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

12th World AIDS Conference

Abstracts 11122-11126 7

11122 The prevalence of the CCR5-A32 gene deletion and
CCR-5 M303 mutation in South African populations
Carolyn Williamson1, B. Brice1, T. Smit2, R. Thomas3, L. Louie5, C. Quillent6,
E. van der Ryst4. 3Medical Microbiology, 1SAIMR Virology University of Cape
Town, Anzio Road, Observatory; 2Department Virology, Medunsa, Pretoria;
4Dept. Virology, University of Free State, Bloemfontein, South Africa; 5School
Public Health, University of California, Berkeley, USA; 6Cirs, Hbpital St Joseph,
Paris, France
Objectives: Resistance to HIV infection has been associated two CCR5 gene
alterations, CCR5-A32 deletion and m303 mutation (Quillent et al., Lancet 1998,
351, 14-18), both of which render the protein non-functional. The aim of this study
was to determine the prevalence of the CCR-5 A32 deletion and m303 mutation
in different South African populations.
Methods: DNA was isolated from 1731 blood samples. For detection of
CCR-A32, a PCR fragment of 189 bp (wild type) or 157 bp (A32) was generated.
For detection of the m303 mutation, a PCR fragment of 350 bp was generated
which was cleaved by Hincll into 120 bp and 230 bp fragments (wild type). This
restriction site is lost when mutated.
Results: Eight individuals were heterozygous and no individuals were homozygous for the A32 CCR5 deletion. Of the 350 black South Africans screened for
the m303 mutation, no individual was identified as having this mutation.
CCR-5 genotype (No. heterozygous/Total no.)
Black African Mixed Race Unknown
HIV pos HIV neg ND HIV pos HIV neg HIV pos HIV neg
Adults: 0/188 2/732 0/521 0 1/9 0 2/5
Infants#: 0/194 1/108 0 1/32 1/29 0/48 0/39
HIV status determined by ELISA; #HIV status determined by PCR
Conclusion: The prevalence CCR5-A32 deletion is low (below 0.2%) in the
black South African populations compared to reported prevalence in Caucasian
populations. The m303 CCR5 mutation was not detected in this group of people.
Larger numbers of individuals of mixed race need to be screened to determine
prevalence of these mutations in this group of people.
F 1123 Prevalence of deletion in the /3-chemokines receptors
(A32 CCR5) sequency codificator in blood donors in Sao
Paulo City (Brazil)
Marcelo C. Silva1, M.A. Rossini2, E.C. Sabino3. 1Av Dr. Arnaldo 355, Lab.
Retrovirus, Sao Paulo; 21nstituto Adolfo Lutz, Sao Paulo; 3Fundagao Pro
Sangue/Hemocentro SR Sao Paulo, SP Brazil
Objective: To investigate the prevalence of 32pb deletion (A32 CCR5) in the
/-Chemokines receptors sequence in blood donnors in Sio Paulo city/Brazil.
Design: Prevalence Study.
Methods: Amplification by PCR with "primers" that flank the coding region for
the 32pb mutation in /3-chemokines receptors CCR5 (A32 CCR5), using isolated DNA from mononuclears periferic blood cells from 200 blood donors of the
Sio Paulo city/Brasil. The differentiation from genotypes was analized on a 2%
agarose gel.
Results: From a total of 200 samples analized, 187 (93.5%) presented wild
type genotype (wt/wt), without deletion for the CCR5 receptor; in 13 samples
(6.5%) we find the heterozygous genotype for the deletion (wt/A32). We didn't
find the homozygous genotype (A32/A32) for the deletion on the CCR5 receptor
in any of the studied samples.
Conclusion: The absence of the homozygous deletion character (A32/A32)
for the CCR5 receptor in Sio Paulo city/Brazil population, can be due to the
small size but can also be related with a large racial interbreeding existing in this
country.
376* 11124 Inhibition of HIV-1 reverse transcription by
actinomycin D (Act D): A potential approach for
AIDS therapy
Judith G. Levin1, J. Guo1, T. Wu1, J. Bess2, L.E. Henderson2. 1Lab of Molecular
Genetics, NICHD NIH, 9000 Rockville Pike Bethesda, MD; 2SAIC-Frederick
NCI-FCRDC, Frederick, USA
Objectives: To investigate the inhibitory effect of Act D on (-) strand transfer, a
unique step in HIV reverse transcription.
Methods: An HIV-1 in vitro system was developed which models the (-) strand
transfer event. This system includes: donor and acceptor RNA templates having
all of R and a portion of U5 or U3, respectively; a 5'-labeled DNA primer; HIV-1
reverse transcriptase (RT); and HIV-1 nucleocapsid (NC) protein, as indicated.
Endogenous RT assays were performed with detergent-treated HIV-1 virions and
products were analyzed by polymerase chain reaction. Act D dose response was
measured in both assays.
Results: Act D inhibits (-) strand transfer with an IC50 value of 0.3 tiM, while

DNA-dependent DNA synthesis catalyzed by RT is much less sensitive (IC50,
60 /iM). The two peptide rings in Act D appear to be needed for the effect on
strand transfer. HIV-1 NC (a nucleic acid chaperone catalyzing rearrangements
of nucleic acids to more stable conformations) dramatically stimulates (-) strand
transfer by transiently destabilizing the complementary TAR structure at the 3'
end of (-) strand strong-stop DNA [(-) SSDNA]. Nevertheless, addition of Act
D to virions or to in vitro reactions with NC leads to only a modest increase in

the IC50 value. Detailed analysis shows that Act D does not inhibit (-) SSDNA
synthesis or RNase H degradation of the donor. However, the actual transfer step
i.e., annealing of (-) SSDNA to acceptor, (requiring NC chaperone activity for
high efficiency, but not RT), is severely limited by Act D, even in the presence of
NC.
Conclusions: Act D inhibits HIV-1 reverse transcription in vitro and in virions
by primarily blocking the nucleic acid chaperone activity of the accessory protein
NC and not by a direct effect on RT activity. To our knowledge, this is the first
example of a drug affecting NC chaperone activity. The potency of Act D inhibition
coupled with its successful use for many years in treatment of certain tumors,
suggest that Act D might also be useful in combination therapy for AIDS patients.
11125 Dominance of the E89G substitution in HIV-1 reverse
transcriptase in regard to increased polymerase
processivity and patterns of pausing
Mark A. Wainberg', Yudong Quan2. 13755 Cote Ste, Catherine Road, Montreal.
Quebec; 2McGill AIDS Center, Montreal, Canada
Objectives: To compare various forms of wild-type (wt) and mutated recombinant reverse transcriptase (RT) molecules, containing drug-resistance-conferring
mutations, in regard to pausing, processivity, and dissociation from an RNA
template/primer.
Methods: The substitution of a glycine for glutamic acid at position 89 in HIV-1
RT (E89G) confers resistance to several nucleoside and nonnucleoside inhibitors
of RT. As residue 89 contacts the template strand, it has been suggested that
this mutation may modulate the conformation of the RT-primer/template complex. In addition, certain mutations in RT that confer resistance to nucleoside
analogs, such as M184V, are located near the polymerase active site. To further
characterize these substitutions, we performed processivity assays alongside an
analysis of pausing profiles with wild-type (wt) RT and recombinant RTs containing
substitutions at E89G, M184V, or both.
Results: We found that E89G RT has higher processivity than wt enzyme as
well as a different pattern of pausing sites. Similar findings were obtained with
the doubly mutated RT, although enzyme containing only the M184V mutation
had lower processivity than wt. Consistent with these observations, and from
a mechanistic standpoint, both E89G-containing as well as doubly mutated RT
had decreased dissociation constants from a complex consisting of RT and template:primer, in comparison with either wt RT or M184V-containing RT. However,
all of the wt and mutant enzymes tested had similar Km values for the RNA template primer used in this work. Viruses containing the E89G mutation synthesized
longer strand DNA products than either wt viruses or viruses containing only the
M184V mutation in endogenous RT assays.
Conclusion: The E89G substitution is a dominant determinant in regard to
each of the koff values from an RTotemplate/primer complex, RT processivity, and
specific patterns of pausing during DNA polymerization.
378*/11126 Compensatory point mutations in the HIV gag
region that are distal from deletion mutations in the
dimerization initiation site can restore viral
replication
Mark A. Wainberg', Chen Liang2, Liwei Rong2. 13755 Cote Ste, Catherine
Road, Montreal, Quebec; 2McGill AIDS Center, Montreal, Canada
Objectives: To determine the effect of a 16 nt deletion termed LD3 within the
dimerization initiation site (DIS), downstream of the LTR, on viral RNA packaging,
gene expression, and viral replication.
Methods and Results: The dimerization initiation site (DIS), downstream of the
LTR within the HIV-1 genome, can form a stem-loop structure (SL1) that has been
shown to be involved in the packaging of viral RNA. Deletion of the sequences
at positions +238-+253, i.e. construct BH10-LD3, can significantly alter this SL1
structure, leading to reductions in viral RNA packaging and viral gene expression,
as well as impaired viral replication. Long-term culture of these mutated viruses
in MT-2 cells, i.e. 18 passages, yielded revertant viruses that possessed similar
infectivity to that of wild-type. Cloning and sequencing showed that these viruses
retained the original 16 nt deletion, but possessed two additional point mutations,
MP2 and MNC, that were located within the P2 and NC regions of the gag
coding region, respectively. Site-directed mutagenesis studies revealed that both
of these point mutations were necessary to compensate for the 16 nt deletion in
BH10-LD3. A construct containing both the 16nt deletion and the MP2 mutation,
i.e. LD3-MP2, produced five times more viral protein than BH10-LD3, while the
MNC mutation reversed the defects in viral RNA packaging. We also deleted nt
+261-+274 within the 3'end of SL1 and showed that the diminished infectivity of
the latter mutated viruses, termed BH10-LD4, could also be restored by the MP2
and MNC point mutations.
Conclusions: Compensatory mutations within the P2 and NC proteins, distal
from deletions within the DIS region of the HIV genome, can restore HIV replication, viral gene expression, and viral RNA packaging to control levels. These
findings illustrate the remarkable versatility of the HIV genome.