Molecular Analysis Of Retroviral Genes And Their Products

National Institute Of Allergy And Infectious Diseases

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Abstract

HIV-1 encodes genes that are crucial for replication in primary cells, exerting functions not provided by the host. Gag, Pol, and Env products represent the main virion components, while Tat and Rev regulate intracellular transcriptional and post-transcriptional events for the controlled expression of viral genes. Of particular interest to us are the HIV accessory proteins Vif, Vpr, Vpu, and Vpx, which are unique to primate lentiviruses. There is strong evidence that these proteins operate in conjunction with specific host factors. Accessory proteins do not have enzymatic activity but function primarily if not exclusively as molecular adaptors to link viral or cellular factors to pre-existing cellular pathways. Over the past decade, our research focus has shifted more and more towards the characterization of host factors and their roles in virus replication. HIV-1 targeted host factors that we have investigated in FY25 were (i) The glutamine transporter ASCT2; (ii) the nuclear pore complex (NPC) cofactor POM121C; and (iii) the myeloid cell specific transcriptional cofactor PU1. (i) The ASCT subfamily of amino acid transporters behaves as a restriction factor of HIV-1. The interplay between HIV-1 and host plasma membrane proteins involved in cell metabolism, signaling, homing, and immunological surveillance, allows HIV-1 to avoid antiviral immune responses and establish a systemic and persistent infection. The ASCT subfamily of amino acid transporters includes the proteins SLC1A4 and SLC1A5, also known as ASCT1 and ASCT2, respectively. These transporters are responsible for the trafficking of neutral amino acids across the cell plasma membrane. Because of their key physiological role, they are expressed at the plasma membrane of a broad set of tissues. In fact, ASCT2 is a major regulator of glutamine transport in HIV-1 primary target cells, such as activated CD4+ T cells and differentiated macrophages. Our previous results suggest that ASCT2 behaves as a restriction factor by promoting the incorporation of the complex formed between ASCT2 and the immature envelope precursor gp160 into the viral particles, leading to a decrease in viral infectivity. We also showed that Vpu targets ASCT2 by affecting the maturation and intracellular trafficking of ASCT2 to the plasma membrane. Our new biochemical and functional studies suggest that ASCT1 behaves similar than ASCT2, by promoting the synthesis and incorporation of ASCT1 into the viral particles, leading to a decrease in viral infectivity. Finally, we discovered that Vpu, a key accessory protein of HIV-1 that has been shown to affect the plasma membrane presence of several transmembrane glycoproteins, also interacts and restricts ASCT1 antiviral activity via a mechanism that we have not elucidated yet. Taken together, not only were we able to identify a subfamily of amino acid transporters that have evolved together to restrict HIV-1 infectivity, but we also demonstrated that Vpu interacts and restrict both members of this subfamily of amino acid transporters. (ii) Pom121C is a mis-localized nuclear pore component that inhibits HIV-1 capsid entry into the nucleus. Nuclear entry of HIV-1 capsids involves the specific interaction with components of the nuclear pore complex. In particular, FG-repeat sequences located in most nucleoporins have been found to play a crucial role in HIV-1 nuclear entry. We previously reported that overexpression of only the FG repeat-containing C-terminal region of the nucleoporin POM121C (614-987) results in significant inhibition of HIV-1 infection. However, the underlying mechanism of this phenomenon remained unclear. We now report the identification of escape mutants of HIV-1 that are resistant to POM121C. We mapped the mutations responsible for POM121C resistance to two amino acid positions in the capsid protein. Furthermore, we demonstrate that inhibition of HIV-1 infection involves an interaction of POM121C and capsid proteins. These findings suggest that targeting the nuclear import process through interference with capsid-nucleoporin interactions could be a potential strategy for developing novel antiviral therapies. (iii) HIV-1 reactivation and cell death in non-T cells with a pharmacological PU.1 inhibitor. Combination antiretroviral therapy (cART) achieves drastic reduction of AIDS-progression risk. However, long-lived HIV-1 latently infected reservoirs such as macrophages, and other non-T cell reservoirs, remain a major obstacle for an HIV-1 cure. The “shock and kill” strategy is being tested as one way to eradicate HIV-1 latently infected reservoirs. The selective and effective “Kill” of HIV-1 latently infected cell depends on a quantitative “Shock” strategy. To improve “Kill” and to induce advanced “Shock”, a deeper understanding of the cell type specific HIV-1 latency mechanism is essential. We previously showed that PU.1, a myeloid cell-specific transcriptional factor, reduces HIV-1 transcriptional activation. However, we also found that acute infection of monocyte-derived-macrophages (MDMs) by HIV-1 induces a reduction of PU.1 expression, and HIV-1 Tat inhibits PU.1 DNA-binding activity in vitro. We now investigated the relationship between PU.1 activity and HIV-1 activation. First, chromatin immunoprecipitation (ChIP)-qPCR analysis showed that PU.1 accumulated at the HIV-1 5’LTR around 1 day post infection in MDMs from healthy donors. However, accumulation of PU.1 at the LTR was significantly reduced at 3 days post infection. At the same time, accumulation of Tat protein at the 5’ LTR was increased. To further investigate the role of PU.1 activity for HIV-1 expression, we used OM10.1 cells, which is a latently infected cell line based on HL-60 myeloid cells. Using PU.1-targeting shRNA, we established stably PU.1 depleted OM10.1 cells to investigate the PU.1 contribution to HIV-1 latency. PU.1 depletion induced HIV-1 reactivation in OM10.1 cells. Interestingly, pharmacological PU.1 inhibitor (PU.1-i) also induced HIV-1 reactivation through PU.1 dissociation from HIV-1 5’LTR in OM10.1 cells. Furthermore, PU.1-i enhanced PKC agonist (Prostratin and PEP005)-mediated HIV-1 reactivation in OM10.1 cells. In addition, combining treatment of PU.1-i and PKC agonists caused selective cell death in OM10.1 cells, but not HIV-1 uninfected HL-60 cells. These data improve our understanding of strategies that could lead to the elimination of latently infected non-T cell reservoirs based on potent “Shock” and selective “Kill” strategies.