Human Immunodeficiency Virus-Type 1 LTR DNA contains an intrinsic gene producing antisense RNA and protein products

Linda B Ludwig,Sanjay Sharma,Julian L Ambrus,Chiu-Bin Hsiao,Stanley A Schwartz,Kristie A Krawczyk,Stephen Brooks

doi:10.1186/1742-4690-3-80

Linda B Ludwig, Sanjay Sharma + Show 5 more

Open Access

https://doi.org/10.1186/1742-4690-3-80

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Abstract

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.

Highlights

While 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
We postulated that if the HIV-1 antisense INR (HIVaINR) functioned, it could indicate the presence of a novel HIV antisense gene responsible for either an RNA or protein product. We demonstrate that this HIVaINR functions as an initiation site for antisense RNA transcription in in vitro transcription reactions and in vivo, in human cells that have stably incorporated the human immunodeficiency virus type 1 (HIV-1) long terminal repeat (LTR) sequences
Using a recombinant HIV-1 antisense gene-FLAG vector transfected into human cells, we show that protein products can be translated

Summary

Introduction

While 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. These promoters are coordinately activated by a shared site for a cellular transcription factor, USF[2] Plant viruses, such as the ssDNA tomato leaf curl virus, can encode multiple, overlapping openreading frames (ORFs) and utilize either strand of the genomic ssDNA or a complementary DNA intermediate as templates for transcription [3]. The human hepatitis D virus, which possesses a single, circular genomic RNA, translates two of its major proteins (hepatitis delta antigen(s)) from a complementary antigenomic RNA intermediate[6]. The strategy of encoding genes on either strand of dsDNA, or genomic RNA or ssDNA and complementary nucleic acid intermediate, enables viruses to greatly enhance coding capability

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