Role of Mason-Pfizer Monkey Virus CA-NC Spacer Peptide-Like Domain in Assembly of Immature Particles

Strohalmová-Bohmová, Karolína; Spiwok, Vojtěch; Lepšı́k, Martin; Hadravová, Romana; Křížová, Ivana; Ulbrich, Pavel; Pichová, Iva; Bednářová, Lucie; Ruml, Tomáš; Rumlová, Michaela

doi:10.1128/jvi.02286-14

Cited by 16 publications

(23 citation statements)

References 72 publications

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“…Finally, in the betaretrovirus Mason-Pfizer monkey virus (MPMV), there is only one cleavage between CA and NC. While the sequence of this region of Gag appears consistent with a helical conformation, careful studies found no evidence for a helix here (55). It is intriguing to note that a region of MPMV Gag on the N-terminal side of CA exhibits behavior somewhat analogous to that of HIV-1 SP1 (56).…”

Section: Fig 10mentioning

confidence: 99%

Dimerization of the SP1 Region of HIV-1 Gag Induces a Helical Conformation and Association into Helical Bundles: Implications for Particle Assembly

Datta

Clark

et al. 2016

J Virol

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HIV-1 immature particle (virus-like particle [VLP]) assembly is mediated largely by interactions between the capsid (CA) domains of Gag molecules but is facilitated by binding of the nucleocapsid (NC) domain to nucleic acid.We previously investigated the role of SP1, a "spacer" between CA and NC, in VLP assembly. We found that small changes in SP1 drastically disrupt assembly and that a peptide representing the sequence around the CA-SP1 junction is helical at high but not low concentrations. We suggested that by virtue of such a concentration-dependent change, this region could act as a molecular switch to activate HIV-1 Gag for VLP assembly. A leucine zipper domain can replace NC in Gag and still lead to the efficient assembly of VLPs. We find that SP1 mutants also disrupt assembly by these Gag-Zip proteins and have now studied a small fragment of this Gag-Zip protein, i.e., the CA-SP1 junction region fused to a leucine zipper. Dimerization of the zipper places SP1 at a high local concentration, even at low total concentrations. In this context, the CA-SP1 junction region spontaneously adopts a helical conformation, and the proteins associate into tetramers. Tetramerization requires residues from both CA and SP1. The data suggest that once this region becomes helical, its propensity to self-associate could contribute to Gag-Gag interactions and thus to particle assembly. There is complete congruence between CA/SP1 sequences that promote tetramerization when fused to zippers and those that permit the proper assembly of full-length Gag; thus, equivalent interactions apparently participate in VLP assembly and in SP1-Zip tetramerization. IMPORTANCEAssembly of HIV-1 Gag into virus-like particles (VLPs) appears to require an interaction with nucleic acid, but replacement of its principal nucleic acid-binding domain with a dimerizing leucine zipper domain leads to the assembly of RNA-free VLPs. It has not been clear how dimerization triggers assembly. Results here show that the SP1 region spontaneously switches to a helical state when fused to a leucine zipper and that these helical molecules further associate into tetramers, mediated by interactions between hydrophobic faces of the helices. Thus, the correct juxtaposition of the SP1 region makes it "association competent." Residues from both capsid and SP1 contribute to tetramerization, while mutations disrupting proper assembly in Gag also prevent tetramerization. Thus, this region is part of an associating interface within Gag, and its intermolecular interactions evidently help stabilize the immature Gag lattice. These interactions are disrupted by proteolysis of the CA-SP1 junction during virus maturation. Immature retrovirus particles are assembled from a single protein, the virus-coded Gag protein. Gag is a multidomain protein in which the N-terminal matrix (MA) domain functions in the association of the protein with the plasma membrane of the virusproducing cell, the capsid (CA) domain contributes most or all of the Gag-Gag interactions in particle as...

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Section: Fig 10mentioning

confidence: 99%

Dimerization of the SP1 Region of HIV-1 Gag Induces a Helical Conformation and Association into Helical Bundles: Implications for Particle Assembly

Datta

Clark

et al. 2016

J Virol

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“…Nevertheless, the 12 and 13 amino acids of the CA C terminus and NC N terminus, respectively, were shown to play a critical role in the assembly of the immature particles (3,4). This part of the M-PMV Gag was therefore termed a spacer peptide-like domain (4). Similarly to M-PMV, gammaretroviruses have no spacer peptide between the CA and NC.…”

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confidence: 99%

“…In contrast, the betaretrovirus Mason-Pfizer monkey virus (M-PMV) has only one cleavage site between the CA and NC. Nevertheless, the 12 and 13 amino acids of the CA C terminus and NC N terminus, respectively, were shown to play a critical role in the assembly of the immature particles (3,4). This part of the M-PMV Gag was therefore termed a spacer peptide-like domain (4).…”

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confidence: 99%

Functional and Structural Characterization of Novel Type of Linker Connecting Capsid and Nucleocapsid Protein Domains in Murine Leukemia Virus

Doležal

Hadravová

Kožíšek

et al. 2016

Journal of Biological Chemistry

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The assembly of immature retroviral particles is initiated in the cytoplasm by the binding of the structural polyprotein precursor Gag with viral genomic RNA. The protein interactions necessary for assembly are mediated predominantly by the capsid (CA) and nucleocapsid (NC) domains, which have conserved structures. In contrast, the structural arrangement of the CA-NC connecting region differs between retroviral species. In HIV-1 and Rous sarcoma virus, this region forms a rod-like structure that separates the CA and NC domains, whereas in Mason-Pfizer monkey virus, this region is densely packed, thus holding the CA and NC domains in close proximity. Interestingly, the sequence connecting the CA and NC domains in gammaretroviruses, such as murine leukemia virus (MLV), is unique. The sequence is called a charged assembly helix (CAH) due to a high number of positively and negatively charged residues. Although both computational and deletion analyses suggested that the MLV CAH forms a helical conformation, no structural or biochemical data supporting this hypothesis have been published. Using an in vitro assembly assay, alanine scanning mutagenesis, and biophysical techniques (circular dichroism, NMR, microcalorimetry, and electrophoretic mobility shift assay), we have characterized the structure and function of the MLV CAH. We provide experimental evidence that the MLV CAH belongs to a group of charged, E(R/K)-rich, single ␣-helices. This is the first single ␣-helix motif identified in viral proteins.

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“…The GPG mutant failed to form immature particles inside the cells; however, a large amount of electron-dense material (presumably Gag) accumulated underneath the cell plasma membrane (Fig. 6D) (55). This compact layer on the membrane did not form any released virus particles or budding structures.…”

Section: Resultsmentioning

confidence: 99%

“…In the GPG mutant, dense layers of accumulated protein are seen underlying the plasma membrane, which are not seen in noninfected cells. Based on the thickness of these layers, and because similar structures have previously been shown to be formed by assembly-incompetent Gag polyproteins (55,60,61), we presume that these extra layers observed under the plasma membrane are formed by accumulated mutant Gag protein.…”

Section: Discussionmentioning

confidence: 99%

Nucleic Acid Binding by Mason-Pfizer Monkey Virus CA Promotes Virus Assembly and Genome Packaging

Füzik

Píchalová

Schur

et al. 2016

J Virol

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The Gag polyprotein of retroviruses drives immature virus assembly by forming hexameric protein lattices. The assembly is primarily mediated by protein-protein interactions between capsid (CA) domains and by interactions between nucleocapsid (NC) domains and RNA. Specific interactions between NC and the viral RNA are required for genome packaging. Previously reported cryoelectron microscopy analysis of immature Mason-Pfizer monkey virus (M-PMV) particles suggested that a basic region (residues RKK) in CA may serve as an additional binding site for nucleic acids. Here, we have introduced mutations into the RKK region in both bacterial and proviral M-PMV vectors and have assessed their impact on M-PMV assembly, structure, RNA binding, budding/release, nuclear trafficking, and infectivity using in vitro and in vivo systems. Our data indicate that the RKK region binds and structures nucleic acid that serves to promote virus particle assembly in the cytoplasm. Moreover, the RKK region appears to be important for recruitment of viral genomic RNA into Gag particles, and this function could be linked to changes in nuclear trafficking. Together these observations suggest that in M-PMV, direct interactions between CA and nucleic acid play important functions in the late stages of the viral life cycle. IMPORTANCEAssembly of retrovirus particles is driven by the Gag polyprotein, which can self-assemble to form virus particles and interact with RNA to recruit the viral genome into the particles. Generally, the capsid domains of Gag contribute to essential proteinprotein interactions during assembly, while the nucleocapsid domain interacts with RNA. The interactions between the nucleocapsid domain and RNA are important both for identifying the genome and for self-assembly of Gag molecules. Here, we show that a region of basic residues in the capsid protein of the betaretrovirus Mason-Pfizer monkey virus (M-PMV) contributes to interaction of Gag with nucleic acid. This interaction appears to provide a critical scaffolding function that promotes assembly of virus particles in the cytoplasm. It is also crucial for packaging the viral genome and thus for infectivity. These data indicate that, surprisingly, interactions between the capsid domain and RNA play an important role in the assembly of M-PMV. The retroviral structural polyprotein Gag contains three conserved domains, matrix (MA), capsid (CA), and nucleocapsid (NC). Gag plays the primary role in immature particle assembly and viral genomic RNA (vRNA) recruitment and packaging.Retroviruses assemble via two different morphogenetic pathways; the first, historically referred to as C-type, wherein particle assembly occurs at the cell membrane, and the second, D-type, assembling in perinuclear regions. The pathogenic human viruses, HIV and human T-cell lymphotropic virus assemble via C-type intermediates, whereas M-PMV is the prototypic D-type retrovirus. The Gag polyprotein of M-PMV is first transported to an intracytoplasmic pericentriolar site, where particle assembly...

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Role of Mason-Pfizer Monkey Virus CA-NC Spacer Peptide-Like Domain in Assembly of Immature Particles

Cited by 16 publications

References 72 publications

Dimerization of the SP1 Region of HIV-1 Gag Induces a Helical Conformation and Association into Helical Bundles: Implications for Particle Assembly

Dimerization of the SP1 Region of HIV-1 Gag Induces a Helical Conformation and Association into Helical Bundles: Implications for Particle Assembly

Functional and Structural Characterization of Novel Type of Linker Connecting Capsid and Nucleocapsid Protein Domains in Murine Leukemia Virus

Nucleic Acid Binding by Mason-Pfizer Monkey Virus CA Promotes Virus Assembly and Genome Packaging

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