Inhibition of HIV-1 replication by delivery of the SRSF1 RNA Recognition Motifs

Caputi, Massimo

Abstract

Transcription of the integrated HIV-1 proviral genome is tightly regulated by the interaction of the viral protein Tat with several cellular factors and he RNA Polymerase II complex. The transcribed viral pre- mRNA is spliced in multiple mRNAs to generate the nine different gene products required for viral replication. HIV has also developed a number of strategies to regulate splicing of its transcripts. Expression of the viral genome is dependent on the interactions between the viral promoter, RNA sequences, viral proteins and host cell factors. Alteration of the mechanisms regulating transcription and splicing of the viral messenger can dramatically affect viral infectivity and pathogenesis. Utilizing a combination of cell-based and biochemical approaches we have isolated a cellular RNA binding protein, SRSF1, which is an inhibitor of both viral transcription and splicing. SRSF1 exerts its antiviral activity by competing with the viral transcriptional transactivator Tat, thus reducing viral transcription, and by binding a series of sequences within the viral messengers, which regulate the choice of multiple splicing sites within the viral transcripts. Over-expression of SRSF1 induces the disruption of both transcription and splicing mechanisms resulting in a strong inhibition of viral replication. The minimal SRSF1 fragment required for its antiviral activity is constituted by the RNA Recognition Motifs (RRMs) 1 and 2, two RNA binding domains (RBDs). Expression of RRM1 and 2, in a stable cell line, can reduce the replication of a number of viral strains up to 3000 fold without altering cell viability. We propose to evaluate the therapeutic potential of the SRSF1 RRM domains. We will create a chimeric protein between the SRSF1 RRMs and the Tat Cell Penetrating Peptide (CPP), a short sequence, which allows for the delivery and internalization of molecular cargoes to eukaryotic cells with high efficiency. We will analyze the efficiency of intracellular delivery and antiviral activity of the CPP-RRMs chimeras in a leukocyte derived cell line and in CD4+ T cells purified from healthy donors and infected with viruses from different subtypes (B, C and D). Next, we will characterize the SRSF1 nuclear localization signal and optimize the nuclear delivery of chimeric proteins carrying the single RRM2, which efficiently binds the target RNA sequences but fails to preferentially localize within the cell nucleus and down-regulate viral replication with efficiency comparable to the RRM1 and 2 combined. Finally, we will utilize a novel endosomolitic agent, named dfTat, which allows for the efficient delivery and internalization of large protein cargoes after simple co-incubation with the target cells. The approach we propose will determine the therapeutic potential of a novel target protein and set-up future studies that utilize animal models.

Public Health Relevance

Current drugs utilized in the treatment of HIV-1 infected individuals cannot completely eliminate the virus. The majority of these drugs targets viral proteins. Unfortunately, because of the high HIV 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 o such factors might provide novel therapeutic targets. Cellular targets have the advantage of not mutating, thus the emergence of resistant viral strains is unlikely. We have identified a cellular factor (SRSF1), which can inhibit viral replication by altering the transcription and processing of the viral messengers. We have shown that a truncated version of the SRSF1 protein, if expressed at high levels, can down-regulate viral replication by over 3000 folds without altering the viability of the cells. We propose to synthesize the SRSF1 truncated protein in bacteria, purify and deliver it to infected cells utilizing the ability of a short peptide, named Cell Penetrating Peptide, to penetrate human cells with high efficiency. We will use this approach to inhibit viral replication ex-vivo in lymphocytes purified from healthy donors and infected by a number of viral strains.