Targeting RNA-protein interactions within the human immunodeficiency virus type 1 lifecycle.
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ABSTRACT:
SUBMITTER: Bell NM
PROVIDER: S-EPMC3928988 | biostudies-literature | 2013 Dec
REPOSITORIES: biostudies-literature
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Project description:Inhibition of virus replication by means of RNA interference has been reported for several important human pathogens, including human immunodeficiency virus type 1 (HIV-1). RNA interference against these pathogens has been accomplished by introduction of virus-specific synthetic small interfering RNAs (siRNAs) or DNA constructs encoding short-hairpin RNAs (shRNAs). Their use as therapeutic antiviral against HIV-1 is limited, because of the emergence of viral escape mutants. In order to solve this durability problem, we tested DNA constructs encoding virus-specific long-hairpin RNAs (lhRNAs) for their ability to inhibit HIV-1 production. Expression of lhRNAs in mammalian cells may result in the synthesis of many siRNAs targeting different viral sequences, thus providing more potent inhibition and reducing the chance of viral escape. The lhRNA constructs were compared with in vitro diced double-stranded RNA and a DNA construct encoding an effective nef-specific shRNA for their ability to inhibit HIV-1 production in cells. Our results show that DNA constructs encoding virus-specific lhRNAs are capable of inhibiting HIV-1 production in a sequence-specific manner, without inducing the class I interferon genes.
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Project description:BackgroundWhile viruses have long been shown to capitalize on their limited genomic size by utilizing both strands of DNA or complementary DNA/RNA intermediates to code for viral proteins, it has been assumed that human retroviruses have all their major proteins translated only from the plus or sense strand of RNA, despite their requirement for a dsDNA proviral intermediate. Several studies, however, have suggested the presence of antisense transcription for both HIV-1 and HTLV-1. More recently an antisense transcript responsible for the HTLV-1 bZIP factor (HBZ) protein has been described. In this study we investigated the possibility of an antisense gene contained within the human immunodeficiency virus type 1 (HIV-1) long terminal repeat (LTR).ResultsInspection of published sequences revealed a potential transcription initiator element (INR) situated downstream of, and in reverse orientation to, the usual HIV-1 promoter and transcription start site. This antisense initiator (HIVaINR) suggested the possibility of an antisense gene responsible for RNA and protein production. We show that antisense transcripts are generated, in vitro and in vivo, originating from the TAR DNA of the HIV-1 LTR. To test the possibility that protein(s) could be translated from this novel HIV-1 antisense RNA, recombinant HIV antisense gene-FLAG vectors were designed. Recombinant protein(s) were produced and isolated utilizing carboxy-terminal FLAG epitope (DYKDDDDK) sequences. In addition, affinity-purified antisera to an internal peptide derived from the HIV antisense protein (HAP) sequences identified HAPs from HIV+ human peripheral blood lymphocytes.ConclusionHIV-1 contains an antisense gene in the U3-R regions of the LTR responsible for both an antisense RNA transcript and proteins. This antisense transcript has tremendous potential for intrinsic RNA regulation because of its overlap with the beginning of all HIV-1 sense RNA transcripts by 25 nucleotides. The novel HAPs are encoded in a region of the LTR that has already been shown to be deleted in some HIV-infected long-term survivors and represent new potential targets for vaccine development.
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Project description:Previous analyses of human immunodeficiency virus type 1 (HIV-1) integration sites generated in infections in vitro or in patients in whom viral replication was repressed by antiviral therapy have demonstrated a preference for integration within protein-coding genes. We analyzed integration sites in peripheral blood mononuclear cells (PBMCs), spleen, lymph node, and cerebral cortex from patients with untreated HIV-1 infections. The great majority of integration sites in each tissue were within genes. Statistical analyses of the frequencies of integration in genes in PBMCs and lymph tissue demonstrated a strong preference for integration within genes. Although the sample size for brain tissue was too small to demonstrate a clear statistical preference for integration in genes, four of the five integration sites identified in brain were within genes. Taken together, our data indicate that HIV-1 preferentially integrates within genes during untreated infection.
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