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 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 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 The Retroviral Capsid Domain Dictates Virion Size, Morphology, and Coassembly of Gag into Virus-Like Particles

2005 ◽  
Vol 79 (21) ◽  
pp. 13463-13472 ◽  
Author(s):  
Danso Ako-Adjei ◽  
Marc C. Johnson ◽  
Volker M. Vogt
Keyword(s):  
Nuclear Export ◽  
Leukemia Virus ◽  
Particle Diameter ◽  
Particle Morphology ◽  
Structural Protein ◽  
Wild Type ◽  
Virus Like Particles ◽  
Gag Proteins ◽  
Hiv 1

ABSTRACT The retroviral structural protein, Gag, is capable of independently assembling into virus-like particles (VLPs) in living cells and in vitro. Immature VLPs of human immunodeficiency virus type 1 (HIV-1) and of Rous sarcoma virus (RSV) are morphologically distinct when viewed by transmission electron microscopy (TEM). To better understand the nature of the Gag-Gag interactions leading to these distinctions, we constructed vectors encoding several RSV/HIV-1 chimeric Gag proteins for expression in either insect cells or vertebrate cells. We used TEM, confocal fluorescence microscopy, and a novel correlative scanning EM (SEM)-confocal microscopy technique to study the assembly properties of these proteins. Most chimeric proteins assembled into regular VLPs, with the capsid (CA) domain being the primary determinant of overall particle diameter and morphology. The presence of domains between matrix and CA also influenced particle morphology by increasing the spacing between the inner electron-dense ring and the VLP membrane. Fluorescently tagged versions of wild-type RSV, HIV-1, or murine leukemia virus Gag did not colocalize in cells. However, wild-type Gag proteins colocalized extensively with chimeric Gag proteins bearing the same CA domain, implying that Gag interactions are mediated by CA. A dramatic example of this phenomenon was provided by a nuclear export-deficient chimera of RSV Gag carrying the HIV-1 CA domain, which by itself localized to the nucleus but relocalized to the cytoplasm in the presence of wild type HIV-1 Gag. Wild-type and chimeric Gag proteins were capable of coassembly into a single VLP as viewed by correlative fluorescence SEM if, and only if, the CA domain was derived from the same virus. These results imply that the primary selectivity of Gag-Gag interactions is determined by the CA domain.

scholarly journals Foreign Glycoproteins Can Be Actively Recruited to Virus Assembly Sites during Pseudotyping

2009 ◽  
Vol 83 (9) ◽  
pp. 4060-4067 ◽  
Author(s):  
Rebecca L. Jorgenson ◽  
Volker M. Vogt ◽  
Marc C. Johnson
Keyword(s):  
Plasma Membrane ◽  
Virus Assembly ◽  
Leukemia Virus ◽  
Viral Assembly ◽  
Surface Glycoprotein ◽  
Viral Budding ◽  
Rous Sarcoma ◽  
Viral Glycoproteins ◽  
Sarcoma Virus ◽  
Hiv 1

ABSTRACT Retroviruses like human immunodeficiency virus type 1 (HIV-1), as well as many other enveloped viruses, can efficiently produce infectious virus in the absence of their own surface glycoprotein if a suitable glycoprotein from a foreign virus is expressed in the same cell. This process of complementation, known as pseudotyping, often can occur even when the glycoprotein is from an unrelated virus. Although pseudotyping is widely used for engineering chimeric viruses, it has remained unknown whether a virus can actively recruit foreign glycoproteins to budding sites or, alternatively, if a virus obtains the glycoproteins through a passive mechanism. We have studied the specificity of glycoprotein recruitment by immunogold labeling viral glycoproteins and imaging their distribution on the host plasma membrane using scanning electron microscopy. Expressed alone, all tested viral glycoproteins were relatively randomly distributed on the plasma membrane. However, in the presence of budding HIV-1 or Rous sarcoma virus (RSV) particles, some glycoproteins, such as those encoded by murine leukemia virus and vesicular stomatitis virus, were dramatically redistributed to viral budding sites. In contrast, the RSV Env glycoprotein was robustly recruited only to the homologous RSV budding sites. These data demonstrate that viral glycoproteins are not in preformed membrane patches prior to viral assembly but rather that glycoproteins are actively recruited to certain viral assembly sites.

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.

scholarly journals Equine Infectious Anemia Virus Utilizes Host Vesicular Protein Sorting Machinery during Particle Release

2003 ◽  
Vol 77 (15) ◽  
pp. 8440-8447 ◽  
Author(s):  
Giancarlo O. Tanzi ◽  
Andrew J. Piefer ◽  
Paul Bates
Keyword(s):  
Equine Infectious Anemia Virus ◽  
Leukemia Virus ◽  
Protein Sorting ◽  
Gag Protein ◽  
Particle Release ◽  
Late Domain ◽  
Equine Infectious Anemia ◽  
Anemia Virus ◽  
Vacuolar Protein ◽  
Hiv 1

ABSTRACT A final step in retrovirus assembly, particle release from the cell, is modulated by a small motif in the Gag protein known as a late domain. Recently, human immunodeficiency virus type 1 (HIV-1) and Moloney murine leukemia virus (M-MuLV) were shown to require components of the cellular vacuolar protein sorting (VPS) machinery for efficient viral release. HIV-1 interacts with the VPS pathway via an association of HIV-1 Gag with TSG101, a component of the cellular complexes involved in VPS. Equine infectious anemia virus (EIAV) is unique among enveloped viruses studied to date because it utilizes a novel motif, YPDL in Gag, as a late domain. Our analysis of EIAV assembly demonstrates that EIAV Gag release is blocked by inhibition of the VPS pathway. However, in contrast to HIV-1, EIAV Gag release is insensitive to TSG101 depletion and EIAV particles do not contain significant levels of TSG101. Finally, we demonstrate that fusing EIAV Gag directly with another cellular component of the VPS machinery, VPS28, can restore efficient release of an EIAV Gag late-domain mutant. These results provide evidence that retroviruses can interact with the cellular VPS machinery in several different ways to accomplish particle release.

scholarly journals Hydrodynamic and Membrane Binding Properties of Purified Rous Sarcoma Virus Gag Protein

2015 ◽  
Vol 89 (20) ◽  
pp. 10371-10382 ◽  
Author(s):  
Robert A. Dick ◽  
Siddhartha A. K. Datta ◽  
Hirsh Nanda ◽  
Xianyang Fang ◽  
Yi Wen ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Rous Sarcoma Virus ◽  
Acyl Chain ◽  
Membrane Binding ◽  
Membrane Association ◽  
Gag Protein ◽  
Rous Sarcoma ◽  
Sarcoma Virus ◽  
Hiv 1

ABSTRACTPreviously, no retroviral Gag protein has been highly purified in milligram quantities and in a biologically relevant and active form. We have purified Rous sarcoma virus (RSV) Gag protein and in parallel several truncation mutants of Gag and have studied their biophysical properties and membrane interactionsin vitro. RSV Gag is unusual in that it is not naturally myristoylated. From its ability to assemble into virus-like particlesin vitro, we infer that RSV Gag is biologically active. By size exclusion chromatography and small-angle X-ray scattering, Gag in solution appears extended and flexible, in contrast to previous reports on unmyristoylated HIV-1 Gag, which is compact. However, by neutron reflectometry measurements of RSV Gag bound to a supported bilayer, the protein appears to adopt a more compact, folded-over conformation. At physiological ionic strength, purified Gag binds strongly to liposomes containing acidic lipids. This interaction is stimulated by physiological levels of phosphatidylinositol-(4,5)-bisphosphate [PI(4,5)P2] and by cholesterol. However, unlike HIV-1 Gag, RSV Gag shows no sensitivity to acyl chain saturation. In contrast with full-length RSV Gag, the purified MA domain of Gag binds to liposomes only weakly. Similarly, both an N-terminally truncated version of Gag that is missing the MA domain and a C-terminally truncated version that is missing the NC domain bind only weakly. These results imply that NC contributes to membrane interactionin vitro, either by directly contacting acidic lipids or by promoting Gag multimerization.IMPORTANCERetroviruses like HIV assemble at and bud from the plasma membrane of cells. Assembly requires the interaction between thousands of Gag molecules to form a lattice. Previous work indicated that lattice formation at the plasma membrane is influenced by the conformation of monomeric HIV. We have extended this work to the more tractable RSV Gag. Our results show that RSV Gag is highly flexible and can adopt a folded-over conformation on a lipid bilayer, implicating both the N and C termini in membrane binding. In addition, binding of Gag to membranes is diminished when either terminal domain is truncated. RSV Gag membrane association is significantly less sensitive than HIV Gag membrane association to lipid acyl chain saturation. These findings shed light on Gag assembly and membrane binding, critical steps in the viral life cycle and an untapped target for antiretroviral drugs.

scholarly journals Equine Infectious Anemia Virus Gag Polyprotein Late Domain Specifically Recruits Cellular AP-2 Adapter Protein Complexes during Virion Assembly

1998 ◽  
Vol 72 (12) ◽  
pp. 10218-10221 ◽  
Author(s):  
Bridget A. Puffer ◽  
S. C. Watkins ◽  
Ronald C. Montelaro
Keyword(s):  
Equine Infectious Anemia Virus ◽  
Protein Complexes ◽  
Adapter Protein ◽  
Virion Assembly ◽  
Late Domain ◽  
Rous Sarcoma ◽  
Equine Infectious Anemia ◽  
Anemia Virus ◽  
Late Assembly Domain

ABSTRACT We have identified an interaction between the equine infectious anemia virus (EIAV) late assembly domain and the cellular AP-2 clathrin-associated adapter protein complex. A YXXL motif within the EIAV Gag late assembly domain was previously characterized as a sequence critical for release of assembling virions. We now show that this YXXL sequence interacts in vitro with the AP-50 subunit of the AP-2 complex, while the functionally interchangeable late assembly domains carried by the Rous sarcoma virus p2b protein and human immunodeficiency virus type 1 p6 protein, which utilize PPPY and PTAPP L domains, respectively, do not bind AP-50 in vitro. In addition, EIAV late domain mutants containing mutations that have previously been shown to abrogate budding also exhibit marked decreases in AP-50 binding efficiencies. A role for AP-2 complex in viral assembly is supported by immunofluorescence analysis of EIAV-infected equine dermal cells demonstrating specific colocalization of the α adaptin subunit of AP-2 with the EIAV p9 protein at sites of virus budding on the plasma membrane. These data provide strong evidence that EIAV utilizes the cellular AP-2 complex to accomplish virion assembly and release.

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