The Noncanonical Gag Domains p8 and n Are Critical for Assembly and Release of Mouse Mammary Tumor Virus

Zábranský, Aleš; Hoboth, Peter; Hadravová, Romana; Štokrová, Jitka; Sakalian, Michael; Pichová, Iva

doi:10.1128/jvi.00652-10

Cited by 8 publications

(9 citation statements)

References 23 publications

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“…To verify the results obtained in human 293T cells, we used the rat mammary cell line RBA, which is suitable for production of MMTV upon transient transfection [ 39 ]. Because the expression level of MMTV proteins is significantly lower in RBA cells than in 293T cells, we examined the accumulation of assembled Gag in culture media by Western blot analysis after 48 h of production in transfected cells (Fig.…”

Section: Resultsmentioning

confidence: 99%

Myristoylation drives dimerization of matrix protein from mouse mammary tumor virus

et al. 2016

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BackgroundMyristoylation of the matrix (MA) domain mediates the transport and binding of Gag polyproteins to the plasma membrane (PM) and is required for the assembly of most retroviruses. In betaretroviruses, which assemble immature particles in the cytoplasm, myristoylation is dispensable for assembly but is crucial for particle transport to the PM. Oligomerization of HIV-1 MA stimulates the transition of the myristoyl group from a sequestered to an exposed conformation, which is more accessible for membrane binding. However, for other retroviruses, the effect of MA oligomerization on myristoyl group exposure has not been thoroughly investigated.ResultsHere, we demonstrate that MA from the betaretrovirus mouse mammary tumor virus (MMTV) forms dimers in solution and that this process is stimulated by its myristoylation. The crystal structure of N-myristoylated MMTV MA, determined at 1.57 Å resolution, revealed that the myristoyl groups are buried in a hydrophobic pocket at the dimer interface and contribute to dimer formation. Interestingly, the myristoyl groups in the dimer are mutually swapped to achieve energetically stable binding, as documented by molecular dynamics modeling. Mutations within the myristoyl binding site resulted in reduced MA dimerization and extracellular particle release.ConclusionsBased on our experimental, structural, and computational data, we propose a model for dimerization of MMTV MA in which myristoyl groups stimulate the interaction between MA molecules. Moreover, dimer-forming MA molecules adopt a sequestered conformation with their myristoyl groups entirely buried within the interaction interface. Although this differs from the current model proposed for lentiviruses, in which oligomerization of MA triggers exposure of myristoyl group, it appears convenient for intracellular assembly, which involves no apparent membrane interaction and allows the myristoyl group to be sequestered during oligomerization.Electronic supplementary materialThe online version of this article (doi:10.1186/s12977-015-0235-8) contains supplementary material, which is available to authorized users.

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Section: Resultsmentioning

confidence: 99%

Myristoylation drives dimerization of matrix protein from mouse mammary tumor virus

et al. 2016

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“…The protein bands between the p15 and p16.5 variants observed on SDS-PAGE might therefore result from alternative phosphorylation, from other post-translational modifications or from cryptic cleavages within the SP1 sequence. The Gag subdomain located between MA and CA of different retroviruses differ in many aspects and have a wide range of functions [ 25 , 45 - 47 ]. In MPMV, RSV and MLV they encode viral late domains and there is even a PTAP motive at the C-terminus of the HERV-K(HML-2) p15 protein.…”

Section: Discussionmentioning

confidence: 99%

“…In the case of MMTV, these are the polypeptides pp21, p8, p3 and n located between MA and CA [ 24 ]. These proteins appear to play a role in Gag folding, intracellular transport, assembly or maturation, although their precise functions are still poorly understood [ 25 ].…”

Section: Introductionmentioning

confidence: 99%

Identification of the protease cleavage sites in a reconstituted Gag polyprotein of an HERV-K(HML-2) element

et al. 2011

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BackgroundThe 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.ResultsBy 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.ConclusionsExpression 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.

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“…Once assembled, immature capsids are transported to the plasma membrane where they are enveloped and released from the cell. The genesis of MMTV virions is directed by the Gag polyprotein, which is composed of seven distinct domains that are proteolytically cleaved: MA, or matrix, the plasma membrane-targeting domain (3); pp21, a phosphoprotein of unknown function; p3, p8, and n, involved in Gag-Gag interactions required for immature capsid assembly (4,5); CA, or capsid, which forms the protein core of the mature virion; and NC, or nucleocapsid, which binds to the viral genomic RNA for encapsidation into virions (reviewed in reference 6).…”

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confidence: 99%

A Murine Retrovirus Co-Opts YB-1, a Translational Regulator and Stress Granule-Associated Protein, To Facilitate Virus Assembly

Bann

Beyer

Parent

2014

J Virol

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The Gag protein of the murine retrovirus mouse mammary tumor virus (MMTV) orchestrates the assembly of immature virus particles in the cytoplasm which are subsequently transported to the plasma membrane for release from the cell. The morphogenetic pathway of MMTV assembly is similar to that of Saccharomyces cerevisiae retrotransposons Ty1 and Ty3, which assemble virus-like particles (VLPs) in intracytoplasmic ribonucleoprotein (RNP) complexes. Assembly of Ty1 and Ty3 VLPs depends upon cellular mRNA processing factors, prompting us to examine whether MMTV utilizes a similar set of host proteins to facilitate viral capsid assembly. Our data revealed that MMTV Gag colocalized with YB-1, a translational regulator found in stress granules and P bodies, in intracytoplasmic foci. The association of MMTV Gag and YB-1 in cytoplasmic granules was not disrupted by cycloheximide treatment, suggesting that these sites were not typical stress granules. However, the association of MMTV Gag and YB-1 was RNA dependent, and an MMTV RNA reporter construct colocalized with Gag and YB-1 in cytoplasmic RNP complexes. Knockdown of YB-1 resulted in a significant decrease in MMTV particle production, indicating that YB-1 plays a role in MMTV capsid formation. Analysis by live-cell imaging with fluorescence recovery after photobleaching (FRAP) revealed that the population of Gag proteins localized within YB-1 complexes was relatively immobile, suggesting that Gag forms stable complexes in association with YB-1. Together, our data imply that the formation of intracytoplasmic Gag-RNA complexes is facilitated by YB-1, which promotes MMTV virus assembly. IMPORTANCECellular mRNA processing factors regulate the posttranscriptional fates of mRNAs, affecting localization and utilization of mRNAs under normal conditions and in response to stress. RNA viruses such as retroviruses interact with cellular mRNA processing factors that accumulate in ribonucleoprotein complexes known as P bodies and stress granules. This report shows for the first time that mouse mammary tumor virus (MMTV), a mammalian retrovirus that assembles intracytoplasmic virus particles, commandeers the cellular factor YB-1, a key regulator of translation involved in the cellular stress response. YB-1 is essential for the efficient production of MMTV particles, a process directed by the viral Gag protein. We found that Gag and YB-1 localize together in cytoplasmic granules. Functional studies of Gag/YB-1 granules suggest that they may be sites where virus particles assemble. These studies provide significant insights into the interplay between mRNA processing factors and retroviruses.

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The Noncanonical Gag Domains p8 and n Are Critical for Assembly and Release of Mouse Mammary Tumor Virus

Cited by 8 publications

References 23 publications

Myristoylation drives dimerization of matrix protein from mouse mammary tumor virus

Myristoylation drives dimerization of matrix protein from mouse mammary tumor virus

Identification of the protease cleavage sites in a reconstituted Gag polyprotein of an HERV-K(HML-2) element

A Murine Retrovirus Co-Opts YB-1, a Translational Regulator and Stress Granule-Associated Protein, To Facilitate Virus Assembly

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