Regulation of HIV-1 Gag expression by SR proteins

HIV infection causes AIDS and is a worldwide pandemic. In 2010, there were 2.7 million new infections, 1.8 million AIDS-related deaths, and 34 million people living with HIV. Currently, there are approved antiretroviral drugs targeting four stages of the HIV life cycle and in 2010 there were over 7 million people receiving antiretroviral therapy. However, drug resistant strains of HIV are a significant problem and there is still a great need for novel drug targets. One promising approach to develop new antiviral drugs is to target cellular proteins. This proposal will investigate how a family of cellular proteins, called SR proteins, regulate expression of the HIV structural protein Gag, which is necessary to make virions. We will identify which of the 12 human SR proteins regulate Gag expression and mechanistically characterise how they do so. This will identify which SR proteins could be targeted by antiretroviral drugs to inhibit HIV Gag expression.

The HIV-1 genomic RNA (gRNA) has several independent functions in the HIV-1 life cycle and its regulation is critical for virus replication. In the nucleus, the gRNA is the pre-mRNA that is spliced into mRNAs for seven different proteins. In the cytoplasm, the unspliced gRNA is the mRNA for Gag and Gag-Pol, which are polyproteins containing the viral structural proteins and enzymatic activities, and is also packaged into virons as the infectious genome. Therefore, the gRNA must avoid splicing to accumulate in the cytoplasm and be translated. In addition, the gRNA has a highly structured 5' UTR and appears to be a poor substrate for translation. While these properties predict that Gag should be poorly expressed, ~2000 molecules of Gag are required for each virion and a large amount of Gag is expressed in an infected cell. Understanding how Gag expression is regulated will fill a critical knowledge gap in the HIV-1 life cycle.

SR proteins are a family of 12 RNA binding proteins in humans that regulate almost all steps of cellular mRNA metabolism. SR proteins also regulate several steps of HIV-1 mRNA metabolism and our preliminary data demonstrate that at least two SR proteins, SRp40 and SRp55, strongly stimulate Gag expression by enhancing gRNA abundance and Gag translation efficiency. In this proposal, we will test the whole SR protein gene family to determine which of these proteins promote Gag expression. To characterise their mechanisms of action, we will perform a structure/function analysis on the active SR proteins to identify the necessary functional domains as well as identify their binding sites in the HIV-1 gRNA and the cellular co-factors they interact with to stimulate Gag expression. Finally, we will determine how SRp40 and SRp55 stimulate Gag translation efficiency. These experiments will increase our knowledge of how HIV-1 Gag expression is controlled and may identify novel antiviral drug targets.

The primary objective of this proposal is to determine how cellular SR proteins regulate HIV-1 Gag expression and this research may have two major impacts. First, this proposal will characterise how HIV-1 utilises specific SR proteins as co-factors for viral replication, which could lead to novel antiretroviral treatments. The development of new anti-HIV drugs remains a critical priority for combating the AIDS pandemic. Targeting cellular factors is an exciting strategy for drug development with Maraviroc, which inhibits viral entry into the cell, as the first approved member of this type of antiretroviral. Small molecules that target one member of the SR protein family, SF2/ASF, inhibit HIV-1 replication in vitro and retroviral replication in a mouse model. A further understanding of how the other 11 human SR proteins regulate HIV-1 gene expression, and in particular Gag expression, may allow the development of novel drugs that prevent the production of viral proteins. We will engage KCL's subsidiary company, KCL Business, to evaluate the commercial potential of any discoveries we make and, if warranted, help foster collaboration with pharmaceutical partners to develop novel therapeutics. Second, identification of new SR protein co-factors and mechanisms that regulate gene expression will be informative to virologists studying gene expression of other viruses and molecular biologists studying cellular gene regulation. Many other viruses use SR proteins and/or their regulatory proteins as co-factors for their gene expression and a further understanding of how SR proteins regulate HIV-1 Gag expression may help elucidate how the other viruses utilise SR proteins. Also, many human diseases involve deregulated cellular mRNA metabolism, including cancer, and a further understanding of SR protein mechanisms of action may aid in the development of novel therapies for those diseases. Therefore, the beneficiaries of this research will potentially include: 1) scientists studying HIV replication, 2) scientists who develop novel anti-HIV therapeutics, 3) scientists who study and develop drugs that target the gene expression of other viruses or aberrant cellular gene expression and 4) patients who have viral infections or deregulated control of gene expression.