Abstracts Vol. 1 [International Conference on AIDS (11th: 1996: Vancouver, Canada)]

Tu.A.393 -Tu.A.492

Tuesday, July 9, 1996

Tu.A.393
RAISED PRODUCTION OF C-C CHEMOKINES BY CD8+T CELLS OF LONG TERM
NON-PROGRESSOR HIV-INFECTED SUBJECTS
Scala E, Aiuti F, Rosso R, D'Offizi GP Ferrara R, Paganelli Roberto. Dept Clinical Medicine,
Ch ar Cline Immunol and Allergy Univ of Rome, "La Sapienza", Rome, Italy.
Objective:The recent description of RANTES, MIP-Ile and MIP-l(3 (C-C chemokines) as
the constituents of the HIVsuppressive factor produced by CD8+ lymphocytes of HIVinfected individuals prompted our study of their production by T cell lines (FCL) and clones
derived from normal and HIV+ subjects at different disease stages.TCL and clones from
healthy donors asymptomatic HIV+ patients with AIDS and Long Term non Progressors
(LTnP) were analysed for surface phenotype, production of C-C chemokines, IL4 and IFNy
production, in order to define association of chemokines secretion with disease stage, lymphocyte subsets, and cyokine profile.
Methods:TCL were established by PHA stimulation and culture with rlL2, and cloned by
limiting dilution. Supernatants obtained after restimulation with PHA were tested for
chernokines, IL4 and IFNy by ELISAs. Surface phenotype was determined by cytofluorimetric analysis with mAbs to TCR, CD4 and CD8.
Results: Moderate to high levels of all three chemokines were produced by TCLs of LTnP
and asymptomatic HIV+, whereas they were low or absent in normal subjects and AIDS. All
these TCLs exihibited a Th I pattern. A TCL from an AIDS hyper -IgE (HIE) patients produced IL4 and RANTES. C[)4 positive clones produced low or moderate levels of these
chemokines in asymptomatic HIV -, but only MIP- I(3 was detected in normal subjects.The
majority of CD8+ clones produced all three chemokines at moderate to high levels, without relationship to IL4 or IFNy secretion, as observed in CD4+ cells. In clones from LTnP
both RANTES and MIP I(3 were produced at levels similar to normal subjects, but higher
than AIDS. Mip- l(a was significantly increased in CD8+ clones of LTnP compared to AIDS,
normal subjects and asymptormatic HIV+. RANTES and MIP-I a were also higher in AIDSHIE compared to asymptomatic HIV+.
Conclusions: RANTES, MIP- Ia and MIP-d I are mainly produced by CD8+ lymphocytes,
irrespective of theirTh pattern. Clones from LTnP secrete higher amounts of all three
chenmokines compared to AIDS patients. Among them, NIP- lea was producted at higher levels by LTnP compared to all other groups, thus pointing to its possible importance in delaying progression of HIV disease.
Roberto Paganelli. Dept. of Clinical Medicine University La SapienzaViale Universita 37
00185 Rome Italy Tel: + 39-644-54941 Fax: + 39-644 54621
Tu.A.394
ON THE MECHANISM OF C-C CHEMOKINES (RANTES, MIP-l( AND MIP- 10)
MEDIATED INHIBITION OF HIV
Garzino-Demo Alfredo*, Gallo RC.*, DeVico AL.*, Cocchi F.*, Lusso P'**, Arya Suresh K.***.
*IHV, MBC, UMBI, Baltimore, MD, USA; **LHV, DIBIT, Hospital S. Raffaele, Milan, Italy;
***LTCB, NCI, NIH, Bethesda, MD, USA;
Objectives: The C-C chemokines RANTES, MIP- Ia and MIP- 13 were identified as the
major HIV suppressive factors released by CD8+ T lymphocytes.To understand the mechanism of action and specifically to explore the possibility that such suppressive factors may
inhibit HIV RNA transcription, the effect of these chemokines on the basal and Tat-induced
HIVI LTR-directed gene expression was studied.
Methods: The effects of recombinant chemokines and/or conditioned medium from CD8+
cells were studied by determining their modulation of HIV- I LTR directed reporter CAT
gene expression in transiently and stably transfected CD4+ Tcell lines, in the presence or
absence of Tat. In addition, their effect on virus expression in CD4 T-cells transfected with
replication competent proviral clones of HIV I, HIV 2, and SIV was measured by a core antigen capture assays.
Results: Treatment of CD'+ Tcells infected with HIV I with the recombinant chemokines
and crude CD8+ Tcell derived conditioned medium lead to a marked suppression of HIV- I
RNA synthesis as analyzed by Northern Blot hybridization. In contrast, virus production in
CD4- cells tr ansfected with molecular clones of HIV, HIV 2. and SIV was not affected.
Further, chemokines and CD8 +-cell conditioned medium did not down modulate LTRdirected CAT gene expression n transiently or stably transfected T-cell lines.
Conclusions: Our results suggest that C C chemokines and conditioned media from CD8+
cells inhibit HIV replication by affecting a step other-than the initiation of transcription.This
is in contrast to the previous reports by others of trascriptional down-modulation of HIV-I
expression by factor(s) secreted by CD8+ cells.
Garziro Demo Alfredo, Ph.D., Institute of Human Virology MBC, UMBI, Baltimore, MD, USA.
Tel. [phone]; Fax [phone]
Tu.A.395
CD8 T LYMPHOCYTE-MEDIATED SUPPRESSION OF HIV-I LTR-MEDIATED
TRANSCRIPTION SHOWS NO CORRELATION WITH CLINICAL STAGE OF
DISEASE OR HEALTH STATUS.
Copeland, K.F.3., Leith, J., Kelleher L., Smaill F., Rosenthal, K.L. Molecular Virology and
Immunology Department of Pathology McMaster University Hamilton, Ontario.
Objectives: CD8 T lymphocytes of HIV - infected individuals efficiently suppress HIV-I
replication in CD1 Tcells and this suppression has been shown to correlate with high
CD4 a cell counts and lick of disease progression This study examines the transcriptional
control of HIV- I [TR mediated expression by CD8 T cells and compares this activity with
chemckine production by CD8 cells and with clinical stage of disease.
Methods: Human jut itT cels were transfected with a vector bearing the HIV I LTR
directing the chloramphenical,cetyl transferase gene (CAT) and a vector expressing HIV I
Tat. CAT activity was measured following culture of the transfected cells with supernatant
of activated CD8 T cells of HlIV infected subjects.The presence of Rantes in supernatants
was measured by E isa.
Results:We have examined the ability of CD8 T cell supernatants of patients of varied
clinical stage to inhibit LTR-mediated gene transcription. Strong suppression of transcription
was not restricted to asymptomatic subjects and in several cases subjects with AIDS were
among the strongest suppressors of transcription. Similarly low level suppressors included
asymptomatic sublects and those who had progressed to AIDS. No correlation of CD8 T
cell mediated suppressive actvwty ouid be found with CD4~ or CD8+ T cell counts, dura
tie of infectin r: iral treatment. CD8+ T cell-mediated suppression was also noted in
patients ex'-.c'ri n iniht loss, Kaposi's sarcoma, pneumocystis carinii and oesophageal
candidiasis. in somee -biect followed longitudinally the suppressive effects of CD8+ T cells
varied dram.i: ltlv; i stveea two-month sampling periods. In addition, the level of inhibition
evoked by cc,: -r apc,c,_rrelated with the amount of Rantes present in the supernatants.
We will prese-t( ata., in e level of suppression of transcription by CD8+T cell supernatants paired \,ith e leveIn' of selected chemokines present in the supernatants. In addition, a comparison of the effect of CD8 T cell mediated suppression on virus replication
and transcription will be presented.
Conclusion: Further investigatlion of CD8+ T cell-mediated suppression is required to
determine its role in disease progression and its effect on other steps in the virus life cycle.
Dr Karen F.T Copeland. McMaster University Dept. of Pathology HSC 3N26, 1200 Main
Street West, Hamilton, Ontario L8N3Z5 [phone] Ext. 22494 Fax: [phone]
Tu.A.490
THE MULTIFACETED FOLE OF NEF IN HIV REPLICATION
Trono, Didier.The Salk Institute for Biological Studies, La Jolla, CA, USA
The HIV I Nef protein is crucial for high level viral replication it vicvo and for AIDS pathogenesis. In vitro, Ne)has been shown i) to downregulate the cell surface expression of
CD4 and, to a lesser extent, of MHC-1. ii) to stimulate proviral DNA synthesis, thereby
enhancing viral replication, and iii) to alter cellular activation pathways. New results will be
presented which tie Nef and an associated cellular serine kinase with the phosphorylation
of a component of the HIV-I nucleoprotein complex. The proposal will be made that this
phenomenon underlies the observed effect of Nef on reverse transcription and, consequently, viral infectivity
DTrono, 10010 N.Torrey Pines Rd., La Jolla, CA, 92037 USA Tel: [phone]
Fax: [phone] E mail: didier_tronolqm.salk.edu
Tu.A.49 I
ROLE OF THE HIV-A NUCLEOCAPSID PROTEIN (NCp7) AND THE A-RICH LOOP IN
CELL-FREE REVERSE TRANSCRIPTASE AND INFECTIVITY ASSAYS
Wainberg, Mark, Li X, Kleiman L, Parniak MA. McGill University AIDS Centre Jewish
General Hospital, Montreal, Canada
Objective: To determine the physiological relevance of NCp and the A-rich loop in
reverse transcription.
Methods: Reactions were performed using recombinant HIV RT in the presence of NCp.
Results: In the presence of tRNALys.3, NCp7 was found to stimulate synthesis of minusstrand strong stop DNA [(-) ss DNA], consistent with previous reports. However, specific
DNA synthesis was only observed at NCp7: RNA ration similar to that predicted to be
present in virions. Moreove, at these concentrations, NCp7 inhibited synthesis of non-specific reverse transcribed DNA products, which are initiated due to self-priming by RNA
templates. In contrast to results obtained with tRNALys.3 as primer, NCp7 inhibited synthesis of (-) ss DNA products primed by a 18 nt ribonucleotide (rPR), complementary to
the PBS, even though rPR can initiate synthesis of such mnaterial in the absence of preannealing with NCp7. Primer placement bandshift assays showed that NCp7 was necessary
for efficient formation of the tRNA/RNA complex. In contrast, NCp7 was found to prevent formation of the rPR/RNA complex. We also investigated the roles of an A-rich loop
upstream of the PBS, a 7nt region immediately downstream of the PBS, and a 54 nt deletion further downstream of the PBS in interactions with tRNALys.3 Only deletions in the
54 nt region, that may prevent formation of the U5/leader stern, but not deletions in the Arich loop or the 7 nt sequence, were found to prevent tRNALys.3 placement and priming
and viral infectivity.
M Wainberg, 3755 Cote Ste Catherine Rd, Montreal, Quebec, H3T I E2 Canada
Tel: [phone] Fax: [phone]
Tu.A.492
THE ROLE OF THE DOUBLE STRANDED RNA DEPENDENT PROTEIN KINASE
PKR IN HIV-I PATHOGENESIS
Koromilas A, Nai, K, DeLuca C, Li S, Wong A, Cuddihy A,Tam N, Hiscott, John. Lady Davis
Institute, McGill Ir'ersit, Montreal, Canada
Objectives: Replication of HIV I is inhibited by interferons (IFNs), and the IFN-induced,
double stranded RNA dependent seine/threonine protein kinase (PKR) is thought to mediate this event by mrodulating protein synthesis. The objecr ctive of the present study was to
examine the role of PKR in HIV-I replication and in moduaton of host signaling via the
NF-KB pathway
Results: Ectopic expression of a mutant of PKR defective in RNA binding elicited intracellu
lar events leading to an induction of HIV I replication in jurkat cells. Specifically, expression
c' CD4, the major HIV I receptor, was induced by the PKR mutant at the transcriptional
level This induction of CD4 correlated with an increase in proviral DNA synthesis upon
HIV I infection. Moreover downmodulation of CD4 mRNA by HIV- I was prevented in
cells expressing the mutant of PKR. Also, to examine the molecular basis of constitutive
NF iB binding activity in HIV I infected cells, we analyzed the turnover of IKBa protein, the
activity of PKR and the intracelular levels of NF KB subunits in myeloid cell models. HIV-I
infection resulted in constitutive, low level expression of type I interferon (IFN) at the
mRNA level. Constitutive PKR activity was also detected in DIV I infected cels, induced as
a result of IFN prcdduction. Increased degradation of IKBa in HIV-I infected cells may
account for the constitutive DNA binding activity Furthermore, a dramatic increase in c
Rel and NF-KB2 p100 was detected in HIV I infected cells relative to uninfected rells.
Conclusions: These findings suggest that modulation of PKR activity has important rmplhcations in HIV I infection through the regulation of CD4 expression and the NF KB pathway
HIV I infection of myeloid cells induces IFN production and PKR activity whch in turn
enhances IKBa phosphorylation and subsequent degradation. Enhanced turnover of IKBa
and the accumulation of NF-KB/Rel proteins may contribute to the chronically activated
state of HIV- I infected cells.
J Hiscott, 3755 Co',e Ste-Catherine, Montreal, Quebec, H3T I E2 Canada Tel: [phone] Fax: 5I I4340-7576 E-mail: [email protected]

227