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

WeA 1006-WeA 1009

TRACK A: BASIC SCIENCE

WeA 1006 ROLE OF TAT AND CELLULAR FACTORS IN HIV-1
EXPRESSION. Gatignol Anne; Chang Y.-N.; Jeang K.-T. Laboratory of
Molecular Microbiology, NIAID, NIH, Bethesda, MD 20892, U.S.A.
Obectives: Induction of HIV expression is due to activation by cellular and viral factors.
Tat, the trans-activator of HIV-1 acts through an RNA target TAR (trans-activator
responsive) to activate DNA elements in the promoter in an appropriate cellular setting.
This suggests important interactions between Tat, DNA/RNA targets and cellular factors in
order to increase transcription. We will describe the role of two cellular proteins that bind
to TAR RNA.
Methods and Results: To obtain cDNAs that encode TAR RNA binding proteins, we
assayed a HeLa cell library using an RNA recognition site probe. Two related cDNAs
code for 37 and 42 kDa proteins called TRBP1. A third one codes for La autoantigen.
We showed by in situ filter binding and gel shift assays that TRBPs and La bind to TAR
RNA. By mutational analysis of the protein, we determined the binding site of TRBP. In
gel retardation assay, the corresponding peptide was sufficient to slow down a TAR RNA
band indicating the formation of an RNA-peptide complex. This complex was competed
away by HeLa cellular RNA, polyl-polyCand homologous RNA but not by DNA,
single stranded or unstructured RNA. This suggests a short, double stranded RNA motif
recognition for TRBP. This site is likely to exist in many copies in HeLa cellular RNA, the
natural TRBP target. In vivo, TRBP activates HIV-1 LTR and other promoters. In cotransfection assays, we observed a synergistic function between Tat and TRBP on HIV-1
LTR.
Conclusions: TRBP may represent one member of a family of cellular proteins that is
capable of influencing gene expression through the binding of RNA. We propose that
TatTAR RNA and cellular factors act in a cooperative manner to activate HIV-1 LTR. The
virus utilizes cellular proteins such as TRBP and La and may co-opt the corresponding
cellular function in order to increase its own expression and/or stability.
(1) Gatignol et al. Science (1991) 251:1597
Gatignol Anne, Laboratory of Molecular Microbiology, NIAID, National
Institutes of Health, Building 4 Rm 310, Bethesda, MD 20892, USA,
Tel: [phone], FAX: [phone]
WeA 1008 THE HIV-1 Vpu PROTEIN INDUCES RAPID DEGRADATION OF CD4.
Willey, R.; Maldarelli, F.; Chen, M.Y.; Martin, M.A.;
and Strebel, K., Laboratory of Molecular Microbiology,
NIAID, NIH; Bethesda, MD 20892, USA.
Objectives: Analysis of the HIV-1 vpu gene product and its effects on
viral and cellular proteins.
Methods: HIV-1 subgenomic and CD4 expression plasmids were generated
using standard molecular biological techniques. Assays were performed
by transfecting HeLa cells followed by metabolic labelling of
transiently expressed proteins and subsequent identification by
immunoprecipitation with specific antisera.
Results: CD4 is a integral membrane glycoprotein that is synthesized
at the endoplasmic reticulum (ER) and subsequently transported to the
cell surface via the golgi system. HIV-infection of CD4 cells leads
to downmodulation of cell surface CD4 due at least in part to the
formation of stable intracellular complexes between CD4 and the HIV-1
Env precursor polyprotein, gpl60. This process "traps" both proteins
in the ER and results in reduced surface expression of CD4 and gpl20/
gp41. We have recently demonstrated that the presence of the 81 amino
acid integral membrane protein, Vpu, can reduce the formation of CD4/
Env complexes resulting in increased gp160 processing and decreased
CD4 stability. We have studied the effect of Vpu on CD4 stability and
found that Vpu induces rapid degradation of CD4 reducing the half-life
of CD4 from 6 hours to 12 minutes. By using defined gp160 and CD4
mutants it can be shown that this Vpu-induced degradation requires
retention of CD4 in the ER which is facilitated through its binding to
gpl60.
Klaus Strebel, NIH, NIAID, 4/310; 9000 Rockville Pike;
Bethesda, MD 20892 (Tel) 301-496-3132 (Fax) 301-402-0226
NOTES

WeA 1007 THE ROLE OF CELLULAR COFACTORS IN TAT-MEDIATED TRANSACTIVATION Shank. Peter R.. Newstein, Michael and Lee, Im-soon.
Brown University Division of Biology and Medicine, Providence, RI,
USA
Objectives: We wished to define the role of various cellular cofactors which intearct
with the Tat protein and the TAR region in the process of trans-activation.
Methods: We have used transient transfection assays of various LTR driven reporter
plasmids along with Tat expression vectors into rodent cells. Rodents are generally
refractory to trans-activation (Hart et al., Science 246:488-491, 1989; Newstein et al., J.
Virol. 64:4564-4567, 1990). We have utalized the chimeric trans-activation system
developed by Selby and Peterlin (Cell 62:769-776, 1990) and various mutations of the TAR
region to define the role of cellular cofactors.
Results: We find that the chimeric trans-activation system in which Tat protein is
artificially tethered near the 5' end of the HIV transcript, by replacing the TAR region
with a heterologous RNA recognition site, is not responsive in rodent cells. If however
chimeric Tat is artificially tethered to the TAR region in constructs in which the normal
Tat binding "bulge" has been replaced by the heterologous RNA recognition site, transactivation occurs efficiently in rodent cells. Furthermore if mutations,which would
inactivate wild-type trans-activation are introduced into the TAR "loop" region into these
constructs the level of trans-activation increases in both rodent and human cells.
Conclusions: Our results suggest that the restriction to Tat-mediated trans-activation in
rodent cells can be overcome by artificially tethering the functional domain of Tat to near
the 5' end of the HIV transcripts if the basic "stem" structure of TAR is retained. We
assume that factors binding to the TAR "stem" region are essential for efficient transactivation.
Peter R. Shank, Division of Biology and Medicine, Brown University,
Providence, R.I. USA 02912, Phone [phone], FAX [phone]
WeA 1009 NATURAL VARIANTS OF THE HIV-1 LONG
TERMINAL REPEAT: ANALYSIS OF PROMOTERS WITH DUPLICATED DNA
REGULATORY MOTIFS. Koken, Sabine; Wamel van J.; Goudsmit J.; Geelen J.L.MC.;
Berkhout B. Human Retrovirus Laboratory, UvA, Amsterdam.
Objectives: The HIV-1 LTR functions as the transcriptional promoter and contains multiple regulatory elements.
Variation within these elements may have profound effects on the viral replication rate and viral pathogenicity. The
goal of this study was to detect and analyze naturally occurring length variations in the HIV-1 promoter region.
Methods: DNA was isolated from peripheral blood mononuclear cells (PBMC) of 16 HIV-1 infected individuals at
different stages of disease, and from brain and spleen tissues of an HIV infected child. A PCR of the HIV-1 LTR
promoter region was used to identify viruses with aberrant LTR structures. Promoter activity of the LTR variants was
assayed upon transfection of LTR-CAT constructs into different cell lines. In order to measure viral replication rates,
the different LTRs were introduced into an HIV-1 molecular clone and transfected into different cell lines. Virus
production was monitored by measuring gag-p24 antigen and RT activity in the culture supernatant
Results: Two classes of LTR size variants were found. When compared to the wildtype HXB2 isolate either an
additional Spl binding site or a duplication of a short DNA motif upstream of the NF-kB site was detected. The
natural variant with 4 Spl sites showed a slightly higher promoter activity and viral replication rate than the wildtype
LTR with 3 Spl sites. In contrast, there was no measurable positive effect of the duplicated motif Nevertheless, this
region does contribute to promoter activity, since deletion of the sequence results in reduced promoter activity. In
order to measure accurately differences in virus production, an equal amount of an LTR size variant and the isogenic
control plasmid was co-transfected into T-cell lines. A small defect in LTR promoter function of one virus is expected
to result in outgrowth of the other. This co-transfection procedure resulted in the outgrowth of viruses with 4 Spl sites
and viruses with 1 copy of the duplicated motif in 35 and 42 days, respectively. A difference in virus production of
approximately 5 to 10% was calculated from the rate of outgrowth of the viruses.
The duplications found in the LTRs of 4 different patients all contained the short sequence motif 5'-ACATG GA-3'.
Electrophoretic mobility shift assays and UV cross-linking experiments were used to analyze protein binding to this
motif. These experiments showed that a family of proteins (50-60 kD) binds to this motif.
Conclusions: Variation in the LTR is detected in patients at different stages of disease. The LTR length polymorphism
results from duplication of promoter-enhancer elements, being either an additional Spl site or a duplication of the 5'ACTGCTGA-3' motif.
Sabine Koken, Human Retrovirus Laboratory, UvA, Meibergdreef 15, 1105 AZ Amsterdam,
the Netherlands, phone [phone], fax [phone]

NOTES

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