility patterns were obtained and compared
with those of the two inoculated strains.
Results: Chimpanzees infected with the clade B HIV- I strain LAI(IIIB) for up to 32 months
readily became infected with the clade E strain CAR/E4002, as shown by increased antibody titers to HIV- I and isolation of the second virus from PBMC 2 weeks after inoculation. HMA of at least 20 clones from PBMC and lymph node cells revealed changes in the
relative proportion of the two HIV-I strains over time and the presence of possible recombir nt viruses. In DNA isolated from lymph node tissue obtained 24 months after infection
with the second virus, two different recombinants were identified in multiple independent
PC.R assays. I hese recombinants were confirmed by DNA sequence analysis.
Conclusions: Infection with and recombination between two diverse strains of HIV- I can
occur in vivo everi though the second strain establishes infection after the first strain.
Although the clade E HIV- I predominated in PBMC initially within 6 weeks after inoculation,
it declined and became the minor viral species suggesting immune-mediated control.These
results indicate there is ongoing active replication of HIV- I in clinically asymptomatic chimpanzees, as i hhusnans.
Patricia N Fultz, Ph.D. University of Alabama at Birmingham Birmingham, AL 35294 UAB
Station, BBRB 51 1, USA. [phone] (telephone)
Tu.A.51 I
CONTINUOUS IN VIVO RECOMBINATION BETWEEN HIV- I STRAINS OF GENETIC
SUBTYPES A AND C
Salminen, Mii _DO, Robertson DL2, Sharp PM2, Hahn, BH3, Burke DS4, McCutchan FE I.
I M. Jackson Foundation and 4 WRAIR, Rockville MD; 2Univ. Nott., UK; 3Univ. Ala., Birm., AL.
Introduction: Recombination between two genetic subtypes of HIV- I can occur, and clades
A through I are known to have participated in these genetic exchanges. Recombinant
fo nmrs are thot aght to result f-om double infections and homologous RNA recombination
between the infecting strains, but it has not yet been established conclusively that recombinants occur i rivoe. We have studied HIV-I genomes firom epidemiologically linked patients
in Zambia fiori whom serial samples were available. Multiple different, but related, A/C
inter-genotypic recombinants were found in both primary and cultured PBMCs.
Methods: Serial samples obtained more than one year apart from the index case and a single sample firom her spouse were analyzed. Complete genomes and/or full-length gag and
en genes were PCR amplified using DNA firom virus cultures on peripheral blood mononuclear cells (PBMCs) or from primary patient PBMCs, and were molecularly cloned and
sequenced. DNA sequences were analyzed for the distribution of recombination breakpoints
by bootscanning, by distance scanning, and by analysis of phylogenetically informative sites.
Results: Analysis of a fill-length HIV- I genome firom the virus culture of PBMCs obtained in
1990 from a Zambian woman established that the provirus was recombinant between subtypes A and C in gag, in pol, and in env.Virtually identical sequences were recovered from
primary and from cultured PBMCs.The gag and env genes of the virus from a 1989 sample,
and troma sample taken at the same time from the woman's husband, were closely related
to lthe 1990 sequences but had different breakpoints. Furthermore, the breakpoints were
not completely shared between the contemporaneous samples of the husband and wife.
Conclusions:T hese results establish that HIV- I inter-genotypic recombination occurs in vivo,
and that multiple recombinant forms can arise over time an infected individual.The predominance of different recombinant forms in serial samples suggests ongoing selection.
MOit Salminen, I 600 East Gude Drive, Rockville, MD 20850 USA
Telephone: + 1-30 I -217-941I0 Fax: [phone] email: [email protected]
Tu.A.512
EVIDENCES FOR AN IMMUNE SELECTION PRESSURE ON THE FIRST
HYPERVARIABLE REGION OF HIV- I AND HIV-2 ENV GENES IN VIVO
Lf Vva, Andrieu JM. Laboratoire d'lmmunologie des Tumeurs, H6pital Laennec, Faculte de
Medecnec Neclken Universite Rend Descartes-Paris V, Paris, France.
Objective: Ta depict the relationship between the variation in length of the first hypervariable region (V I) of HIV env gene and isolate-specific neutralizating antibody response along
tIe course of the infection.
Methods and Results: A viro-immunological study was conducted on four HIV-I infected
individuals and one HIV2 infected subject from <10 weeks to 3-6 years after their serocoe rsion Polyrmerase chain reaction (PCR) demonstrated that near the time of serovonversion a sirnie band of the V I ftragment was observed in the serum-derived HIV RNA of
all individuals; its length was however different in each patient. It was intriguing that one
HIV- I seroconverter who failed to mount neutralizing antibodies and progressed to AIDS
rapidly (<2 years) exhibited a constant singleVI band over time, although HIV-specific cytotoxic t1 lyinphocytes (CTLs) activity was steadily detected during the course of the infection.
In the rerraining three HIV- I infected patients who developed isolate-specific neutralizing
response shortly after seroconversion and remained asymptomatic for 4-6 years, theirV I
genes evolved rapidly from a single band to multiple bands. Fluctuation in the length of multiple\/i bands was associated with temporal change in sensitivity of the viral isolates to
ccc ccti ai it mcn by autologous sera.The cessation of thisVI-length fluctuation was associated
oihm errreumnece of neutralizatioresistant variants that preceded the accelerated CD4+ Tell depletion ard disease progression. In a 6-year follow-up of one seroconverter with
s~yn u rt,1r111c 1HIV 2 infection, fluctuation in the length of a single VI band and dynamic isolate specifc rnetralizing antibody response were observed in parallel. In contrast, when the
Ili,elites talerr frorn PBMk of ill five patients at a time close to their seroconversion
-eme schism. on over I year (> 10 passages) in donor PBMC (i.e. without the selection poetuce of the irmrnune system), their VI band remained constantly unchanged.
Conclusions: These findings denote that neutralizing antibody response to HIV constitutes
n selective forces to drive viral diversity in vivo and that absence or loss of
lIrsus l rc presure predict the virus spread and disease progression.
I c:eu Lu,1, aboraoire dlhrmmnologie des iumeurs, H6pital Laennec, 42 rue de Svres,
/50nn7 -sis. Iu ance. el: 33 (I) 4439-6407 - Fax: 33 (I) 4439-6465

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CHEMOKINES AND HIV INFECTION
Levy ay A, Mackewicz C, Baker E, Strarnford
Medicine, University of 5(alifotr ria, San F rancisc
The c and 3 chemokines have been shown to
peripheral blood CD4+ cells and established
products required for efficienrt suppression of
endogenously produced by CD8 -cells. More
chemokines inclrding (1)4 I cells. the ability )
CD4+ cells is currently under srtmdy. Physicoch
prodution and levels of chenmokinre.secretion
slances are not the CD8+ cell ntiviral factor
CAF production correlate; wihin aymptolm
tion by (C )8+ cells are similarm n 1 ifected
T+bodies to these (insss and ml i e t l:irest tltl- trc
in vitro Finally, CAF p roducti byIn tno svirus s
correlate with levels of 13 chemline pniodIuctic
Ie, MIP-II3 and other cherlmn vil - 1 is

o, CA, USA
Sblock replicatior
ell Ines. -Ioweve
virus is above the
over other cells ca
)f these chemokirn
oemical analyses, ti
by lymphocytes n
((At) that we ha
atic sltate while Iindividuals manic '
nin CAF likewise0
cbelow those show
sum linir -transformed
ron. The potentlit inr
tuicer further s;tu.,f (, rtr ains of HIV in
the concerrt- ation of these, - n(,onnal's found
n pr oduce thei
-s to influence rinfection of
e Pl kW c)a chemokine
)Ailte that thesne sub-. W nbed. Moreover,!(, of hnommmne pmroduc,ting diffuerent clinical stages.
nre 1,01 n ifficted by antin to hc re a lviral activity
I8,1 II unes does not
tc-, tin r f RAN TES, MIP
Cancer Reseach Irntimute and Dept. of

JA Levy M.Il). ance Rrerch Institute Uni el ity of c(al to I -, n o. CA, 94143
0128, USA. tel.: 4 15 47, 407 Fix: -1m 476 82,6' L il:
Tu.A.510
INVIVO RECOMBINATION BETWEEN TWO HIV-I STRAINS FROM DIFFERENT
CLADES FOLLOWING SUPERINFECTION OF CHIMPANZEE
Fultz, Patricia N, Wei Q*,Yuce I, Barre Sinoussi F', iard M i Alabama
at Birmingha Birminhamtr, Al, ISA:' Iii it l P,tsie Paris,
Objective:-o determine s tll' m( iiF etween two us hrmr different
Clades occurs in vivc n n ufT -u Jc lu- of cl r hl ii z 's.
Methods: Chimpanrzees inlet tlfo sire thr r I n v< wint HsIV ' u1(lll,:re noculitled
IV with a subtype (dade) l sltin, A 11/E10(1.Proviral DNA il,,.!f n i i PBMl and
lymph node biopsie s atdfe l i fl inx lalir. etr,4 i nobiity ssays
(LIMA) were mm ci 5ci '1cmmcnrt lpins I_ n ~ "t01ie romsl'( I' Indiii;,' ~dcs