Abstract Book Vol. 2 [International Conference on AIDS (14th: 2002: Barcelona, Spain)]

350 Abstracts ThOrA1l432-ThOrA1l435

XIV International AIDS Conference

suit in slow progression to AIDS, increased time for transmission, and increased
prevalence in the epidemic.
Methods: Six subtype C and nine B HIV-1 isolates were used to infect PBMC and
other primary human cells in pairwise competition experiments. A heteroduplex
tracking technique was then used to measure the relative production in a dual
HIV-1 infection.
Results: All subtype C HIV-1 isolates were less fit than subtype B isolates (p <
0.0001). However, intrasubtype variations in fitness were not significant. Subtype
C isolates also appear less fit than subtype A and D isolates. Increased replication of subtype B over subtype C HIV-1 isolates was also observed in primary
macrophages and CD4+ T cells. However, subtype C isolates were more fit in
skin-derived Langerhan cells (LC). Ultimately, efficiency of host cell entry predicted the winner of each HIV-1 competition. Furthermore, phylogenetic analyses
based on a fitness matrix suggest that ex vivo fitness maps to the env and not the
gag or pol genes.
Discussion: Subtype C isolates are less fit than other subtypes but predominates
in the HIV epidemic suggesting increased in vivo fitness. This dichotomy may be
due to slower disease progression in subtype C-infected individuals resulting in
an increased transmission time. In addition, subtype C may still be efficient at infecting LC, or cells thought to be the primary target for heterosexual transmission.
Presenting author: Eric Arts, 10900 Euclid Ave., Div. ID, BRB1034, Case Westen
Reserve University, Cleveland, OH, 44106, United States, Tel.: [phone],
Fax: [phone], E-mail: [email protected]
ThOrA1432 A glycine to serine mutation at position 140 of
integrase confers resistance to integrase inhibitors
and defective integration
W.E. Robinson, PRJ. King, D.J. Lee, R.A. Reinke, J.G. Victoria. University of
California, D440 Med Sci I, University of California, Irvine, California,
92697-4800, United States
Background: Culture of HIV with increasing concentrations of L-chicoric acid
(L-CA), an inhibitor of integrase (IN), resulted in a single point mutation within
IN that conferred resistance of the virus to L-CA. The effects of this mutation
on integration and susceptibility to other IN inhibitors were unknown. It was hypothesized that the mutation would cause resistance of the protein to L-CA and
cross-resistance to L-731,988, a diketoacid.
Methods: IN containing the G140S mutation (ING140S) was purified. Susceptibility to both L-CA and L-731,988 was determined. The enzymatic activities of the
IN proteins were measured under steady-state and non-steady-state conditions.
Binding of the mutant protein to LTR substrate was measured. The effect of the
mutation on HIV replication kinetics was measured using reverse transcriptase
release, immunofluorescence, and quantitative real-time polymerase chain reaction (PCR). The ratio of two LTR circle DNA to cDNA in HIV- infected cells was
used to determine effects of the mutation on integration.
Results: Using real-time PCR, a growth defect secondary to failure of integration
was demonstrated. The ING140S protein was attenuated approximately 4-fold
for catalysis under equilibrium conditions compared to wildtype IN (INWT) and
attenuated 5-fold in steady-state kinetic analysis of disintegration. Fifty-percent
inhibitory concentration assays were performed with IN inhibitors against both
ING140S and INWT in disintegration and strand transfer reactions. ING140S was
resistant to both L-CA and L-731,988. HIV containing the mutation was resistant
to L-731,988 and L-CA.
Conclusions: Glycine 140 is important for susceptibility of IN to both L-CA and
a diketoacid. Mutation of this residue to serine confers cross-resistance to both
inhibitors and a defect in catalysis mediated by IN. Therefore, both L-731,988 and
L-CA are inhibitors of IN and may interact with the same drug binding pocket on
IN.
Presenting author: W. Edward Robinson, D440 Med Sci I, University of California, Irvine, California, 92697-4800, United States, Tel.: [phone], Fax:
[phone], E-mail: [email protected]
ThOrA 433 Mechanisms of HIV-1 escape from a CCR5-specific
small molecule entry inhibitor
S. Kuhmann', A. Trkola2, P. Pugach3, J. Strizki4, B. Baroudy4, J.P. Moore5.
'Well Medical College of Cornell University Dept of Microbiology & Immunology
New York, United States; 2University Hospital Zurich, Div. of Infectious Diseases,
Dept. of Medicine, Zurich, Switzerland; 3 Weill Medical College of Cornell
University Department of Microbiology & Immunology New York, United States;
4 Schering Plough Research Institute, Kenilworth, United States; 5 Weill Medical
College of Cornell University Wefi Medical College of Cornell University 1300
York Avenue, Box 62, New York, NY 10021, United States
CCR5-specific inhibitors of viral entry are now under development as new therapies to treat HIV-1 infection. One such compound, the CCR5 antagonist SCH-C, is
presently in Phase I clinical trials (Strizki et al., PNAS 98, 12718, 2001). We have

studied how HIV-1 escapes in vitro from a closely related entry inhibitor, AD101,
from the same general chemical family as SCH-C (Trkola et al., PNAS 99, 395,
2002). To develop an escape mutant, the R5 primary isolate CC1/85 was passaged in PBMCs with increasing concentrations of AD101. By 19 passages, a
virus emerged with >20,000-fold resistance to AD101. The escape mutant was
cross-resistant to SCH-C, but remained sensitive to CCR5-specific MAbs and partially sensitive to the CC-chemokine, RANTES. Its phenotype was stable in that

it did not revert to AD101 sensitivity upon passage for 9 more weeks in PBMC,
in the absence of AD101. The escape mutant continues to exclusively use CCR5
as a co-receptor; it cannot replicate in cells from humans homozygous for the
CCR5-delta
32 allele which lack CCR5. Its persistence in using CCR5 contrasts markedly
with the proven ability of the parental isolate to evolve in vivo to use CXCR4,
and occurs despite the availability of CXCR4 in the PBMC cultures. Escape from
AD101 proceeds by a two step process: first, viruses are selected from the isolate that can more efficiently use low levels of CCR5, then mutations arise de
novo in the V3 loop of gpl120 which result in full resistance. The molecular mechanisms by which these sequential mutations in env alter the interactions of the
envelope glycoproteins with CCR5 are under investigation, using pseudotyped reporter viruses with specific mutations in the cloned env genes, and site-directed
mutants of CCR5. The sensitivity of the escape mutants to neutralizing Abs is
also under evaluation, as is an assessment of whether a tropism expansion has
occurred.
Presenting author: John Moore, Weill Medical College of Cornell University,
1300 York Avenue, Box 62, New York, NY 10021, United States, Tel.: [phone], Fax: [phone], E-mail: ern2001 @med.cornell.edu
ThOrA1434 Differential effects of HIV-1 protease mutations
outside of the active site on nelfinavir and ritonavir
binding
J.C. Clemente, R. Hemrajani, T Scordino, B.M. Dunn. University of Florida, 3301
SW 13th St, Gainesville fl. 32608, Apt k196, United States
Background: The biggest obstacle facing the therapy of Human Immunodeficiency Virus infection is the occurrence of drug resistant viruses. The development of cross-resistance by enzymatic targets further decreases the ability to
provide adequate therapy for patients. Of special interest is the effect mutations
outside of the active site of the HIV-1 protease have on inhibitor binding and virus
viability
Methods: Using site directed mutagenesis we have engineered protease variants containing the active site mutation D30N in the presence and absence of the
non-active mutations M361 and A71V. We have also engineered a variant containing the mutations M361 and A71V. Using a chromogenic substrate mimicking the
CA/p2 cleavage site we have determined the Ki binding constants for Nelfinavir
and Ritonavir, and the catalytic efficiency of the variants.
Results: The variants with the mutations D30N alone and M361/A71V together
show a decrease in the catalytic constant by 70 and 80% respectively, and both
showed a 90% decrease in the specificity constant when compared to wild type.
The D30N/M361/A71V variant showed a 9% increase in its catalytic constant
and a 30% decrease in its specificity constant. The wild type enzyme and the
M361/A71V mutant both bind Nelfinavir with subnanomolar Ki values. The addition
of D30N greatly decreased the binding affinity of Nelfinavir. The D30N/M361/A71V
mutant bound Nelfinavir with less affinity than the wild type enzyme, but no less
than the D30N mutant. In contrast, the D30N mutation had no effect of Ritonavir
binding. The addition of M361 and A71V mutations to the D30N mutant decreased
the binding affinity of Ritonavir.
Conclusions: These data suggest that the non-active site mutations M361 and
A71V in the presence of the active site mutation D30N function to enhance catalytic activity Surprisingly, although they originate in response to Nelfinavir and
Ritonavir, they decrease the binding affinity of Ritonavir but not Nelfinavir.
Presenting author: Jose Clemente, 3301 SW 13th St, Gainesville fl. 32608,
Apt k196, United States, Tel.: [phone], Fax: [phone], E-mail:
[email protected]
ThOrAl 435 In vitro effects of HIV infection on ABC transporter
expression and antiretroviral drug efficacy
S. Jorajuria, N. Dereuddre-Bosquet, D. Dormont, P. Clayette. CEA, service de
neurovirologie, drm/dsv, commissariat a I'energie atomique (cea), 60, 68 avenue
de la division leclerc-bp6, 92 265 fontenay-aux-roses cedex, France
Background: Intracellular concentrations of anti-HIV drugs are determinant for
their pharmacological efficacy in peripheral blood as well as in sanctuary sites
e.g. lymph nodes and CNS. ABC transporters such as P-glycoprotein (P-gp) and
multidrug resistance-associated proteins (MRP) are reported to limit protease inhibitor (IP) and nucleoside reverse transcriptase inhibitor (NRTI) access to their
cellular and tissular targets. Moreover, HIV may regulates the expression and activity of these host cell factors. Therefore, the present study aimed to evaluate in
primary cultures of human monocyte-derived macrophages (MDM) and lymphocytes, effects 1) of retroviral infection and HAART on the expression and activity of
P-gp and MRP and 2) of specific inhibitors of these host proteins on antiretroviral
activities of NRTI, Non-NRTI and IP
Methods: The mRNA expression of these different host cell factors were measured by RT-PCR, cell surface expression by flow cytometry, and activity using

specific subtrates e.g. rhodamine.
Results: On the one hand, we evidenced a transitory increase of P-gp mRNA
expression in lymphocytes and MDM in response of in vitro HIV infection. This
was correlated to an increased P-gp cell surface expression and activity, and
an increased TNF-alpha and IL-6 production and mRNA. In contrast, no significant modulation of MRP was observed, and in parallel, treatments with AZT
and indinavir do not influence the P-gp activity. On the other hand, PSC833 and