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Detection of the HIV-1 minus-strand-encoded antisense protein and its association with autophagy.

ABSTRACT: HIV-1 proteins are synthesized from a single transcript in an unspliced form or following splicing, but the existence of an antisense protein (ASP) expressed from an antisense polyadenylated transcript has been suggested. Difficulties linked to the detection of this protein in mammalian cells led us to codon optimize its cDNA. Codon-optimized ASP was indeed efficiently detected in various transfected cell lines following flow cytometry and confocal microscopy analyses. Western blot analyses also led to the detection of optimized ASP in transfected cells but also provided evidence of its instability and high multimerization potential. ASP was mainly distributed in the cytoplasm in a punctate manner, which was reminiscent of autophagosomes. In agreement with this observation, a significant increase in ASP-positive cells and loss of its punctate distribution was observed in transfected cells when autophagy was inhibited at early steps. Induction of autophagy was confirmed by Western blot analyses that showed an ASP-mediated increase in levels of LC3b-II and Beclin 1, as well as colocalization and interaction between ASP and LC3. Interestingly, Myc-tagged ASP was detected in the context of proviral DNA following autophagy inhibition with a concomitant increase in the level and punctate distribution of LC3b-II. Finally, 3-methyladenine treatment of transfected or infected U937 cells decreased extracellular p24 levels in wild-type proviral DNA and to a much lesser extent in ASP-mutated proviral DNA. This study provides the first detection of ASP in mammalian cells by Western blotting. ASP-induced autophagy might explain the inherent difficulty in detecting this viral protein and might justify its presumed low abundance in infected cells.

SUBMITTER: Torresilla C

PROVIDER: S-EPMC3624327 | biostudies-literature | 2013 May

REPOSITORIES: biostudies-literature

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Detection of the HIV-1 minus-strand-encoded antisense protein and its association with autophagy.

Torresilla Cynthia C Larocque Émilie É Landry Sébastien S Halin Marilène M Coulombe Yan Y Masson Jean-Yves JY Mesnard Jean-Michel JM Barbeau Benoit B

Journal of virology 20130220 9

HIV-1 proteins are synthesized from a single transcript in an unspliced form or following splicing, but the existence of an antisense protein (ASP) expressed from an antisense polyadenylated transcript has been suggested. Difficulties linked to the detection of this protein in mammalian cells led us to codon optimize its cDNA. Codon-optimized ASP was indeed efficiently detected in various transfected cell lines following flow cytometry and confocal microscopy analyses. Western blot analyses also ...[more]

PMID: 23427159

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Project description:BACKGROUND: Recent evidence proposes a novel concept that mammalian natural antisense RNAs play important roles in cellular homeostasis by regulating the expression of several genes. Identification and characterization of retroviral antisense RNA would provide new insights into mechanisms of replication and pathogenesis. HIV-1 encoded-antisense RNAs have been reported, although their structures and functions remain to be studied. We have tried to identify and characterize antisense RNAs of HIV-1 and their function in viral infection. RESULTS: Characterization of transcripts of HEK293T cells that were transiently transfected with an expression plasmid with HIV-1NL4-3 DNA in the antisense orientation showed that various antisense transcripts can be expressed. By screening and characterizing antisense RNAs in HIV-1NL4-3-infected cells, we defined the primary structure of a major form of HIV-1 antisense RNAs, which corresponds to a variant of previously reported ASP mRNA. This 2.6 kb RNA was transcribed from the U3 region of the 3' LTR and terminated at the env region in acutely or chronically infected cell lines and acutely infected human peripheral blood mononuclear cells. Reporter assays clearly demonstrated that the HIV-1 LTR harbours promoter activity in the reverse orientation. Mutation analyses suggested the involvement of NF-?? binding sites in the regulation of antisense transcription. The antisense RNA was localized in the nuclei of the infected cells. The expression of this antisense RNA suppressed HIV-1 replication for more than one month. Furthermore, the specific knockdown of this antisense RNA enhanced HIV-1 gene expression and replication. CONCLUSIONS: The results of the present study identified an accurate structure of the major form of antisense RNAs expressed from the HIV-1NL4-3 provirus and demonstrated its nuclear localization. Functional studies collectively demonstrated a new role of the antisense RNA in viral replication. Thus, we suggest a novel viral mechanism that self-limits HIV-1 replication and provides new insight into the viral life cycle.

Dataset Information

Detection of the HIV-1 minus-strand-encoded antisense protein and its association with autophagy.

Publications

Detection of the HIV-1 minus-strand-encoded antisense protein and its association with autophagy.

Similar Datasets

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