Bridging the Gap: Conference Record [Abstract book, International Conference on AIDS (12th: 1998: Geneva, Switzerland)]

14 Abstracts 11158-11162

12th World AIDS Conference

the evolutionary pathway back to the naturally occurring 215T variant is a least
favoured option. Our data would fit in the hypothesis that, after a bottleneck
transmission, the evolution of zidovudine resistant RT is moving in a distinctive
direction as a Muller's ratchet.
111588 HIV-1 cellular RNA load arise in late stage of HIV-disease
but do not correlate with the proviral sequence variability:
Cross-sectional study representing all stages of infection
Abderrazzak Merzouki1, L. Bouhdoud1, F. Mandy2, M. Arella1. Institut Armand
Frappier 531 boul des Prairies, Laval, Quebec; 2Health and Welfare Canada,
Ottawa ON, Canada
Objective: To study the relationship between the ongoing viral replication in
PBMCs (measure of the differences in the relative abundance of HIV-1 regulatory
and structural mRNAs) of 30 patients at different stages of the infection and the
proviral sequence variability.
Methods: We describe a cross sectional study of multiply spliced (tat, rev, and
nef mRNA) and unspliced (gag-pol mRNA) HIV-1 RNA expression using a reverse
transcriptase-quantitative, competitive PCR (RT-qcPCR) assay in a cohort of 30
patients (10 in stage II, 10 in stage III and 10 in stage VIC1). We used the
nested-PCR to amplify the V3, C4 and C5-C6 env gene regions. Amplicons were
cloned and up to 10 from each sample were sequenced. Phylogenetic analysis
and calculations of intra- and inter-patient variability were performed by using the
software PHYLIP version 3.5.
Results: mRNA quantitation data are expressed as copy number per 1000
PBMC. Patients in stage II showed intermediate levels of RNA expression (between 10 and 300 copies) with a mean ratio of total regulatory to gag-pol mRNAs
of 6.2 ~ 4.8. Patients in stage III and IVC1 showed a high level of total regulatory
mRNA (between 20 and 600 copies) and a rising level of gag-pol mRNA (between
50 and 500 copies) resulting in a 2.3 fold reduction of the mean splicing ratio, from
6.2 ~ 4.8 to 2.7 ~ 1.4 (stage III) and 2.8 ~ 1.7 (stage IVC1). These RNA patterns
are unrelated with sequence proviral variability. Both intra-/ and inter-patient V3,
C4 and C5-C6 sequences displayed great variability (ranging from 6.1% to 12.5%
for the V3, 1.8% to 8.6% for the C4 and 2.0% to 6.8% for the C5-C6 region)
irrespective of the degree of infection progression or the ongoing viral replication
in PBMCs.
Conclusions: We showed that the ratio of multiply spliced to unspliced RNA
declines by threefold over the course of HIV-1 infection while viral RNA expression in PBMCs of infected individuals have a rise in total viral RNA in late stage
HIV-disease. Such rise is unrelated with the level of proviral sequences variability
but follows the appearance of variants with predicted SI/T-cell tropic phenotype
(rapidly. replicating and cytopathic phenotype) that map the V3 region.
|11159 Characterization of a new long-term T-cell line SCHT
persistently infected with HIV-1
Galina G. Miller1, A.A. Kushch2, L.A. Glukhowa2, R.R. Klimova2, I.V. Titova3,
L.N. Pokidicheva3. 1Gamaleya Str. 18 123098 Moskow; 2lnst of Virology; 3The
Gamaleya Inst for Epid & Microbiol Moskow, Russia
Objectives: The persistently infected T-lymphoblastoid cell lines are the manageable experimental models for in vitro studying molecular and cellular mechanisms
involved in the occurrence and development of the persistent form of HIV infection
and pave the way to controlling the foci of persistent HIV infection in people.
Method: An investigation of SCHT in dynamic of productive persistent infection.
Western blot (WB) analysis of the SCHT viral proteins. Chromosome analysis
and Karyotype reconstruction. Detection of a site of HIV genome integration on
chromosomes by in situ hybridization. Evaluation of receptor activity of SCHT by
monoclonal antibodies (MAB). Estimation of expression and accumulation of HIV
structural proteins directed by genes env and pol.
Results: SCHT cell line is able cultivating in vitro unprecedently long period
of time without any destruction and without adding uninfected cells to support of
infection. Abundant virl production and long-term stable expression of the full-size
major structural proteins were detected. Chromosome analysis carry out every
2-3 years was revealed a great stability of its karyotype. All cells are contain of
integrated proviral DNA HIV in chromosomal DNA.
Conclusion: New type of T-cell line SCHT with high infective viral production
and high-level expression of the viral genes without cell death was described. We
speculate possibility to development of a non-lytic form of HIV infection in vivo.
SCHT cell line is offer as a useful model to search fundamental problem of HIV
persistence.
| 11160 Interaction of HIV-1 and hepatitis B virus (HBV) in mixed
infection
Svetlana V. Antonenko1'2, E.V. Barbasheva2, E.V. Murashko2,
A.M. Scherbinskaya2. 14 Uzviz Protasiv Yar, 252038 Kiev; 2Res. Inst.
Epidemiology & Infection Disease, Kiev, Ukraine

Objectives: To study some events conserning of HIV-1 and HBV interaction in in
vivo mixed infection.
Methods: The blood samples of HIV-infected patients with chronic hepatitis B
were tested for HIV-1 proviral DNA level in PBMCs, HBV DNA level in plasma
using a quantitative PCR, HIV-1 antigen p24 level in plasma by ELISA. The results
were compared with the clinical stages of disease. Some biological properties

of clinical HIV-1 isolates were detected by indirect immunofluorescence, reverse
transcriptase reactions, PCR, light microscopy.
Results: HIV-infected patients with chronic hepatitisB who had high amounts
of HBV DNA had significant higer amounts HIV-1 proviral DNA. They were more
frequently HIV-1 p24 antigen positive compared to patients with low amounts of
HBV DNA. Comparative analisys of biological patterns HIV-1 isolates from HIV-1
positive patients in the periods activation of chronic HBV infection shows this
isolates to posses significantly higer infective, replicative and cytocide activity.
The data obtained testifly to the mutual influence of the HIV-1 and HBV upon
the mixed infections.
11161 1 Infectious molecular clones with the non-homologous
dimer initiation sequence found in different subtypes of
HIV-1 can recombine and initiate a spreading infection
Daniel St. Louis, D. Gotte1, E. Sanders-Buell1, M.O. Salminen2, J.K. Carr1,
F.E. McCutchan1. 8901 Wisconsin Ave., B-17 Rm. 10, Bethesda, MD; 1 Henry M.
Jackson Foundation, Rockville, MD, USA; 2National Public Health Institute,
Helsinki, Finland
Background: The direct involvement of recombination in the generation of HIV-1
diversity has emerged from analysis of viruses from multiple geographic locales.
The formation of RNA dimers within the virion is essential for the high level of
recombination exhibited by retroviruses. Sequences responsible for in vitro HIV-1
RNA dimerization, dimerization initiation site, DIS, vary among HIV-1 subtypes
and may be an obstacle for recombination.
Method: DIS loop sequences was determined in 75 viral isolates of subtypes
A, B, C, D, E, F, G, and group O. We constructed deletion mutants of an infectious
molecular clone of HIV-1, NL4-3, containing DIS variants identified in the HIV-1
subtypes and an artificial, AAAAAA DIS sequence and evaluated the effect of
DIS variants on virus replication and recombination.
Results: Two forms of the DIS occur among HIV-1 isolates examined to date:
DIS (CG) in HIV-1 subtypes B and D and DIS (TA) in other subtypes and in
group O. Unexpectedly, a naturally occurring but apparently rare DIS that is not
palindromic and should form a less stable duplex, and an artificial, AAAAAA sequence that should be unable to form a duplex, both supported virus replication in
vitro when introduced into the infectious molecular clone NL4-3. Furthermore, the
recombination frequency between deletion mutants of NL4-3 was not significantly
affected by DIS loop inhomology.
Conclusion: The different DIS found in HIV-1 subtypes B and D is not a primary
impediment to RNA dimer formation, and recombination, with other subtypes and
that A/D and B/F recombinants, already identified in the global epidemic may be
an example of recombination between virus with different DIS.
S11162 Biology and molecular biology of HIV coinfection with
divergent HIV strains
Bin Wang1, R.B. Lal2, D.E. Dwyer1, A.L. Cunningham1, N.K. Saksena1.
1Retroviral Genetics Lab CVR, WIHR R = Z145 Westmead Hospital, 2nd Floor
Westmead NSW 2145, Australia; 2Centers for Disease Control, Atlanta Georgia,
USA
Objectives: To analyze the biology and molecular viral strains (recombinant and
non-recombinant) from HIV-1 co-infected and multiply exposed individual.
Design: Systematic study of viral sequences of different infecting strains, their
replication kinetics on diffrent cell types, and coreceptor usage by different strains
in vivo.
Methods: The HIV-1 viral quasispecies from the PBMCs of an HIV-1 infected
IVDU was analyzed by PCR amplifying proviral DNA amplified in the gp120
env and the vpr gene from peripheral blood mononuclear cells (PBMCs). Fulllength gp120 and Vpr gene fragments were cloned into PGEM-T vector, and
sequencedon automated sequencer. Viral strains isolated by coculture were further analyzed for tropism and replication kinetics on PBMCs, monocytes, and
monocyte-derived macrophages. In addition, the phenotypic detrmination was
carriedout on MT-2 cells, and the coreceptor usage on HOS cells.
Results: These data provide evidence in favor of coinfection of the patient with
two divergent strains of HIV-1. Based on gp120 env region, the two groups differed
by 14-15%, with more than 25% difference between various variable domains
(V1-V5) of the gp120 region. Coculture experiments on different cell types further
provided evidence in favour of co-passaging of two strains suggesting complementation between two strains present in the patient. Molecular analysis further
revealed that complementation occurred between intact and defective strains,
with one of the viruses acting as a helper virus for the other strain to survive. The
study of viral replication kinetics in PBMCs, monocytes and monocyte derived
macrophages (MDMs) revealed the virus to be T-cell tropic. Coreceptor usage
data shows that one of the viral strains from the patient may use CCR-5 or CCR-1,
2, 3 and CXCR4 independent pathway for its entry into the cell. Such strains
are extremely rare in nature and suggest a usage of some unknown coreceptor
molecule by this HIV-1 strain.
Conclusions: This study demonstrates for the first time that complementation

between defective and intact HIV-1 strains can provide a mechanism for the
continuation of defective/attenuated viral lineages in vivo. Molecular changes in
the gp120 gene may lead to differential tropism and co-receptor usage in IHV-1
strains.