The integrated HIV-1 proviral genome is transcribed by the host transcription machinery into a single 9.2 kb primary transcript. Transcription is tightly regulated by the interaction of the viral protein Tat with several cellular factors. To express the nine different gene products required for viral replication the virus has developed a number of strategies to regulate splicing of its transcript. Alteration of the delicate balance between spliced and unspliced RNAs can dramatically affect HIV-1 infectivity and pathogenesis. Transcription and processing of the viral genome involves interactions between viral RNA sequences, viral proteins and host cell factors, which may constitute novel therapeutic targets. We have identified a number of cellular factors and mechanisms regulating the splicing of specific viral mRNAs. We have altered the expression levels of several RNA processing factors in a cell line expressing HIV-1. Over-expression of the cellular protein hnRNP A1 led to a dramatic decrease in viral replication due to a marked reduction in the processing of mRNAs coding for the viral proteins Env, Tat and Rev. Viral transcription was severely reduced by the decrease in the amount of the viral transactivator Tat expressed. hnRNP A1 up-regulation induced a decrease in viral replication of over 200 fold in a stable cell line without affecting cell viability. We propose to evaluate the therapeutic potential of hnRNP A1 expression in cultures of Peripheral Blood Mononuclear Cells (PBMCs). We will utilize a VSV-G pseudotyped retroviral system to transduce primary PBMC cultures with an hnRNP A1 expression cassette fused to the fluorescent protein eGFP. PBMCs will be infected with different viral strains and the effects of hnRNP A1 expression on viral replication and cell viability will be analyzed. We will also develop a delivery system that specifically targets cells infected by HIV-1 to eliminate potential secondary effects due to hnRNP A1 over-expression in uninfected cells. Finally we will identify the minimal hnRNP A1 sequence required for viral inhibition creating a library of random N- and C- terminus truncation. The efficacy and toxicity of the active fragment will be compared with the full-length proteins in PBMCs challenged with divergent viral strains.
Current drugs utilized in the treatment of HIV-1 infected individuals cannot completely eliminate the virus, furthermore new multidrug resistant HIV strains are appearing with growing frequency. The great majority of these drugs target viral proteins. Unfortunately, because of the HIV-1 high mutation rate new multidrug resistant strains are appearing with growing frequency. Recent setbacks in the development of vaccines have accentuated the need for the development of drugs with novel mechanisms of action. Since the virus utilizes a multitude of host cellular proteins to replicate efficiently the identification of such factors might provide for novel therapeutic targets. Cellular targets have the advantage of not mutating, thus making unlikely the emergence of resistant viral strains. We have identified a cellular factor (hnRNP A1), which, if expressed at high levels in human cells, down-regulates viral replication over 200 folds without altering the viability of the cells. We propose to create to deliver this gene, or portions of it, in leukocytes, the primary target of the virus, to inhibit viral replication and block the progression of the infection.
|Paz, Sean; Lu, Michael L; Takata, Hiroshi et al. (2015) SRSF1 RNA Recognition Motifs Are Strong Inhibitors of HIV-1 Replication. J Virol 89:6275-86|
|Paz, Sean; Krainer, Adrian R; Caputi, Massimo (2014) HIV-1 transcription is regulated by splicing factor SRSF1. Nucleic Acids Res 42:13812-23|
|Jean-Philippe, Jacques; Paz, Sean; Lu, Michael L et al. (2014) A truncated hnRNP A1 isoform, lacking the RGG-box RNA binding domain, can efficiently regulate HIV-1 splicing and replication. Biochim Biophys Acta 1839:251-8|