Final Program and Oral Abstracts [International Conference on AIDS (8th: 1992: Amsterdam, Netherlands)]

MoA 0072-MoA 0076

MONDAY, 20 JULY 1992

MoA 0072 Small amino acid change in the HIV-1 V3 loop can affect viral
infectivity and not cytopathicity. Cheng-Mayer, Cecilia
Cancer Research Institute, Univ. of California, San Francisco,
San Francisco, CA
Objective: To examine whether single amino acid change in env. specifically a region of gpl20 that
encompasses the V3 loop affects infectivity and cytopathicity of HIV-1 in vitro.
Methods: Biologic properties of site-directed mutants of HIV-1 were determined. Sequence analyses
confirmed the nature of the mutations and two independent clones of each mutant were studied with
similar results.
Results: Replication in Syncytia formation in
HIV-1 306 V3 Sequence 333 PMC HUT78 HUT78
SF13wt TRKGIHIGPGRAVYTTGRIIGDIRQAHC + + ++
mul ---S-Y------FH------ ---- + + ++
mu2 ------------H------------ +* NA
mu3 -------------H---- ---- - - NA
SF2wt TRKSIYIGPGRAFHTTGRIIGDIRQAHC + + +
mul ---G-H------VY----- ----- +* +* +
mu2 -----H-------Y----------- + +* +
mu3 -----H----------------- - - NA
mu5 ---G-H------VY--------T----- NA
*delay in peak RT by at least 7 days; NA, not applicable
Conclusions: These studies of SF13 and SF2 mutants showed (1) amino acid (aa) change in the V3 loop
had no effect on CPE (e.g., SF13mul,SF2mul) (2) complementarity of amino acids at positions 311
and 319 within the V3 loop (e.g., histidine/tyrosine in WT vs histidine/histidine in SF13mu3, SF2mu3)
influences HIV-1 infectivity (3) a single aa change in the conserved DIRQAHC sequence of the V3 loop
abolishes infectivity (e.g., SF2mul vs SF2mu5) (4) a single aa change outside the V3 loop has been
found to affect viral infectivity. All these results indicate that the structure of the V3 loop, influenced by
amino acid changes within and outside of the loop region, governs HIV-1 infectivity and cytopathicity.
Cheng-Mayer, Cecilia Cancer Research Institute, Univ. of Calif.,
San Francisco, San Francisco, CA 94143-0128
M oA 0074 Structure-Function Relationships of HIV1 Envelope V3 Loop in Regulation of Host
Cell Tropism. Christoph Ebenbichler, Ruili Gu, Timothy Henkel, Peter Westervelt
and Lee Ratner, Departments of Medicine and Molecular Microbiology,
Washington University School of Medicine, St.Louis, USA
Objective: To identify the determinants within env that regulate host cell tropism, and analyse their
-5ructural features and mechanism of action. Results: Chimeric viruses were constructed with
sequences from a monocyte-tropic isolate (ADA) or those from brain or spleen tissues of AIDS patients
together with sequences from isolates incapable of macrophage infection (NLHX). In each case, the
tropism determinant mapped to a 94 amino acid portion of the gpl20 envelope protein (tropism domain)
that includes the principal neutralization domain (V3 loop). Comparisons of tropism domain sequences of 3
non-macrophage tropic and 8 macrophage-tropic clones demonstrated that residues in (275,283,287) or
agjacent to (313) the V3 loop were different between clones with different tropism. Mutation of residue
287 from K (in HX) to E (in ADA) conferred partial activity. Each of the chimeric envelope proteins were
expressed in recombinant vaccinia viruses in a monkey kidney cell line and in primary human
lymphocytes and macrophages. The envelope proteins capable of conferring macrophage infection showed
less cleavage in the V3 loop by a cell surface protease than did those envelope proteins which did not
allow macrophage infection. Similar results were obtained in thrombin or tryptase cleavage studies of
purified envelope proteins produced in an insect cell system. Antibody reactivity studies demonstrated a
different conformation for the V3 loop of the macrophage tropic envelope proteins than the
non-permissive envelopes. Summary: A macrophage tropism domain for envelope has been mapped to a
94 amino acid segment including the V3 loop; residue 287 was shown to be partially responsible for this
activity. Altered antibody reactivity and limited susceptibility for V3 loop cleavage correlate closely
with macrophage tropism. These findings suggest that a host cell protease which cleaves V3 may be
responsible for regulating HIV1 infection of macrophages.
Ebenbichler Christoph, Department of Medicine and Molecular Microbiology, Washington
University School of Medicine, Box 8125, 660 S. Euclid, St. Louis, MO 63110, Tel [phone], Fax [phone]
M oA 0076 NOVEL DETERMINANTS OF TROPISM OF HIV-1 IN gpl20 AND gp41
Peden, Keith Silver, Jonathan; Fujita, Kazunobu; Laboratory of Molecular
Microbiology, NIAID, NIH, Bethesda, MD 20892, USA.
Objective: To ascertain the sequence changes that account for the enhanced replicative capacity of the
ELI strain of HIV-1 passaged in H9 cells.
Methods: Virus stocks prepared from HeLa cells transfected with molecular clones of HIV-lEu were
used to infect peripheral blood mononuclear cells (PBMC) and CD4-positive cell lines. DNA isolated
from infected cells was analyzed by PCR-SSCP (Single Strand Conformation Polymorphism) to locate
sequence changes in the HIV-1 genome. These identified mutations were introduced into the parent
genome, and the growth properties of the resulting viruses were characterized.
Results: Virus derived from a molecular clone of the ELI strain of HIV-1 exhibits delayed replication
kinetics on H9 cells and is unable to infect productively U937 cells. When the virus that emerged from
H9 cells was used to infect different CD4-positive cell lines, it was found to have acquired altered
growth characteristics, viz., it exhibited accelerated growth kinetics on H9 cells and was able to infect
U937 cells. Sequence changes in the genomes of the passaged virus were mapped to the env gene using
PCR-SSCP. Clones were found with sequence changes in the N-terminus of gp41, a region involved
with fusion, and with changes in both the N-terminus of gp41 and in the CD4-binding region of gpl20.
No changes were found in the immunodominant V3 loop region, a known determinant of viral tropism.
Mutations in gp41 alone conferred upon ELI an enhanced replication capacity in H9 cells, since the
mutant viruses infected these cells without the delay in virus production seen with the parent virus. The
additional change in the CD4-binding site of gpl20 was necessary before ELI was capable of infecting
U937 cells. None of these mutations affected the ability of ELI to replicate in PBMC.
Conclusions: These results demonstrate that HIV-1 can adapt to growth in certain tissue culture cell
lines by selecting for variants with mutations in the env gene. These data also show that the CD4 -binding region of gpl20 and the fusogenic region of gp41 are determinants of HIV-1 tropism. While
the mechanism of the enhanced replicative capacity remains to be elucidated, the locations of the
mutations strongly suggest that increased virus uptake is involved.

MOA 0073 IDENTIFICATION OF INDIVIDUAL AMINO ACIDS
WITHIN THE V3 REGION DETERMINING SYNCYTIUM FORMATION AND
REPLICATION RATE OF VIRUSES CHIMERIC FOR V3. De Jon. Jacques: De Ronde, A.;
Keulen, W.; Tersmette, M.; Goudsmit, J. HRL, UvA, Amsterdam, the Netherlands
Objectives: Determination of position and kind of amino acids within the V3 region associated with the syncytium
forming capacity and replication rate of V3-chimeric viruses.
Methods: V3 regions differing in four amino acids derived from a non-syncytium inducing (168.1/NSI) and a syncytium
inducing isolate (168.10/SI) of one individual, were cloned into a full-length HXB-2 molecular clone. Transfection in
SupTl-cells generated HXB-2 viruses, only differing in the V3 region. The phenotype of each chimeric virus
corresponded with the phenotype of the virus from which its V3 region was derived.
position: 1 11 22 25 29 35
S4 4 4 4 4
168.1 /NSI CTRPNNNTRKSIHIGPGRAFYATGDIIGDIRQAHC
168.10/ SI ----------R----------T--Q---N------
To determine the minimal amino acid changes responsible for the observed phenotype change, a set of chimeric viruses
(24) covering all possible combinations of the four amino acid changes was constructed by PCR. The complete set of
molecular clones was transfected by electroporation in SupTl-cells; syncytia formation as well as p24 core antigen
production were monitored for 14 days after transfection.
Results: Of the molecular clones containing single amino acid changes only the R (1681/R) at position 11 was able
to confer the syncytium inducing/fast replicating phenotype to 168.1. Transfection of the 168.10 molecular clone
resulted in the killing of all SupTl-cells by massive syncytia formation. Transfection of the 168.1/R molecular clone
did not cause the killing of all the transfected cells, because the initially formed syncytia disappeared after 12 days of
cultivation. The complete 168.10 phenotype was obtained when, in addition to the R mutation, one extra mutation,
with the exception of the T at position 22, was generated.
Conclusions: The R amino acid at position 11 is the most important contributor to the switch from the 168.1/NSI to
the 168.10/SI phenotype. However, to obtain the complete 168.10/SI phenotype one additional mutation, at position
25 or at the not previously described position 29, is necessary.
Jacques de Jong, Human Retrovirus Laboratory, UvA, Meibergdreef 15, 1105 AZ Amsterdam,
the Netherlands, phone [phone], fax [phone]
MOA 0075 PHENOTYPE ASSOCIATED ENV GENE VARIATION AMONG 8 RELATED HIV-1
CLONES: EVIDENCE FOR IN VIVO RECOMBINATION AND DETERMINANTS OF
CYTOTROPISM OUTSIDE THE V3 DOMAIN. Groenink. Martijn; Andeweg, AC."; van
der Jagt, R.C.M.'; Schuitemaker, H.'; Bosch, M.L; Huisman, J.G.; and Tersmette, M.'.
Centr. Lab. Netherlands Red Cross Blood Transf. Serv., Amsterdam. Lab. Immunobiology,
Nat. Inst. Public Health and Environmental Prot., Bilthoven, The Netherlands.
Obiectives: To genetically compare phenotypically heterogeneous HIV-1 variants and to analyze
regions of the HIV-1 env gene previously identified as determinants of biological properties.
Methods: Eight phenotypically heterogeneous HIV-1 clones, recovered directly from a single patient
peripheral blood mononuclear cells under limiting dilution conditions, were compared for the
nucleotide sequences of their env genes. In addition, the accessory gene sequences were obtained
for four of these clones.
Results: Four clones with intermediate phenotypes were the product of genetic recombination events.
In contrast to the accessory genes, all 8 HIV-1 clones demonstrated extensive phenotype-associated
sequence variation dispersed non-randomly over the env gene. Sequence variation associated with
syncytium inducing (SI) capacity and tropism for the MT2 T-cell line was concentrated in the C1-V4
region of the gpl20 glycoprotein. In contrast, sequence variation associated with tropism for the H9
and Sup T1 T-cell lines was completely limited to the gp120-V5 region and the gp41 glycoprotein.
The fusion domain, and amino acids involved in gp120-CD4 and gp120-gp41 interaction and their
directly surrounding residues were completely conserved. The SI clones, which differed in their
tropism for primary monocytes and T-cell lines possessed identical V3 sequences.
Conclusion: Three clones most likely resulted from genetic recombination events in vivo, indicating
that this phenomenon may account for the emergence of proviruses with novel phenotypic properties
during the course of the HIV-1 infection. Variation among most accessory genes was limited. The
finding of identical V3 sequences in clones differing in tropism for primary monocytes and T-cell
lines demonstrated the existence of determinants of tropism outside the env V3 region, most
probably located within the gpl20-V5 region and the gp41 molecule.
Groenink. Martijn: Central Laboratory of the Netherlands Red Cross Blood
Transfusion Service, Plesmanlaan 125, 1066 CX Amsterdam, The Netherlands.
Telephone: [phone]. Fax: [phone].
NOTES

Peden, Keith, Building 4, Room 310, NIAID, NIH, Bethesda, MD 20892, USA
Telephone: [phone], FAX: [phone]

Mo20