scholarly journals The Structure of Immature Virus-Like Rous Sarcoma Virus Gag Particles Reveals a Structural Role for the p10 Domain in Assembly

2015 ◽  
Vol 89 (20) ◽  
pp. 10294-10302 ◽  
Author(s):  
Florian K. M. Schur ◽  
Robert A. Dick ◽  
Wim J. H. Hagen ◽  
Volker M. Vogt ◽  
John A. G. Briggs
Keyword(s):  
Rous Sarcoma Virus ◽  
Electron Tomography ◽  
Virus Particles ◽  
Terminal Domain ◽  
Rous Sarcoma ◽  
Sarcoma Virus ◽  
Subtomogram Averaging ◽  
Immature Virus ◽  
Infectious Form ◽  
Hiv 1

ABSTRACTThe polyprotein Gag is the primary structural component of retroviruses. Gag consists of independently folded domains connected by flexible linkers. Interactions between the conserved capsid (CA) domains of Gag mediate formation of hexameric protein lattices that drive assembly of immature virus particles. Proteolytic cleavage of Gag by the viral protease (PR) is required for maturation of retroviruses from an immature form into an infectious form. Within the assembled Gag lattices of HIV-1 and Mason-Pfizer monkey virus (M-PMV), the C-terminal domain of CA adopts similar quaternary arrangements, while the N-terminal domain of CA is packed in very different manners. Here, we have used cryo-electron tomography and subtomogram averaging to studyin vitro-assembled, immature virus-like Rous sarcoma virus (RSV) Gag particles and have determined the structure of CA and the surrounding regions to a resolution of ∼8 Å. We found that the C-terminal domain of RSV CA is arranged similarly to HIV-1 and M-PMV, whereas the N-terminal domain of CA adopts a novel arrangement in which the upstream p10 domain folds back into the CA lattice. In this position the cleavage site between CA and p10 appears to be inaccessible to PR. Below CA, an extended density is consistent with the presence of a six-helix bundle formed by the spacer-peptide region. We have also assessed the affect of lattice assembly on proteolytic processing by exogenous PR. The cleavage between p10 and CA is indeed inhibited in the assembled lattice, a finding consistent with structural regulation of proteolytic maturation.IMPORTANCERetroviruses first assemble into immature virus particles, requiring interactions between Gag proteins that form a protein layer under the viral membrane. Subsequently, Gag is cleaved by the viral protease enzyme into separate domains, leading to rearrangement of the virus into its infectious form. It is important to understand how Gag is arranged within immature retroviruses, in order to understand how virus assembly occurs, and how maturation takes place. We used the techniques cryo-electron tomography and subtomogram averaging to obtain a detailed structural picture of the CA domains in immature assembled Rous sarcoma virus Gag particles. We found that part of Gag next to CA, called p10, folds back and interacts with CA when Gag assembles. This arrangement is different from that seen in HIV-1 and Mason-Pfizer monkey virus, illustrating further structural diversity of retroviral structures. The structure provides new information on how the virus assembles and undergoes maturation.

scholarly journals Structure of the mature Rous sarcoma virus lattice reveals a role for IP6 in the formation of the capsid hexamer

2020 ◽  
Author(s):  
Martin Obr ◽  
Clifton L. Ricana ◽  
Nadia Nikulin ◽  
Jon-Philip R. Feathers ◽  
Marco Klanschnig ◽  
...  
Keyword(s):  
Rous Sarcoma Virus ◽  
Electron Tomography ◽  
Structural Mechanism ◽  
Rous Sarcoma ◽  
Sarcoma Virus ◽  
Subtomogram Averaging ◽  
Opposing Effects ◽  
Hiv 1

AbstractInositol hexakisphosphate (IP6) is an assembly cofactor for HIV-1. We report here that IP6 is also used for assembly of Rous sarcoma virus (RSV), a retrovirus from a different genus. IP6 was ∼100-fold more potent at promoting RSV mature CA assembly than observed for HIV-1 and removal of IP6 in vivo reduced infectivity by 100-fold. By cryo-electron tomography and subtomogram averaging, mature virus-like particles (VLPs) showed an IP6-like density in the CA hexamer, coordinated by rings of six lysines and six arginines. Phosphate and IP6 had opposing effects on CA in vitro assembly, inducing formation of T=1 icosahedrons and tubes, respectively, implying that phosphate promotes pentamer and IP6 hexamer formation. Subtomogram averaging and classification optimized for analysis of pleomorphic retrovirus particles revealed that the heterogeneity of mature RSV CA polyhedrons results from an unexpected, intrinsic CA hexamer flexibility. In contrast, the CA pentamer forms rigid units organizing the local architecture. These different features of hexamers and pentamers determine the structural mechanism to form CA polyhedrons of variable shape in mature RSV particles.

scholarly journals Structure of the mature Rous sarcoma virus lattice reveals a role for IP6 in the formation of the capsid hexamer

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Martin Obr ◽  
Clifton L. Ricana ◽  
Nadia Nikulin ◽  
Jon-Philip R. Feathers ◽  
Marco Klanschnig ◽  
...  
Keyword(s):  
Rous Sarcoma Virus ◽  
Electron Tomography ◽  
Structural Mechanism ◽  
Rous Sarcoma ◽  
Cryo Electron Tomography ◽  
Sarcoma Virus ◽  
Subtomogram Averaging ◽  
Opposing Effects ◽  
Hiv 1

AbstractInositol hexakisphosphate (IP6) is an assembly cofactor for HIV-1. We report here that IP6 is also used for assembly of Rous sarcoma virus (RSV), a retrovirus from a different genus. IP6 is ~100-fold more potent at promoting RSV mature capsid protein (CA) assembly than observed for HIV-1 and removal of IP6 in cells reduces infectivity by 100-fold. Here, visualized by cryo-electron tomography and subtomogram averaging, mature capsid-like particles show an IP6-like density in the CA hexamer, coordinated by rings of six lysines and six arginines. Phosphate and IP6 have opposing effects on CA in vitro assembly, inducing formation of T = 1 icosahedrons and tubes, respectively, implying that phosphate promotes pentamer and IP6 hexamer formation. Subtomogram averaging and classification optimized for analysis of pleomorphic retrovirus particles reveal that the heterogeneity of mature RSV CA polyhedrons results from an unexpected, intrinsic CA hexamer flexibility. In contrast, the CA pentamer forms rigid units organizing the local architecture. These different features of hexamers and pentamers determine the structural mechanism to form CA polyhedrons of variable shape in mature RSV particles.

scholarly journals Effect of Small Polyanions on In Vitro Assembly of Selected Members of Alpha-, Beta- and Gammaretroviruses

Viruses ◽  
2021 ◽  
Vol 13 (1) ◽  
pp. 129
Author(s):  
Alžběta Dostálková ◽  
Barbora Vokatá ◽  
Filip Kaufman ◽  
Pavel Ulbrich ◽  
Tomáš Ruml ◽  
...  
Keyword(s):  
Equine Infectious Anemia Virus ◽  
Electron Tomography ◽  
Leukemia Virus ◽  
Virus Like Particles ◽  
Rous Sarcoma ◽  
Sarcoma Virus ◽  
Anemia Virus ◽  
Immature Virus ◽  
Hiv 1

The assembly of a hexameric lattice of retroviral immature particles requires the involvement of cell factors such as proteins and small molecules. A small, negatively charged polyanionic molecule, myo-inositol hexaphosphate (IP6), was identified to stimulate the assembly of immature particles of HIV-1 and other lentiviruses. Interestingly, cryo-electron tomography analysis of the immature particles of two lentiviruses, HIV-1 and equine infectious anemia virus (EIAV), revealed that the IP6 binding site is similar. Based on this amino acid conservation of the IP6 interacting site, it is presumed that the assembly of immature particles of all lentiviruses is stimulated by IP6. Although this specific region for IP6 binding may be unique for lentiviruses, it is plausible that other retroviral species also recruit some small polyanion to facilitate the assembly of their immature particles. To study whether the assembly of retroviruses other than lentiviruses can be stimulated by polyanionic molecules, we measured the effect of various polyanions on the assembly of immature virus-like particles of Rous sarcoma virus (RSV), a member of alpharetroviruses, Mason-Pfizer monkey virus (M-PMV) representative of betaretroviruses, and murine leukemia virus (MLV), a member of gammaretroviruses. RSV, M-PMV and MLV immature virus-like particles were assembled in vitro from truncated Gag molecules and the effect of selected polyanions, myo-inostol hexaphosphate, myo-inositol, glucose-1,6-bisphosphate, myo-inositol hexasulphate, and mellitic acid, on the particles assembly was quantified. Our results suggest that the assembly of immature particles of RSV and MLV was indeed stimulated by the presence of myo-inostol hexaphosphate and myo-inositol, respectively. In contrast, no effect on the assembly of M-PMV as a betaretrovirus member was observed.

scholarly journals Visualizing Rous Sarcoma Virus Genomic RNA Dimerization in the Nucleus, Cytoplasm, and at the Plasma Membrane

Viruses ◽  
2021 ◽  
Vol 13 (5) ◽  
pp. 903
Author(s):  
Eunice C. Chen ◽  
Rebecca J. Kaddis Maldonado ◽  
Leslie J. Parent
Keyword(s):  
Plasma Membrane ◽  
Single Molecule ◽  
Rous Sarcoma Virus ◽  
Genomic Rna ◽  
Virus Particles ◽  
Rna Dimerization ◽  
Rous Sarcoma ◽  
Sarcoma Virus ◽  
Genome Dimerization ◽  
Hiv 1

Retroviruses are unique in that they package their RNA genomes as non-covalently linked dimers. Failure to dimerize their genomes results in decreased infectivity and reduced packaging of genomic RNA into virus particles. Two models of retrovirus genome dimerization have been characterized: in murine leukemia virus (MLV), genomic RNA dimerization occurs co-transcriptionally in the nucleus, resulting in the preferential formation of genome homodimers; whereas in human immunodeficiency virus (HIV-1), genomic RNA dimerization occurs in the cytoplasm and at the plasma membrane, with a random distribution of heterodimers and homodimers. Although in vitro studies have identified the genomic RNA sequences that facilitate dimerization in Rous sarcoma virus (RSV), in vivo characterization of the location and preferences of genome dimerization has not been performed. In this study, we utilized three single molecule RNA imaging approaches to visualize genome dimers of RSV in cultured quail fibroblasts. The formation of genomic RNA heterodimers within cells was dependent on the presence of the dimerization initiation site (DIS) sequence in the L3 stem. Subcellular localization analysis revealed that heterodimers were present the nucleus, cytoplasm, and at the plasma membrane, indicating that genome dimers can form in the nucleus. Furthermore, single virion analysis revealed that RSV preferentially packages genome homodimers into virus particles. Therefore, the mechanism of RSV genomic RNA dimer formation appears more similar to MLV than HIV-1.

scholarly journals A Possible Role for the Asymmetric C-Terminal Domain Dimer of Rous Sarcoma Virus Integrase in Viral DNA Binding

PLoS ONE ◽  
2013 ◽  
Vol 8 (2) ◽  
pp. e56892 ◽  
Author(s):  
Ke Shi ◽  
Krishan K. Pandey ◽  
Sibes Bera ◽  
Ajaykumar C. Vora ◽  
Duane P. Grandgenett ◽  
...  
Keyword(s):  
Dna Binding ◽  
Rous Sarcoma Virus ◽  
Viral Dna ◽  
Terminal Domain ◽  
Rous Sarcoma ◽  
Sarcoma Virus

scholarly journals Mutations in the Spacer Peptide and Adjoining Sequences in Rous Sarcoma Virus Gag Lead to Tubular Budding

2008 ◽  
Vol 82 (14) ◽  
pp. 6788-6797 ◽  
Author(s):  
Paul W. Keller ◽  
Marc C. Johnson ◽  
Volker M. Vogt
Keyword(s):  
Rous Sarcoma Virus ◽  
Mutational Analysis ◽  
Leukemia Virus ◽  
Alpha Helix ◽  
Insertion Mutagenesis ◽  
Structural Protein ◽  
Enveloped Virus ◽  
Rous Sarcoma ◽  
Sarcoma Virus ◽  
Hiv 1

ABSTRACT All orthoretroviruses encode a single structural protein, Gag, which is necessary and sufficient for the assembly and budding of enveloped virus-like particles from the cell. The Gag proteins of Rous sarcoma virus (RSV) and human immunodeficiency virus type 1 (HIV-1) contain a short spacer peptide (SP or SP1, respectively) separating the capsid (CA) and nucleocapsid (NC) domains. SP or SP1 and the residues immediately upstream are known to be critical for proper assembly. Using mutagenesis and electron microscopy analysis of insect cells or chicken cells overexpressing RSV Gag, we defined the SP assembly domain to include the last 8 residues of CA, all 12 residues of SP, and the first 4 residues of NC. Five- or two-amino acid glycine-rich insertions or substitutions in this critical region uniformly resulted in the budding of abnormal, long tubular particles. The equivalent SP1-containing HIV-1 Gag sequence was unable to functionally replace the RSV sequence in supporting normal RSV spherical assembly. According to secondary structure predictions, RSV and HIV-1 SP/SP1 and adjoining residues may form an alpha helix, and what is likely the functionally equivalent sequence in murine leukemia virus Gag has been inferred by mutational analysis to form an amphipathic alpha helix. However, our alanine insertion mutagenesis did not provide evidence for an amphipathic helix in RSV Gag. Taken together, these results define a short assembly domain between the folded portions of CA and NC, which is essential for formation of the immature Gag shell.

Reconstitution of the Rous sarcoma virus transforming protein pp60v-src into phospholipid vesicles

1988 ◽  
Vol 8 (5) ◽  
pp. 1896-1905
Author(s):  
M D Resh
Keyword(s):  
Lipid Bilayers ◽  
Rous Sarcoma Virus ◽  
Membrane System ◽  
Phospholipid Bilayer ◽  
Phospholipid Vesicles ◽  
Amino Terminal ◽  
Terminal Domain ◽  
Rous Sarcoma ◽  
Sarcoma Virus ◽  
Amino Terminal Domain

An artificial membrane system was developed to study the molecular basis for interaction of pp60v-src, the Rous sarcoma virus transforming protein, with lipid bilayers. pp60v-src was extracted from cell membranes by detergent solubilization and reincorporated into phospholipid vesicles. Reconstituted pp60v-src retained tyrosine kinase activity and was integrally associated with the liposome through a 10-kilodalton (kDa) amino-terminal domain. The same 10-kDa domain was shown to anchor pp60v-src to the plasma membrane of transformed cells. Reconstitution experiments performed with nonmyristylated pp60v-src proteins revealed that these polypeptides did not interact with phospholipid vesicles. In contrast, myristylated, soluble pp60v-src molecules (including a highly purified pp60v-src preparation) could be reconstituted into liposomes, but their interaction with the liposomal bilayer was not mediated by the 10-kDa amino-terminal domain. When membrane proteins were included during reconstitution of purified pp60v-src, binding through the 10-kDa anchor was restored. A model is presented to accommodate the different types of interactions of pp60v-src with liposomes; the model postulates the existence of an additional membrane component that anchors the pp60v-src polypeptide to the phospholipid bilayer.

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