Vesicular Stomatitis Virus Matrix Protein Mutations That Affect Association with Host Membranes and Viral Nucleocapsids

Viral matrix (M) proteins bind the nucleoprotein core (nucleocapsid) to host membranes during the process of virus assembly by budding. Previous studies using truncated M proteins had implicated the N-terminal 50 amino acids of the vesicular stomatitis virus M protein in binding both membranes and nucleocapsids and a sequence from amino acids 75-106 as an additional membrane binding region. Structure-based mutations were introduced into these two regions, and their effects on membrane association and incorporation into nucleocapsid-M protein complexes were determined using quantitative assays. The results confirmed that the N terminus of M protein is involved in association with plasma membranes as well as nucleocapsids, although these two activities were differentially affected by individual mutations. Mutations in the 75-106 region affected incorporation into nucleocapsid-M complexes but had only minor effects on association with membranes. The ability of site-specific mutant M proteins to complement growth of temperature-sensitive M mutant virus did not correlate well with the ability to associate with membranes or nucleocapsids, suggesting that complementation involves an additional activity of M protein. Mutants with similar abilities to associate with membranes and nucleocapsids but differing in complementation activity were incorporated into infectious cDNA clones. Infectious virus was repeatedly recovered containing mutant M proteins capable of complementation but was never recovered with mutant M proteins that lacked complementation activity, providing further evidence for a separate activity of M protein that is essential for virus replication. Viral matrix (M) proteins bind the nucleoprotein core (nucleocapsid) to host membranes during the process of virus assembly by budding. Previous studies using truncated M proteins had implicated the N-terminal 50 amino acids of the vesicular stomatitis virus M protein in binding both membranes and nucleocapsids and a sequence from amino acids 75-106 as an additional membrane binding region. Structure-based mutations were introduced into these two regions, and their effects on membrane association and incorporation into nucleocapsid-M protein complexes were determined using quantitative assays. The results confirmed that the N terminus of M protein is involved in association with plasma membranes as well as nucleocapsids, although these two activities were differentially affected by individual mutations. Mutations in the 75-106 region affected incorporation into nucleocapsid-M complexes but had only minor effects on association with membranes. The ability of site-specific mutant M proteins to complement growth of temperature-sensitive M mutant virus did not correlate well with the ability to associate with membranes or nucleocapsids, suggesting that complementation involves an additional activity of M protein. Mutants with similar abilities to associate with membranes and nucleocapsids but differing in complementation activity were incorporated into infectious cDNA clones. Infectious virus was repeatedly recovered containing mutant M proteins capable of complementation but was never recovered with mutant M proteins that lacked complementation activity, providing further evidence for a separate activity of M protein that is essential for virus replication. Most viruses that have a membrane or envelope as part of their structure acquire their envelopes by budding from the plasma membrane of the host cell. For budding to occur, the nucleoprotein core of the virus (nucleocapsid) must interact with the cytoplasmic surface of the host membrane. For many viruses this interaction is mediated by a matrix (M) 2The abbreviations used are: M, matrix; aa, amino acid; NCM, nucleocapsid-M protein complex; pfu, plaque-forming units; rwt, recombinant wild-type; VLP, virus-like particle; VSV, vesicular stomatitis virus; VV-T7, vaccinia virus that expresses T7 RNA polymerase; tsM, temperature-sensitive M protein. protein that binds to both the nucleocapsid and the host membrane (1Pornillos O. Garrus J.E. Sundquist W.I. Trends Cell Biol. 2002; 12: 569-579Abstract Full Text Full Text PDF PubMed Scopus (240) Google Scholar, 2Jayakar H.R. Jeetendra E. Whitt M.A. Virus Res. 2004; 106: 117-132Crossref PubMed Scopus (102) Google Scholar). Despite the similarity in the functions of viral M proteins, there is little structural or sequence similarity among the M proteins of different virus families (3Timmins J. Ruigrok R.W. Weissenhorn W. FEMS Microbiol. Lett. 2004; 233: 179-186Crossref PubMed Google Scholar). Thus, understanding the relationship of structure to function must be undertaken for individual M proteins before the general principles involved in virus budding can be understood. The goal of the experiments described here was to determine sequences in the M protein of vesicular stomatitis virus (VSV) involved in binding to membranes and nucleocapsids. VSV is the prototype member of the Rhabdoviridae family and has been widely studied to determine mechanisms involved in virus budding (2Jayakar H.R. Jeetendra E. Whitt M.A. Virus Res. 2004; 106: 117-132Crossref PubMed Scopus (102) Google Scholar). The core of the virus contains an ∼11-kilobase negative-stranded RNA genome covered by 1300 copies of a single nucleocapsid protein (4Thomas D. Newcomb W.W. Brown J.C. Wall J.S. Hainfeld J.F. Trus B.L. Steven A.C. J. Virol. 1985; 54: 598-607Crossref PubMed Google Scholar). The nucleocapsid also contains lesser amounts of two proteins, P and L, which constitute the viral RNA-dependent RNA polymerase. The envelope contains a single species of transmembrane glycoprotein (G protein) that mediates virus attachment and entry into host cells. The virion contains ∼2000 copies of the M protein (4Thomas D. Newcomb W.W. Brown J.C. Wall J.S. Hainfeld J.F. Trus B.L. Steven A.C. J. Virol. 1985; 54: 598-607Crossref PubMed Google Scholar), which binds the nucleocapsid to the envelope and condenses the nucleocapsid into a tightly coiled helical nucleocapsid-M protein (NCM) complex that gives the virion its bullet-like shape (5Barge A. Gaudin Y. Coulon P. Ruigrok R.W. J. Virol. 1993; 67: 7246-7253Crossref PubMed Google Scholar, 6Lyles D.S. McKenzie PubMed Scopus Google Scholar, W.W. Brown J.C. J. Virol. PubMed Google Scholar, W.W. Brown J.C. J. Virol. PubMed Google Scholar). with VSV and in that M protein in the of VSV M protein is both in a and to the cytoplasmic surface of the host plasma membrane J. Virol. 1993; 67: PubMed Google Scholar, McKenzie D.S. J. Virol. PubMed Scopus Google Scholar, D.S. PubMed Scopus Google Scholar, McKenzie D.S. PubMed Scopus Google Scholar, D. J. Virol. PubMed Google Scholar, D.S. Virus Res. PubMed Scopus Google Scholar, D.S. J. Virol. PubMed Google Scholar, J. Virol. PubMed Google Scholar, J. Virol. PubMed Google Scholar, W. P. D. J. Virol. PubMed Google Scholar). and membrane experiments have that a of and M protein binding to membranes by binding on the surface of the host plasma membrane J. PubMed Scopus Google Scholar). of M protein to nucleocapsids is well its binding to membranes. Most of the M protein in complexes is in a D.S. McKenzie PubMed Scopus Google Scholar). M protein not bind to nucleocapsids from which of the M protein has been or to nucleocapsids that have never been with M protein D.S. PubMed Scopus Google Scholar, D.S. McKenzie PubMed Scopus Google Scholar). that binding of M protein to nucleocapsids in must be in a separate which of the M protein is into the complex the binding M protein not have that binding to membranes nucleocapsids. The M protein contains a N terminus that is to The of M protein is to a core J. Virol. PubMed Google Scholar, Gaudin Y. PubMed Scopus Google Scholar, Gaudin Y. J. 2002; PubMed Scopus Google Scholar, D.S. J. Virol. PubMed Google Scholar). The ability to of M protein of both the N-terminal sequence and a sequence to M protein Gaudin Y. PubMed Scopus Google Scholar, Gaudin Y. J. 2002; PubMed Scopus Google the structure a for the of the on two of the M protein structure that had been to be involved in binding to membranes or had implicated the N-terminal sequence in binding to both nucleocapsids and membranes J. Virol. 1993; 67: PubMed Google Scholar, McKenzie D.S. J. Virol. PubMed Scopus Google Scholar, J. Virol. PubMed Google Scholar, Gaudin Y. J. 2002; PubMed Scopus Google Scholar, D.S. J. Virol. PubMed Google Scholar, B.L. McKenzie D.S. J. Virol. 1993; 67: PubMed Google Scholar, J. J. Virol. PubMed Google and studies had implicated an additional region from 75-106 in membrane binding J. Virol. PubMed Google Scholar). the experiments described M protein sequence were using a in the N-terminal and were on the structure in the region. were used to determine the amino acids involved in these The results that the N terminus of M protein is involved in association with plasma membranes as well as nucleocapsids, although these two activities differentially affected by individual mutations. Mutations in the region affected incorporation into complexes but had only minor effects on association with membranes. the ability of mutant M proteins to function in the of virus that a activity of M protein that is separate from its ability to associate with membranes or complexes is for virus and M of the VSV was into as described McKenzie D.S. J. Virol. PubMed Scopus Google Scholar). that was during was to using the The mutations described in were using the protein using the The M protein sequences were by sequence were with vaccinia virus T7 a of of for and using with or with the M protein the of the T7 for the in of the McKenzie D.S. J. Virol. PubMed Scopus Google Scholar). and were a of in the were with a of of of VSV to that were or with and with of or mutant M protein. or were with for and for as described D.S. J. Virol. PubMed Scopus Google Scholar). was with of were used to the plasma membrane and cytoplasmic with in the shape of an were cell. were for The of the plasma membrane was the the the plasma membrane. cytoplasmic were the and the plasma membrane and the in of which to the of the was in the of the the and of M were a of in and The were with a of of and of and the were with of the determine of M protein were with containing for M protein was from as described D.S. J. Virol. PubMed Scopus Google Scholar), or were by and among mutant M proteins in were in these For of M protein were to M protein for The were for for and with with for The was with and and the was and M protein was from as described D.S. J. Virol. PubMed Scopus Google Scholar). of M protein association with membranes by in was as described McKenzie D.S. J. Virol. PubMed Scopus Google Scholar). a of in were with VV-T7, and the were with of the were with for and in Cell were to and were on a by for were from the and M protein was and by and were with VV-T7, with the M and with containing for The were a and to the as described D.S. McKenzie PubMed Scopus Google Scholar). and were by and or mutant M protein was by in and the ability to with M protein in virion complexes was as described D.S. PubMed Scopus Google Scholar). or mutant M protein was to in containing was to a of to the viral was to be as as the used The was for to M protein were using a The and were by and of M ability of and mutant M protein to complement growth of virus was as described D.S. McKenzie PubMed Scopus Google Scholar). were with virus and with or mutant M protein of the T7 were with virus and the of infectious virus were determined by of M Mutations into protein mutations were introduced into containing a infectious cDNA of VSV, and recombinant viruses were as described D.S. J. Virol. PubMed Scopus Google Scholar). studies of VSV matrix function have used M protein or M protein to the sequences involved in binding to nucleocapsids or membranes. the studies here was used to the binding the that the effects of were to were on was from the the structure did not the N amino acids the were in of with the sequence The sequence has the ability to different an or a on the protein in which A. 1985; PubMed Scopus Google Scholar). The in structure has been used in in of to the of A.C. Biol. PubMed Scopus Google Scholar). The to by their in the amino for is The sequence was not as is to the function of M protein that results in virus (2Jayakar H.R. Jeetendra E. Whitt M.A. Virus Res. 2004; 106: 117-132Crossref PubMed Scopus (102) Google Scholar). Mutations the plasma membrane binding were on the structure of M protein. The mutations were to the of amino acids to in the binding region. to by the which were with and Mutations in the 75-106 of M and of M protein were for their of in using the vaccinia virus T7 as this is the only in that have to of M protein with in D.S. McKenzie PubMed Scopus Google Scholar, B.L. McKenzie D.S. J. Virol. 1993; 67: PubMed Google Scholar, D.S. J. Virol. PubMed Scopus Google Scholar, D.S. J. Virol. PubMed Scopus Google Scholar). of the mutant M proteins were the as M as determined by for and of by and for of the containing in the 75-106 and which were that of M protein experiments were used to determine the of was to of the mutant M were with recombinant vaccinia virus that expresses T7 RNA and were with the mutant M protein or M protein for were with in for and Cell were with protein and by and The only mutant M proteins that had that were that of were the and as in The of these be to these mutant M proteins ability to associate with membranes or nucleocapsids their were in the of the of of and among M proteins of of the Rhabdoviridae family were and and as well as these The of of these mutant M proteins were determined by with for by as the of the in with M of or in with the M Mutants with with M association with host plasma membranes of and mutant M proteins was determined in by different The was quantitative of have that this is in quantitative in plasma membrane association of M protein that a of D.S. J. Virol. PubMed Scopus Google Scholar). The was of membranes and has been used for many to determine the membrane association of M protein J. Virol. 1993; 67: PubMed Google Scholar, McKenzie D.S. J. Virol. PubMed Scopus Google Scholar, D.S. PubMed Scopus Google Scholar, McKenzie D.S. PubMed Scopus Google Scholar, D. J. Virol. PubMed Google Scholar, W. P. D. J. Virol. PubMed Google Scholar). The was of of D.S. McKenzie PubMed Scopus Google Scholar, Y. J. Virol. 1993; 67: PubMed Google Scholar). M protein is in in the of viral association with the plasma membrane results in of membrane to as D.S. McKenzie PubMed Scopus Google Scholar, Y. J. Virol. 1993; 67: PubMed Google Scholar). is of the association of the sequence with host proteins, which the function of M protein that results in virus (2Jayakar H.R. Jeetendra E. Whitt M.A. Virus Res. 2004; 106: 117-132Crossref PubMed Scopus (102) Google Scholar). Thus, the is a that both association of M protein with plasma membranes and association of the sequence of M protein with host proteins (2Jayakar H.R. Jeetendra E. Whitt M.A. Virus Res. 2004; 106: 117-132Crossref PubMed Scopus (102) Google Scholar). For were with by with or mutant M protein for The B.L. McKenzie D.S. J. Virol. 1993; 67: PubMed Google Scholar), which is the N terminus of M was used as a for membrane The were with and with protein and were with to the of the also vaccinia virus in the were with the of the in was little of M protein in the of the of the envelope by D.S. J. Virol. PubMed Google Scholar). M protein was in both the plasma membrane and in the with a in the membrane. M protein not be the plasma membrane and the The of the M proteins was in the plasma membrane the that have the ability to associate with host plasma the M protein with and an results with the mutant in which association with the membrane is membrane and cytoplasmic were in the shape of an cell. of a in The of the plasma membrane was the the the plasma membrane. cytoplasmic were the and the plasma membrane and the of the were from The of membrane to cytoplasmic were for cell. For mutant M protein as well as the and were in of separate experiments the of M protein plasma for the mutant M The mutant M proteins and were different from M protein a association with membranes. results with that both the N-terminal region and the 75-106 region association with membranes. association of and mutant M proteins was also determined by of membranes and were with VV-T7, with or mutant M with and in Cell were with and to in to membranes. M protein in was by and of the mutant M proteins with host membranes to an similar to that of M protein. to results of there was and mutant M proteins in the of M protein in the membrane For results and mutant M proteins in separate was and mutant in the of M protein in the membrane The for the quantitative the and membrane were not in but a is that the membrane association with membranes in to the plasma the plasma membrane M a in of ability of mutant M proteins to was by the of M into the from cells. were with and with or M protein the of the T7 M protein was used as a and the mutant M which membrane binding activity, was used as a The were with for and into the were by and by and The amounts of or mutant M protein in and were and as a of M protein in M proteins and had different with M protein. The that were different from M protein in were that had different membrane association as determined by with the of mutant a budding sequence in M protein which little in budding of with H.R. Whitt M.A. P. J. Virol. 2004; PubMed Scopus Google Scholar), but to from cells. a separate of the single and of the sequence were for their ability to the single mutant and mutant to a with that of M of the was M Mutants a in the to with determine the of M protein mutations on the ability to into used M protein the of M protein for M protein in complexes from McKenzie D.S. J. Virol. PubMed Scopus Google Scholar, D.S. PubMed Scopus Google Scholar, McKenzie D.S. PubMed Scopus Google Scholar, D.S. McKenzie PubMed Scopus Google Scholar). this or mutant M protein with by in was with and the virion envelopes were with to the a to the of M protein with M complexes were and by and the M protein is capable of with a of the protein be in the the M protein that is not in the amounts of M protein be in the results the assembly into complexes of and mutant M For the results into separate results for the N-terminal and as well as the and and M proteins, of the had binding activity with the M but the abilities of of the to be incorporated into complexes were with M protein in the The N-terminal mutant M proteins and into complexes as as M protein results the from that the N terminus of M protein is involved in binding to nucleocapsids D.S. J. Virol. PubMed Google Scholar). for M proteins containing mutations in the 75-106 region. The association of the mutant with complexes was similar to that of M protein in the mutant was in its ability to associate with that this region of M protein is involved in interaction with nucleocapsids. The and were to have a of M protein in the in the of complexes in and the association with complexes did not this The is of of the mutant M proteins the of the which M protein is D.S. J. Virol. PubMed Google which be further evidence of of these mutant M proteins in to their and to the of and and the association with complexes of the single and of the sequence in a separate of The single mutant had a of M protein and an of association with that this protein was to be The had of and were incorporated into complexes to although had activity with that of M protein. that the region of M protein is involved in interaction with nucleocapsids. Most M Mutants M mutant viruses containing nucleocapsids but in M protein the D.S. McKenzie PubMed Scopus Google Scholar). has been that the in assembly of these is in the of association of M protein with nucleocapsids McKenzie D.S. PubMed Scopus Google Scholar). The site-specific mutant M proteins in were for their ability to complement a mutant virus to determine complementation is with the ability to bind membranes or the ability to bind nucleocapsids. were with virus and with or mutant M protein of the T7 were with virus and the of infectious virus were determined by as a complementation which is virus in the of the M protein by virus in the of the M protein of the N-terminal the growth of virus to a similar to that of M with the of the the in the 75-106 the and virus but the and the did not with were not that the ability to complement virus growth did not correlate with the ability to associate with plasma membranes or The with the in association with membranes or complexes and were to complement virus growth as as M protein. The that did not complement virus growth and were in their ability to associate with complexes but not as as the of a Virus the M mutant and the two in the sequence that have function and an have similar abilities to and associate with complexes to similar in their ability to complement virus further their effects on virus these mutations were incorporated into containing infectious VSV of infectious virus from was in M mutant virus was recovered in with an similar to M infectious virus was never recovered from containing the or mutations. were to viruses containing the and mutations in the Virus were from virus containing the The of the in the recovered virus was confirmed by were with virus or the recombinant virus a of of for growth and for growth and the of infectious was determined by was little and viruses in or growth The incorporation of viral proteins into virus was determined by with were with or viruses and for The were and in for or The and were and by and was and viruses in the of viral protein in the protein of the or the of viral proteins incorporated into experiments similar to were and there was in the of viral protein or assembly into the of M protein for incorporation into complexes results that the of M protein incorporation into complexes is not a of the of virus a of the of the mutant M proteins the in studies using and of M protein that N-terminal sequences association with both host plasma membranes and viral nucleocapsids J. Virol. 1993; 67: PubMed Google Scholar, J. Virol. PubMed Google Scholar, Gaudin Y. J. 2002; PubMed Scopus Google Scholar, D.S. J. Virol. PubMed Google Scholar, B.L. McKenzie D.S. J. Virol. 1993; 67: PubMed Google Scholar, J. J. Virol. PubMed Google Scholar). there is the sequences plasma membrane association and N-terminal incorporation into can be by the of the mutant on membrane association and with of the mutant on incorporation into with the these the of M protein that separate these two functions of M protein in virus The to this was to have quantitative for these individual of of mutant M M in a is similar N-terminal sequences can bind to both the plasma membrane and nucleocapsids. on with and J. J. Virol. PubMed Google that the N terminus of M protein with the region of the envelope the was the and the the N that the N terminus of M protein an in which the with and the with is that in an interact with on both the membrane and the nucleocapsid protein. the is not there as many M proteins as N proteins in virion complexes (4Thomas D. Newcomb W.W. Brown J.C. Wall J.S. Hainfeld J.F. Trus B.L. Steven A.C. J. Virol. 1985; 54: 598-607Crossref PubMed Google Scholar). Thus, is that the N of M protein interact with the and the N of M protein interact with the a M protein amino acids 75-106 were as a membrane binding on J. Virol. PubMed Google Scholar). in the in the region had only in membrane of M protein. The and were in plasma membrane and binding these is that the studied mutations have had effects on the mutant that the region is involved in binding to but this had little on membrane association and the mutant M proteins containing in the sequence that activity in and were in their for incorporation into the association with membranes the ability to be incorporated into complexes well with the ability of the site-specific mutant M proteins to complement mutant virus different mutant viruses have been and the amino for their have been determined Y. J. J. Virol. 1985; PubMed Google Scholar, J. J. Virol. PubMed Google Scholar). these in the of the M protein structure and to the of M protein the Gaudin Y. J. 2002; PubMed Scopus Google Scholar). the protein in the the is to be incorporated into complexes in M protein that of the protein is not McKenzie D.S. PubMed Scopus Google Scholar). the protein to be in that from McKenzie D.S. PubMed Scopus Google Scholar). The in assembly that is in with mutant viruses to be in the of assembly of M protein with nucleocapsids, as with these viruses the that nucleocapsids but of M protein D.S. McKenzie PubMed Scopus Google Scholar). Most of the site-specific mutant M proteins growth of mutant virus as as M with in membrane binding and and as well as with in binding to complexes that of these functions is for be with the that the protein is both to bind to membranes and to be into complexes but is in a function different from of these two the ability to binding to nucleocapsids McKenzie D.S. PubMed Scopus Google Scholar). The site-specific M proteins that to complement growth of mutant virus and the in the sequence that had in membrane or association with although not as as of the that did complement virus For the virus was incorporated into complexes the and of these was in evidence that these two were different from to these mutations into recombinant viruses containing the were recovered and had in virus were never to a virus containing the this is not on its with the results of the complementation these results that the mutant M protein is in an essential in virus The recombinant virus containing the mutant M protein had in virus assembly an in for association with complexes binding experiments have that the binding in the M protein is for the complex into the tightly coiled that gives the virion its bullet-like shape (5Barge A. Gaudin Y. Coulon P. Ruigrok R.W. J. Virol. 1993; 67: 7246-7253Crossref PubMed Google Scholar, W.W. Brown J.C. J. Virol. PubMed Google Scholar, W.W. Brown J.C. J. Virol. PubMed Google Scholar). is and with a with the of M protein D.S. McKenzie PubMed Scopus Google Scholar). a in the for incorporation into as that with the not have a on the of virus a of the in assembly of M protein into and the mutations that have the on M protein is as a which the and the plasma membrane is that association of M protein with nucleocapsids is the plasma membrane and involves nucleocapsids with the membrane of M protein of this have the of nucleocapsids in that with membrane that the viral envelope glycoprotein (G but not M and have that these nucleocapsids to virus budding D.S. J. Virol. PubMed Scopus Google Scholar). Thus, of assembly of M protein with these nucleocapsids be the in of a virus budding and is this that is to be in with mutant viruses the the assembly of M protein with nucleocapsids has been additional M protein is from the membrane and to the complex this not to be for virus is for the budding that is of the in and in the on VSV M protein. The here that association of M protein with membranes and complexes is for virus the in virus assembly that to be is the that assembly of M protein with nucleocapsids, have that this in assembly of M protein with nucleocapsids host to the of the host in the budding mediated by M protein D.S. PubMed Scopus Google Scholar). The for interaction with host proteins for the of of the sequence among M proteins, as this sequence be involved in the as well as in the of M protein. the M protein mutant be a in the as this mutant M protein also to complement the mutant virus and be in of assembly with nucleocapsids. the for with and the for also for of the The and the in part by of to the of with

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