Abstract
BackgroundRetroviral gene expression generally depends on a full-length transcript that initiates in the 5' LTR, which is either left unspliced or alternatively spliced. We and others have demonstrated the existence of antisense transcription initiating in the 3' LTR in human lymphotropic retroviruses, including HTLV-1, HTLV-2, and HIV-1. Such transcripts have been postulated to encode antisense proteins important for the establishment of viral infections. The antisense strand of the HIV-1 proviral DNA contains an ORF termed asp, coding for a highly hydrophobic protein. However, although anti-ASP antibodies have been described to be present in HIV-1-infected patients, its in vivo expression requires further support. The objective of this present study was to clearly demonstrate that ASP is effectively expressed in infected T cells and to provide a better characterization of its subcellular localization.ResultsWe first investigated the subcellular localization of ASP by transfecting Jurkat T cells with vectors expressing ASP tagged with the Flag epitope to its N-terminus. Using immunofluorescence microscopy, we found that ASP localized to the plasma membrane in transfected Jurkat T cells, but with different staining patterns. In addition to an entire distribution to the plasma membrane, ASP showed an asymmetric localization and could also be detected in membrane connections between two cells. We then infected Jurkat T cells with NL4.3 virus coding for ASP tagged with the Flag epitope at its C-terminal end. By this approach, we were capable of showing that ASP is effectively expressed from the HIV-1 3' LTR in infected T cells, with an asymmetric localization of the viral protein at the plasma membrane.ConclusionThese results demonstrate for the first time that ASP can be detected when expressed from full-length HIV-1 proviral DNA and that its localization is consistent with Jurkat T cells overexpressing ASP.
Highlights
Retroviral gene expression generally depends on a full-length transcript that initiates in the 5’ long terminal repeat (LTR), which is either left unspliced or alternatively spliced
Construction and characterization of an human immunodeficiency virus type 1 (HIV-1) ASP mutant proviral clone In order to study ASP, we first generated a mutated proviral clone in which a stop codon was inserted in frame to the asp open reading frames (ORF)
This mutation resulted in termination of ASP at amino acid 12 of the published sequence [2] without altering the amino acid composition of the Env protein encoded on the sense strand. This resulting pNL4.3ASPmut12 construct was transfected in 293T cells and compared for p24 production to 293T cells transfected with wild type (WT) pNL4.3
Summary
Retroviral gene expression generally depends on a full-length transcript that initiates in the 5’ LTR, which is either left unspliced or alternatively spliced. We and others have demonstrated the existence of antisense transcription initiating in the 3’ LTR in human lymphotropic retroviruses, including HTLV-1, HTLV-2, and HIV-1 Such transcripts have been postulated to encode antisense proteins important for the establishment of viral infections. The objective of this present study was to clearly demonstrate that ASP is effectively expressed in infected T cells and to provide a better characterization of its subcellular localization Human lymphotropic retroviruses, such as human T-cell leukemia virus type 1 (HTLV-1) or human immunodeficiency virus type 1 (HIV-1), have evolved multiple strategies to direct the synthesis of a complex proteome from a small genome, which involves alternative splicing, internal ribosomal entry sites, ribosomal frameshifting, and leaky scanning [1]. Asp codes for a highly hydrophobic protein [2] (Figure 1A) that has been found associated with virions released from infected cells [22]
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