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Analysis of early HIV-1 induced changes in host gene expression following infection of primary cells

Final Report Summary - GIFHIVIPC (Analysis of early HIV-1 induced changes in host gene expression following infection of primary cells)

Human immunodeficiency virus type one (HIV-1) infection induces the depletion of CD4+ T cells in infected individuals, leading to AIDS. The currently available treatments for HIV-1 infection do not cure the infection, display many side effects, lead to drug resistance, and are expensive. Consequently, there is a dire need to further define the cell host/virus interactions that are either required for viral replication, or are capable of controlling infection. Accordingly, recent work has identified four cell autonomous factors, the Apobec3g, TRIM5a, Tetherin and SAMHD1 proteins (4, 8, 9, 11, 13), that have the capacity to suppress HIV-1 replication: collectively, these host-encoded proteins are called intrinsic (or innate) resistance factors. It is anticipated that the identification of such factors will facilitate the development of new classes of anti HIV therapeutics.

In this context, the project aimed to determine if cells are able to respond rapidly to HIV-1 infection through the induced expression of proteins that are then capable of limiting replication. A genome wide analysis of gene expression was conducted in primary CD4+ T cells (obtained from 16 different healthy donors) following HIV-1 infection, by using microarrays. Time-course experiments were performed with CD4+ T cells following infection with the IIIB strain of HIV-1 or incubation with an envelope-deleted virus. The expression of several hundred genes was regulated more than two fold following HIV-1 infection (529 genes were differentially expressed at 12h, and 326 at 48h, respectively). Importantly no significant gene expression changes were observed following incubation with a virus devoid of Envelope (i.e. unable to enter the cells). This showed that gene regulation was specifically due to true HIV-1 infection. An extensive gene ontology and pathway analysis on the microarrays data showed that there was a specific enrichment for chromatin and nucleosome organisation functions early post-infection (i.e. 12h) whereas immune response-, cell activation- and apoptosis-related functions were significantly affected at a later stage (i.e. 48h post infection). Interestingly, among the differentially expressed genes, many interferon (IFN)-stimulated genes (ISGs) were upregulated both at 12 and 48h post infection, whereas type I IFN was usually not detected in the cell supernatant using ELISA or a reporter cell assay. The microarrays data were validated using RNAs obtained from independent time courses, cDNA synthesis and quantitative real time polymerase chain reaction (qRT-PCR). 15 genes were validated by qRT-PCR including nine ISGs (e.g. ZBP1, IRF7, Mx1, OAS1, TRIM25) and 6 other genes (e.g. HIST1H4H, PTK2, and ZNF791). Importantly, the study was then extended to other HIV-1 strains, namely NL4-3 and BK132 (a primary isolate), and similar data were obtained. This strongly suggests that global gene expression changes are induced by HIV-1 infection of primary CD4+ T cells. A last batch of microarrays is currently being processed to complete the project. This work should then provide a global view of gene expression changes induced by HIV-1 and will lead to a peer-reviewed publication.

This work showed that HIV-1 infection induced the expression of numerous ISGs, some of them well known for being anti-viral against other viruses. Interestingly, it has been known for a long time that type I IFN-treatment (hence ISGs induction) renders some cell types resistant to HIV-1 replication (1, 7, 10, 12). However the actual step at which viral replication was affected was not clearly defined. Hence we sought to better define the effect(s) of IFNa on HIV-1 infection of primary human macrophages and CD4+ T cells, as well as several monocytic and T cell lines. We demonstrated that IFNa treatment of macrophages, monocytic THP-1 cells, and to a lesser extent primary CD4+ T cells, markedly inhibited infection, whereas the effects were minimal in CD4+ T cell lines. Virus entry was essentially unaffected by IFNa, but substantial decreases in nascent cDNA accumulation correlated closely with losses in infectivity. We also found that diverse primate and non-primate retroviruses were susceptible to suppression by IFNa. Importantly, all the primary and immortalised cells used were proficient at responding to IFNa, as judged by the induced expression of prototypic ISGs, including PKR and OAS1, indicating that a general deficiency in IFNa-responsiveness did not underlie IFNa's inability to elicit an anti-viral state in CD4+ T cell lines. This work led to a publication in Journal of Virology in 2010 (3).

The potential hallmark for Apobec3 mediated editing in IFNa-treated macrophages was investigated. Indeed, Apobec3 proteins, and particularly APOBEC3A, are IFNa-inducible (6) and therefore it was interesting to study their potential role in the IFNa-mediated block of HIV-1 infection. Infrequent editing of HIV-1 reverse transcripts was observed in IFNa-treated macrophages. Based on the local sequence contexts of these mutations and the established characteristics of Apobec3 protein expression in myeloid cells, it seemed likely that Apobec3a could be responsible for a substantial proportion of this activity. This work led to a publication is Journal of Virology in 2011 (5).

Finally, some of the most interesting genes have been chosen for further analysis. This analysis was focused on the ISGs induced by HIV-1 infection, as these genes are coding either known or potential anti-viral factors. A number of about 15 candidate ISGs were cloned and their activity against HIV-1 infection was analysed in a model cell line. Preliminary data showed that the overexpression of one of these genes was specifically affects the early steps of HIV-1 infection whereas its silencing in IFN-treated cells partially rescues the IFNa mediated block to HIV-1 infection. Its effect is now being characterised in more details. This work led to the identification of a new IFNa-inducible anti-HIV-1 cellular factor and will lead to a peer-reviewed publication.

References
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