scholarly journals Late Domain Function Identified in the Vesicular Stomatitis Virus M Protein by Use of Rhabdovirus-Retrovirus Chimeras

1999 ◽  
Vol 73 (4) ◽  
pp. 3359-3365 ◽  
Author(s):  
Rebecca C. Craven ◽  
Ronald N. Harty ◽  
Jason Paragas ◽  
Peter Palese ◽  
John W. Wills
Keyword(s):  
Vesicular Stomatitis Virus ◽  
Rous Sarcoma Virus ◽  
M Protein ◽  
Enveloped Viruses ◽  
Gag Proteins ◽  
Rous Sarcoma ◽  
M Proteins ◽  
Sarcoma Virus ◽  
Vesicular Stomatitis

ABSTRACT Little is known about the mechanisms used by enveloped viruses to separate themselves from the cell surface at the final step of budding. However, small sequences in the Gag proteins of several retroviruses (L domains) have been implicated in this process. A sequence has been identified in the M proteins of rhabdoviruses that closely resembles the PPPPY motif in the L domain of Rous sarcoma virus (RSV), an avian retrovirus. To evaluate whether the PPPY sequence in vesicular stomatitis virus (VSV) M protein has an activity analogous to that of the retroviral sequence, M-Gag chimeras were characterized. The N-terminal 74 amino acids of the VSV (Indiana) M protein, including the PPPY motif, was able to replace the L domain of RSV Gag and allow the assembly and release of virus-like particles. Alanine substitutions in the VSV PPPY motif severely compromised the budding activity of this hybrid protein but not that of another chimera which also contained the RSV PPPPY sequence. We conclude that this VSV sequence is functionally homologous to the RSV L domain in promoting virus particle release, making this the first example of such an activity in a virus other than a retrovirus. Both the RSV and VSV motifs have been shown to interact in vitro with certain cellular proteins that contain a WW interaction module, suggesting that the L domains are sites of interaction with unknown host machinery involved in virus release.

Viral products in cells infected with Vesicular Stomatitis virus and superinfected with Rous Sarcoma virus

1988 ◽  
Vol 100 (1-2) ◽  
pp. 121-129 ◽  
Author(s):  
M. Semmel ◽  
G. Mercier ◽  
N. Pavloff ◽  
G. Dambrine ◽  
F. Gay ◽  
...  
Keyword(s):  
Vesicular Stomatitis Virus ◽  
Rous Sarcoma Virus ◽  
Rous Sarcoma ◽  
Sarcoma Virus ◽  
Vesicular Stomatitis ◽  
In Cells

scholarly journals Membrane Binding of the Rous Sarcoma Virus Gag Protein Is Cooperative and Dependent on the Spacer Peptide Assembly Domain

2015 ◽  
Vol 90 (5) ◽  
pp. 2473-2485 ◽  
Author(s):  
Robert A. Dick ◽  
Marilia Barros ◽  
Danni Jin ◽  
Mathias Lösche ◽  
Volker M. Vogt
Keyword(s):  
Plasma Membrane ◽  
Nucleic Acid ◽  
Rous Sarcoma Virus ◽  
Membrane Binding ◽  
Membrane Interaction ◽  
Gag Proteins ◽  
Peptide Assembly ◽  
Rous Sarcoma ◽  
Sarcoma Virus

ABSTRACTThe principles underlying membrane binding and assembly of retroviral Gag proteins into a lattice are understood. However, little is known about how these processes are related. Using purified Rous sarcoma virus Gag and Gag truncations, we studied the interrelation of Gag-Gag interaction and Gag-membrane interaction. Both by liposome binding and by surface plasmon resonance on a supported bilayer, Gag bound to membranes much more tightly than did matrix (MA), the isolated membrane binding domain. In principle, this difference could be explained either by protein-protein interactions leading to cooperativity in membrane binding or by the simultaneous interaction of the N-terminal MA and the C-terminal nucleocapsid (NC) of Gag with the bilayer, since both are highly basic. However, we found that NC was not required for strong membrane binding. Instead, the spacer peptide assembly domain (SPA), a putative 24-residue helical sequence comprising the 12-residue SP segment of Gag and overlapping the capsid (CA) C terminus and the NC N terminus, was required. SPA is known to be critical for proper assembly of the immature Gag lattice. A single amino acid mutation in SPA that abrogates assemblyin vitrodramatically reduced binding of Gag to liposomes.In vivo, plasma membrane localization was dependent on SPA. Disulfide cross-linking based on ectopic Cys residues showed that the contacts between Gag proteins on the membrane are similar to the known contacts in virus-like particles. Taken together, we interpret these results to mean that Gag membrane interaction is cooperative in that it depends on the ability of Gag to multimerize.IMPORTANCEThe retroviral structural protein Gag has three major domains. The N-terminal MA domain interacts directly with the plasma membrane (PM) of cells. The central CA domain, together with immediately adjoining sequences, facilitates the assembly of thousands of Gag molecules into a lattice. The C-terminal NC domain interacts with the genome, resulting in packaging of viral RNA. For assemblyin vitrowith purified Gag, in the absence of membranes, binding of NC to nucleic acid somehow facilitates further Gag-Gag interactions that lead to formation of the Gag lattice. The contributions of MA-mediated membrane binding to virus particle assembly are not well understood. Here, we report that in the absence of nucleic acid, membranes provide a platform that facilitates Gag-Gag interactions. This study demonstrates that the binding of Gag, but not of MA, to membranes is cooperative and identifies SPA as a major factor that controls this cooperativity.

scholarly journals Role of the Rous Sarcoma Virus p10 Domain in Shape Determination of Gag Virus-Like Particles Assembled In Vitro and within Escherichia coli

2000 ◽  
Vol 74 (21) ◽  
pp. 10260-10268 ◽  
Author(s):  
Swati M. Joshi ◽  
Volker M. Vogt
Keyword(s):  
Escherichia Coli ◽  
Rous Sarcoma Virus ◽  
Gag Protein ◽  
Virus Like Particles ◽  
Gag Proteins ◽  
Rous Sarcoma ◽  
Crude Extracts ◽  
Sarcoma Virus ◽  
Shape Determination

ABSTRACT Purified retrovirus Gag proteins can assemble in vitro into virus-like particles (VLPs) in the presence of RNA. It was shown previously that a Rous sarcoma virus Gag protein missing only the protease domain forms spherical particles resembling immature virions lacking a membrane but that a similar protein missing the p10 domain forms tubular particles. Thus, p10 plays a role in spherical particle formation. To further study this shape-determining function, we dissected the p10 domain by mutagenesis and examined VLPs assembled within Escherichia coli or assembled in vitro from purified proteins. The results identified a minimal contiguous segment of 25 amino acid residues at the C terminus of p10 that is sufficient to restore efficient spherical assembly to a p10 deletion mutant. Random and site-directed mutations were introduced into this segment of polypeptide, and the shapes of particles formed in E. coliwere examined in crude extracts by electron microscopy. Three phenotypes were observed: tubular morphology, spherical morphology, or no regular structure. While the particle morphology visualized in crude extracts generally was the same as that visualized for purified proteins, some tubular mutants scored as spherical when tested as purified proteins, suggesting that a cellular factor may also play a role in shape determination. We also examined the assembly properties of smaller Gag proteins consisting of the capsid protein-nucleocapsid protein (CA-NC) domains with short N-terminal extensions or deletions. Addition of one or three residues allowed CA-NC to form spheres instead of tubes in vitro, but the efficiency of assembly was extremely low. Deletion of the N-terminal residue(s) abrogated assembly. Taken together, these results imply that the N terminus of CA and the adjacent upstream 25 residues play an important role in the polymerization of the Gag protein.

Functional domains of the pp60v-src protein as revealed by analysis of temperature-sensitive Rous sarcoma virus mutants

1984 ◽  
Vol 4 (8) ◽  
pp. 1508-1514
Author(s):  
A W Stoker ◽  
P J Enrietto ◽  
J A Wyke
Keyword(s):  
Rous Sarcoma Virus ◽  
Kinase Activity ◽  
Temperature Sensitive ◽  
Tyrosine Kinase Activity ◽  
Rous Sarcoma ◽  
Heat Labile ◽  
Src Gene ◽  
Sarcoma Virus

Four temperature-sensitive (ts) Rous sarcoma virus src gene mutants with lesions in different parts of the gene represent three classes of alteration in pp60src. These classes are composed of mutants with (i) heat-labile protein kinase activities both in vitro and in vivo (tsLA27 and tsLA29), (ii) heat-labile kinases in vivo but not in vitro (tsLA33), and (iii) neither in vivo nor in vitro heat-labile kinases (tsLA32). The latter class indicates the existence of structural or functional pp60src domains that are required for transformation but do not grossly affect tyrosine kinase activity.

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