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

12th World AIDS Conference

Abstracts 11133-11137 9

mission of HIV-1 by cell-free virus and by co-cultivation with uninfected cells were
evaluated by the MAGI assay for Tat-mediated expression of /-galactosidase, by
detection of HIV-1 antigens by immunofluorescence staining or by Elisa for p24,
and by analysis of viral protein structure by electrophoresis and Western blotting.
Alternatively, cell-free and cell-cell transmission of an HIV-1 mutant in the viral
PR (SVC-P2) were similarly evaluated.
Results: When viral PR was inhibited by Ro 31-8959 or was inactivated by
mutation, HIV-1 transmission by cell-free virus was reduced 100-350 fold. However transmission of HIV-1 by cocultivation in the absence of PR activity was
reduced only 2-20 fold. Western blot analysis of preintegration complexes in cell
nuclei confirmed the absence of mature viral matrix protein during transmission
by cocultivation in the presence of Ro 31-8959.
Conclusion: HIV-1 synthesized during PR inhibition or inactivation retains
infectivity and can be transmitted by cell-cell contact.
111133 HIV maturation and stability in the presence of protease
inhibitors in vitro
Richard M. Donovan1, D.A. Friedman2, H.J. Caceres2, D.M. Baxa2, C.E. Bush2,
N.P. Markowitz2. I Infectious Diseases Henry Ford Hospital, 2799 West Grand
Blvd. Detroit MI.; 2Henry Ford Hospital, Detroit, Ml, USA
Background: Protease inhibitors (PIs) act as competitive inhibitors of the HIV viral protease. The objective of this study was to compare virucidal versus virustatic
properties of the currently used Pis by examining the ability of immature HIV
virions to mature, and the stability of RNA in immature virions.
Methods: HeLa/LAV cells constitutively producing HIV were grown in the
presence or absence of saquinavir, indinavir, nelfinavir or ritonavir to generate
immature HIV virions. Immature HIV virions were incubated for time periods ranging from minutes to several days, under a variety of conditions of temperature
and pH. The resultant HIV Gag protein forms were then analyzed by western
blot, and a novel antigen capture ELISA for Pr55Gag. HIV RNA was measured
by RT-PCR.
Results: Western blots showed that all cultures containing 1 /iM or more of
any of the 4 PIs had a strong Pr55Gag band, and a fainter high molecular weight
Gag-Pol band. Cultures without drugs had strong p24 and p17 bands and faint
or absent bands of immature forms. Quantitative results from antigen capture
ELISA on immature virions incubated at 37 C for 48 hours showed that immature
Gag forms remained at 85%, 84%, 98%, and 96% of initial values for saquinavir,
indinavir, nelfinavir, and ritonavir, respectively. Removal of residual PI by repeated
pelleting of the virion at 23,000 x g, or by ultrafiltration did not cause the immature
HIV virions to mature. Incubation of the virions under conditions optimized for the
activity of the HIV protease similarly did not lead to detectable maturation of the
viral Gag proteins. The average HIV RNA copy number in immature virions after
incubation for 48 hours at 37 C was 80% of their initial value, while the average
of mature virions was 90%. HIV virions disrupted by detergent immediately had
RNA copy numbers fall to less than 1% of their initial value.
Conclusions: We found no evidence that immature HIV virions produced in
the presence of any of the drugs used as protease inhibitors were able to undergo
maturation in vitro. However, the HIV RNA genome in immature virions does
appear to be stable for relatively long time periods.
111134 Identification of a minimum HIV-1 gag sequence required
for particle assembly
Chin-Tien Wang. Taipei 112, No 201 Sec 2, Shih-Pai Road Shih-Pai, Taipei,
Taiwan
Objectives: To identify a minimum HIV-1 gag coding sequence capable of particle
assembly and release from mammalian cells.
Methods: We constructed a series of C-terminally truncated HIV gag mutants
and mutants derived from combination of the C-terminal truncation mutants and a
MA deletion (AMA) mutant. The abilities of these mutants to assemble and release
virus particles were assessed by Western immunoblotting and sucrose density
gradient fractionation experiments. Localization of the mutant Gag proteins in
expressing cells were revealed by indirect immunofluorescence experiments, and
mutant particle-associated RT activities were tested by in vitro RT assays.
Results: Our analysis indicated that truncated Gag precursors lacking most
of the C-terminal gag gene products assembled and were released from 293T
cells. A mutant with a combined deletion of the MA (AMA) and p6 domain even
produced particles at levels comparable to that of wild-type virus. Furthermore,
most mutants derived from combination of the AMA and the C-terminal truncation
mutations did not prevent particle production completely, provided the CA-p2
region remained intact. Sucrose density gradient fractionation analysis indicated
that most mutants exhibited a wt retrovirus particle density. Exceptions to this
rule were mutants with an intact MA domain, by deleted downstream of the p2
domains. These C-terminal truncations mutants possessed particle densities of
1.13-1.15 g/ml, lower than that of wt.
Conclusion: Our smallest HIV gag gene product capable of particle formation
was a 28 kDa protein which consists of a few MA amino acids and the CA-p2

domain. The N-terminal portions of the CA domain, which have been shown to
be dispensable for core assembly, became critical when most of the MA domain
was deleted, suggesting a requirement of an intact CA to assemble and release
particles.

I11135] Role of HIV-1 capsid phosphorylation in viral infection
Christine Cartier, P. Sivard, C. Tranchat, C. Desgranges, V. Boyer. INSERM
Unite 271, 151 Cours Albert Thomas, 69424 Lyon Cedex 03, France
Background: Our previous study showed that two cellular protein kinases are
incorporated into HIV-1 virions. One of those protein kinases is the ERK2 Mitogen-Activated protein kinase. We also detected the phosphorylation of the p24
capsid protein in the viral particles.
Objectives: To study the role of p24 phosphorylation during viral life cyle.
Methods: In vitro phosphorylation assays using GST viral fusion proteins and
recombinant kinases. Site-directed mutagenesis of each serine residue of p24.
Characterisation of those mutant viruses in transfection and infection assays.
Study of viral proteins maturation process by Western-blot analysis.
Results: We showed by in vitro binding and phosphorylation experiments that
the viral capsid was not phosphorylated by ERK2 MAPK. By site-directed mutagenesis using pNL4-3 infectious clone, we generated 9 mutants corresponding to the
substitution of each serine residue by alanine. One of those showed a reduced
Reverse Transcriptase activity and p241evel compared to wild type virus after
transfection of 293 cells. By Western-blot experiments, the maturation process of
viral proteins was analysed. Moreover, two additionnal mutants demonstrated a
very weak capacity to induce syncitia formation in the C8166 cell infection assay.
Conclusion: Those preliminary results suggest a direct link between p24
phosphorylation and viral infectivity.
11136 Production and characterization of core like particles
(CLP) by expression of the HIV-1 gag gene in a
Baculovirus system
Eric Saman1, I. Ottevaere1, M. Vanden Haeseveldel, M. Cornelissen2,
K. Devos1. 'Innogenetics, Industriepark 7B4; 2University Gent, Gent, Belgium
Objective: Expression of the HIV-1 gag gene in a Baculovirus insect system to
generate particles which resemble the native viral core structure. Physical and
immunochemical characterization of these structures.
Methods: The gag gene of the HIV-1 isolate Ant166 was introduced in the
baculovirus expression vector pAcUW51 under the control of the polyhedrin promoter. This construct was introduced into the baculovirus genome via homologous
recombination and the recombinant virus was used to infect Sf9 cells in different
culture media. The culture supernatant and the cell lysate were analysed for the
presence of high molecular weight particles by sucrose gradient centrifugation,
western blot and p24 capturing ELISA. Budding of the particles was visualized by
transmission electron microscopy of infected cells. The stability of the particles
was investigated by treatment with different detergents.
Results: Sucrose gradient (20-60% sucrose) analysis of the Sf9 cell culture
supernatant revealed the presence of high density material (1.15 g/cm3) reactive
in a p24 capturing ELISA. When cells were grown in serum containing medium,
a broad distribution of the p24 containing material was seen over the gradient
whereas in serum free medium a sharp and symmetrical antigen profile was
found at 1.15 g/cm3. Treatment of the high density material with SDS or nonionic
detergents (e.g. NP40) resulted in disappearance of the high density antigen
peak, indicating the disruption of the particulate structure of the antigen. Electron
microscopy confirmed the presence of budding particles at the surface of the
infected Sf9 cells, giving additional evidence for the presence of highly ordered
CLP.
Conclusions: Production of the HIV-1 gag precursor in Sf9 insect cells generates CLP which bud from the cell membrane into the culture medium. Treatment
of this material with NP40 detergent disrupts these structures, which is in contrast
to published findings. The relative lower stability of our CLP may be attributed
to a point mutation in the N-terminal part of the matrix protein. This possibility is
being investigated.
11137 The role of the cytoskeleton of HIV-infected cells as the
transportation system of viral structural proteins
Ichiro Takahashi1, M. Takama1, K. Ushijima2, Y. Nonomura1, H. Hoshino3.
'Kaga 2-11-1, Itabashi- KU, Tokyo 173 Lab. EM. Teikyo Univ. Sch. Med.; 2Tokyo
Univ. Sch. Med. Tokyo;3 Gunma Univ. Sch. Med. Maebashi, Japan
Objectives: To clarify the role of cytoskeleton of HIV-infected cells that would
play an important part to transport viral structural proteins during viral producing
processes.
Methods: Lymphocyte strains (Molt-4/HTLV-III B, TALL-1/KB-1) and monocyte strains (U937/HTLV-III B) were used and fixed for the immuno-electron
microscopy. Eeach thin sections were immuno-stained with 1st Abs (antigag MoAbs: p17/p24, anti-env MoAbs: gp41/gp120, anti-cytoskeleton MoAbs:
actin/intermediate filament(IF) etc.) and with colloidal golds as 2nd Abs.
Results: Each strains revealed common features, i.e., the viral structural proteins were synthesized on the surfaces of rough endoplasmic reticulum etc. and
then via long fibrous structures those were transported to just under the plasma
membrane of infected cell. These fibrous structures were identified as IF that were

located from around nuclei to plasma membranes. On the other hand contrary
to expectations, actin filaments were depolimerized that were no more able to
observe as usual filamentous structures.
Conclusion: From these results, it was revealed that among cytoskeletons
just intermediate filament played a role to transport viral structural poteins in
HIV-infected cells.