Dataset Information

Biochemical analysis of the complex between the tetrameric export adapter protein Rec of HERV-K/HML-2 and the responsive RNA element RcRE pck30.

ABSTRACT: The RNA export adaptor protein Rec, encoded for by the human endogenous retrovirus HERV-K/HML-2 elements, binds to the Rec responsive element (RcRE) located in the 3' untranslated region of HERV-K/HML-2 transcripts. Binding allows the nucleocytoplasmic export of unspliced viral RNA, thereby overcoming host restriction. Chemical probing of the secondary structure of the RcRE corroborated the theory that the RcRE forms a complex folded structure with seven stem-loop regions. Laser-induced liquid beam ion desorption mass spectrometry revealed that Rec forms stable tetramers, which are further stabilized upon RNA binding. The RNA protein complex consists of three Rec tetramers, which bind to multiple sites on the RcRE-preferentially to purine-rich motifs-which represent several low-affinity binding sites. Mutated RcREs, with one to three purine-rich motifs deleted, were still bound and exported by Rec, indicating that the complex folded structure of the RcRE is important for Rec binding. This suggests a binding model where up to three Rec tetramers bind to the complex folded structure of the RcRE and the binding seems to be tightened by recognition of the purine-rich motifs.

SUBMITTER: Langner JS

PROVIDER: S-EPMC3416163 | biostudies-literature | 2012 Sep

REPOSITORIES: biostudies-literature

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Biochemical analysis of the complex between the tetrameric export adapter protein Rec of HERV-K/HML-2 and the responsive RNA element RcRE pck30.

Langner Janina S JS Fuchs Nina V NV Hoffmann Jan J Wittmann Alexander A Brutschy Bernhard B Löwer Roswitha R Suess Beatrix B

Journal of virology 20120613 17

The RNA export adaptor protein Rec, encoded for by the human endogenous retrovirus HERV-K/HML-2 elements, binds to the Rec responsive element (RcRE) located in the 3' untranslated region of HERV-K/HML-2 transcripts. Binding allows the nucleocytoplasmic export of unspliced viral RNA, thereby overcoming host restriction. Chemical probing of the secondary structure of the RcRE corroborated the theory that the RcRE forms a complex folded structure with seven stem-loop regions. Laser-induced liquid b ...[more]

PMID: 22696641

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Project description:BACKGROUND: The human genome harbors several largely preserved HERV-K(HML-2) elements. Although this retroviral family comes closest of all known HERVs to producing replication competent virions, mutations acquired during their chromosomal residence have rendered them incapable of expressing infectious particles. This also holds true for the HERV-K113 element that has conserved open reading frames (ORFs) for all its proteins in addition to a functional LTR promoter. Uncertainty concerning the localization and impact of post-insertional mutations has greatly hampered the functional characterization of these ancient retroviruses and their proteins. However, analogous to other betaretroviruses, it is known that HERV-K(HML-2) virions undergo a maturation process during or shortly after release from the host cell. During this process, the subdomains of the Gag polyproteins are released by proteolytic cleavage, although the nature of the mature HERV-K(HML-2) Gag proteins and the exact position of the cleavage sites have until now remained unknown. RESULTS: By aligning the amino acid sequences encoded by the gag-pro-pol ORFs of HERV-K113 with the corresponding segments from 10 other well-preserved human specific elements we identified non-synonymous post-insertional mutations that have occurred in this region of the provirus. Reversion of these mutations and a partial codon optimization facilitated the large-scale production of maturation-competent HERV-K113 virus-like particles (VLPs). The Gag subdomains of purified mature VLPs were separated by reversed-phase high-pressure liquid chromatography and initially characterized using specific antibodies. Cleavage sites were identified by mass spectrometry and N-terminal sequencing and confirmed by mutagenesis. Our results indicate that the gag gene product Pr74Gag of HERV-K(HML-2) is processed to yield p15-MA (matrix), SP1 (spacer peptide of 14 amino acids), p15, p27-CA (capsid), p10-NC (nucleocapsid) and two C-terminally encoded glutamine- and proline-rich peptides, QP1 and QP2, spanning 23 and 19 amino acids, respectively. CONCLUSIONS: Expression of reconstituted sequences of original HERV elements is an important tool for studying fundamental aspects of the biology of these ancient viruses. The analysis of HERV-K(HML-2) Gag processing and the nature of the mature Gag proteins presented here will facilitate further studies of the discrete functions of these proteins and of their potential impact on the human host.

Project description:BackgroundHuman endogenous retroviruses (HERVs) comprise approximately 8% of the human genome and while the majority are transcriptionally silent, the most recently integrated HERV, HERV-K (HML-2), remains active. During HIV infection, HERV-K (HML-2) specific mRNA transcripts and viral proteins can be detected. In this study, we aimed to understand the antibody response against HERV-K (HML-2) Gag in the context of HIV-1 infection.ResultsWe developed an ELISA assay using either recombinant protein or 164 redundant "15mer" HERV-K (HML-2) Gag peptides to test sera for antibody reactivity. We identified a total of eight potential HERV-K (HML-2) Gag immunogenic domains: two on the matrix (peptides 16 and 31), one on p15 (peptide 85), three on the capsid (peptides 81, 97 and 117), one on the nucleocapsid (peptide 137) and one on the QP1 protein (peptide 157). Four epitopes (peptides 16, 31, 85 and 137) were highly immunogenic. No significant differences in antibody responses were found between HIV infected participants (n = 40) and uninfected donors (n = 40) for 6 out of the 8 epitopes tested. The antibody response against nucleocapsid (peptide 137) was significantly lower (p < 0.001), and the response to QP1 (peptide 157) significantly higher (p < 0.05) in HIV-infected adults compared to uninfected individuals. Among those with HIV infection, the level of response against p15 protein (peptide 85) was significantly lower in untreated individuals controlling HIV ("elite" controllers) compared to untreated non-controllers (p < 0.05) and uninfected donors (p < 0.05). In contrast, the response against the capsid protein (epitopes 81 and 117) was significantly higher in controllers compared to uninfected donors (p < 0.001 and <0.05 respectively) and non-controllers (p < 0.01 and <0.05). Peripheral blood mononuclear cells (PBMCs) from study participants were tested for responses against HERV-K (HML-2) capsid recombinant peptide in gamma interferon (IFN-γ) enzyme immunospot (Elispot) assays. We found that the HERV-K (HML-2) Gag antibody and T cell response by Elispot were significantly correlated.ConclusionsHIV elite controllers had a strong cellular and antibody response against HERV-K (HML-2) Gag directed mainly against the Capsid region. Collectively, these data suggest that anti-HERV-K (HML-2) antibodies targeting capsid could have an immunoprotective effect in HIV infection.

Dataset Information

Biochemical analysis of the complex between the tetrameric export adapter protein Rec of HERV-K/HML-2 and the responsive RNA element RcRE pck30.

Publications

Biochemical analysis of the complex between the tetrameric export adapter protein Rec of HERV-K/HML-2 and the responsive RNA element RcRE pck30.

Similar Datasets

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