Unit: Molecular Retrovirology
Director: WAIN-HOBSON Simon
Work is centred around the immunopathology of SIV infection. Projects aims to illustrate the role of antigenic stimulation, the dynamic of infiltration of CTLs into inflammatory sites, SIV latency and strain differences among HIV-1 subtypes. Novel attenuated SIV vaccines are being developed.
1. Mechanism of APOBEC3G induced retroviral G->A hypermutation Rodolphe SUSPENE, Michel HENRY, Denise GUETARD et Jean-Pierre VARTANIAN
This host death mechanism is circumvented by the HIV Vif protein which prevents APOBEC3G from getting into the virion. It is postulated that cytidine bases in nascent DNA synthesis are lethally edited by the host cell APOBEC3G molecule. The principal objective was to determine the mechanism of APOBEC3G associated G->A hypermutation.
One of the more singular traits among the wealth of genetic variation provided by HIV is G->A hypermutation - that is monotonous substitution of G residues for A when compared to the positive strand. for some hypermutated segments up to 60% of Gs can be substituted. Hypermutation only occurs during reverse transcription and mostly during minus DNA strand synthesis. Mechanistically therefore Cs are substituted on the minus strand which gives rise to G->A on the reference plus strand.
In a normal setting the HIV Vif protein interacts with APOBEC3G and prevents it from becoming incorporated into the virion. Conversely in the absence of Vif, APOBEC3G is packaged into the virion. APOBEC3G can bind RNA, but the role of this interaction, if any, in targeting of APOBEC3G to virions remains to be defined. Given the kinship to cytidine deaminases, we have demonstrated that cytidine residues on the neo-synthesized minus DNA strand are massively deaminated yielding uracil independently of reverse transcriptase. Hence, APOBEC3G is a single stranded DNA cytidine deaminase capable of restrincting retroviral replication.
2. Stoichiometry of HIV infection /Quantitating HIV DNA from reverse transcription to integration in vivo Rodolphe SUSPENE, Michel HENRY, Denise GUETARD et Jean-Pierre VARTANIAN
By studying splenocytes from two HIV-1 infected individuals, it was possible to show by fluorescent in situ hybridization (FISH) that, on average, the proviral copy number per cell was 3-4 with a range of 1-8. Greater than 75% of infected cells harboured two or more proviruses. By laser microdissecting individual FISH+ nuclei, followed by PCR, cloning and sequencing, it was possible to show that a single cell harboured a genetically diverse collection of genomes with up to 29% amino acid variation in the V1V2 hypervariable regions of the envelope protein. Hence the work may not only provide insights into the infection process in vivo but also highlight the tempo of recombination and its impact on HIV evolution.
Building on the observation that in vivo cells harbour multiple proviruses, the question we ask here is how many viruses infect a cell in vivo? It is known that proviruses are accompanied by unintegrated forms in the nucleus. By extensive sequencing of cloned PCR material from microdissected nuclei from human splenocytes, the number of discrete sequences asymptote to a finite number. The ratio of unintegrated/integrated DNA is ~ 10-20. Using infected PBMCs, as opposed to established T cell lines, and the proteasome inhibitor epoxomycin we have shown by TaqMan quantitation of nascent DNA formation that the proteasome degrades up to 75% of incoming virions. Combining the two studies the fraction of HIV RNA that gets converted into a provirus could be as low as 1:100. Hence, as a splenocyte may harbour 3-4 proviruses in vivo, this means that the cell was originally infected by ~300-400 virions. The high ratio and wide range in the number of virions making it to the provirus indicates a substantial stochastic component to the infection process.
3. A novel means to attenuate SIV by exchanging the viral promoter Nicole CHENCINER, Philippe BLANCOU, Denise GUETARD
Among the many HIV/SIV immunogens, only some attenuated live viral vaccines have afforded strong protection against intravenous challenge with a pathogenic SIV isolate. They have invariably been obtained by deleting gene segments. To date there has been a general inverse correlation between the degree of attenuation and protection. The exchange of the SIV promoter by other viral or cellular promoters may confer novel properties to the chimera, notably attenuation. Alternatively if viral expression is shifted away from the crucially important CD4+ T lymphocytes to other cells, such as macrophages and dentritic cells, this may preserve sufficient help to allow the immune system to contain infection.
We have worked extensively on one particular chimera where the core SIV promoter just 3' of nef nd 5' of TAR has been replaced by the powerful immediate early promoter of human cytomegalovirus (CMV-IE). The chimera (SIVmegalo) grows to very low titres in vivo - median titres for 15 animals were <400 copies/ml. This represents >1000 fold reduction in peak viremia compared to the parental virus. When challenged by the pathogenic virus SIVmac251, viremia was contained by >1000 compared to naive controls. In view of this, novel promoter chimeras are being tested in pilot studies (Blancou et al., J. Virol;, Feb 2004). We hope to greatly improve the safety of SIVmegalo in protecting against SIVmac251 by the intravenous, vaginal and intrarectal routes. We would like to know where SIVmegalo is replicating, why it is so attenuated and learn as much as possible about the nature of the immune responses it induces. The long term course of SIVmegalo infection will become clear. We should have an answer to the physiological relevance of HIV core promoter polymorphism, as some insight into whether shifting tropism into macrophages and dentritic cells spares CD4+T lymphocytes.
4. Construction of SIV chimeras. Compartmentalisation of viral replication throughout specific promoters usage. Mireille CENTLIVRE, Marie MICHEL and Monica SALA-SCHAEFFER
The aim of this project is to analyse the influence of different promoters on viral transcription, replication, cellular tropism and pathogenesis of immunodeficiency viruses. To develop this study in vivo, SIV chimeras have been constructed where the SIV genome presents non-overlapping Nef and LTR elements (STR). In the STR clone, the SIV homologous region has been replaced by either the enhancer/core portion of the HIV-1 B, C and E subtypes promoters, or the human promoters for the IL-2, IL-4 and INF-γ genes. These chimeras allow the phenotypic analysis of subtype-specific enhancer/core promoter HIV-1 regions. Moreover, compartmentalisation of viral replication in Th1 (IL-2 and INF-γ promoters) and Th2 (IL-4 promoter) cells will clarify the impact on pathogenesis of viral replication into a specific cellular subset.
5. Contribution of different cell compartments to persistence and pathogenesis of SIV in vivo Peter SOMMER
The molecular mechanisms leading to post-integration latency of HIV/SIV as well as the contribution of different cellular compartmenats to viral persistence and AIDS pathogenesis remain poorly understood. These important aspects of HIV/SIV infection rely on viral gene expression and replication, which are regulated by promoter elements located within the viral LTR. The function of these elements depends on the presence of cellular transcription factors. Our laboratory has concentrated on the construction of chimeric SIVs with promoters modified by the insertion of constitutive and tissue-specific regulatory elements. This novel approach will allow to dissect the cell tropism of SIV in vivo and to address experimentally the impact of different cellular compartments on viral persistence and pathogenesis in the macaque model.
Keywords: SIV, HIV, Vaccine, multi-infection