Characterization of the major capsid proteins of myxoma virus particles using MALDI-TOF mass spectrometry

Zachertowska, Alicja; Brewer, Dyanne; Evans, David H.

doi:10.1016/j.jviromet.2005.08.015

Cited by 22 publications

(28 citation statements)

References 25 publications

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“…Furthermore, we showed that A6 was not a virion membrane protein, which is consistent with the lack of a predicted transmembrane domain in A6. Our conclusion that A6 is tightly packaged in the virion core correlates with a recent mass spectrometry analysis of the myxoma virus virion, which showed that M095L, the A6 ortholog in myxoma virus, was part of the virion core (26). Although A6 is packaged in the virion core, we did not find A6 protein to be preferentially localized to the viral factories, where immature virions are assembled.…”

Section: A6 Is Expressed Postreplicatively In Vaccinia Virus-infectedsupporting

confidence: 88%

Vaccinia Virus A6L Encodes a Virion Core Protein Required for Formation of Mature Virion

Meng

Embry

Sochia

et al. 2007

J Virol

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Vaccinia virus A6L is a previously uncharacterized gene that is conserved in all sequenced vertebrate poxviruses. Here, we constructed a recombinant vaccinia virus encoding A6 with an epitope tag and showed that A6 was expressed in infected cells after viral DNA replication and packaged in the core of the mature virion. Furthermore, we showed that A6 was essential for vaccinia virus replication by performing clustered charge-to-alanine mutagenesis on A6, which resulted in two vaccinia virus mutants (vA6L-mut1 and vA6L-mut2) that displayed a temperature-sensitive phenotype. At 31°C, both mutants replicated efficiently; however, at 40°C, vA6L-mut1 grew to a low titer, while vA6L-mut2 failed to replicate. The A6 protein expressed by vA6L-mut2 exhibited temperature-dependent instability. At the nonpermissive temperature, vA6L-mut2 was normal at viral gene expression and viral factory formation, but it was defective for proteolytic processing of the precursors of several major virion proteins, a defect that is characteristic of a block in virion morphogenesis. Electron microscopy further showed that the morphogenesis of vA6L-mut2 was arrested before the formation of immature virion with nucleoid and mature virion. Taken together, our data show that A6 is a virion core protein that plays an essential role in virion morphogenesis.Poxviruses are a family of large, complex, double-stranded DNA viruses that replicate entirely in the cytoplasm of infected cells ( (VACWR125) is one of about 20 conserved open reading frames of WR that has not been previously characterized. The amino acid sequence of A6 gives no hint to its function as it has no recognizable motif or any homolog outside the poxvirus family. To determine the role that A6 plays in viral life cycle, we constructed recombinant VACV encoding A6 with an epitope tag and temperature-sensitive (ts) mutants with a lesion in A6. We characterized these recombinant viruses and report here that A6 is a virion core protein playing an essential role in virion morphogenesis.The morphogenesis of the VACV virion goes through a series of stages that can be distinguished by electron microscopy (recently reviewed in reference 2). Electron-dense virosomes consisting of viral proteins appear first. Then, crescentshaped precursors of the virion membrane develop at the periphery of the virosome and subsequently circularize to form the spherical immature virions (IVs). As IVs encapsidate the viral genome, they appear as IVs with an electron-dense nucleoid (IVNs). Eventually, IVNs evolve into the brick-shaped intracellular mature virions (MVs), which represent the majority of infectious particles produced during VACV infection. Concomitant with this change, precursor forms of several virion proteins, including p4a and p4b, were proteolytically processed into mature proteins (12, 21). We present data here suggesting that A6 is essential for the transition from IV to IVN and MV. MATERIALS AND METHODSCells and viruses. BS-C-1 cells were maintained in minimum essential medium with Earle's s...

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Section: A6 Is Expressed Postreplicatively In Vaccinia Virus-infectedsupporting

confidence: 88%

Vaccinia Virus A6L Encodes a Virion Core Protein Required for Formation of Mature Virion

Meng

Embry

Sochia

et al. 2007

J Virol

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“…Most of the MYXV proteins identified in that study were orthologs of either VACV structural proteins or virion-associated enzymes, suggesting that leporipoxviruses utilize the same conserved core assembly and protein cleavage pathways employed by orthopoxviruses (180). One difference was that M151R, an ortholog of the VACV B13R protein that was not previously known to be a capsid component, was also detected in MYXV core fractions.…”

Section: Vol 73 2009 Poxvirus Proteomics and Virus-host Protein Intmentioning

confidence: 86%

“…One difference was that M151R, an ortholog of the VACV B13R protein that was not previously known to be a capsid component, was also detected in MYXV core fractions. Moreover, M093L, a protein of unknown function that lacks any known orthopoxvirus ortholog, was shown to associate with membrane fractions (180). In conclusion, it is clear that these comprehensive proteomic analyses of poxvirus virions have enabled the identification of many virion components, some of which are likely to be involved in the earliest stages of infection and pathogenesis.…”

Section: Vol 73 2009 Poxvirus Proteomics and Virus-host Protein Intmentioning

confidence: 95%

“…In contrast to VACV, little effort has been devoted to proteomic analysis of infectious MV particles from other chordopoxviruses; however, recently, the protein composition of MV particles of the Lausanne strain (Lau) of myxoma virus (MYXV) (MYXV-Lau), a member of the genus Leporipoxvirus, was determined (180). Using both reverse-phase highperformance LC and SDS-PAGE in combination with MALDI-TOF MS, 17 different MYXV-Lau capsid proteins a ssRNA, single-stranded RNA; ssDNA, single-stranded DNA; ND, not determined; VETF, vaccinia early transcription factor; VTF, vaccinia termination factor; TIR, Toll/IL-1 receptor; ITR, inverted terminal repeat; TFIIS, transcription factor IIS; iActA, Listeria ivanovii protein involved in actin tail formation.…”

Section: Proteomic Studies Of Poxvirus Virionsmentioning

confidence: 99%

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Poxvirus Proteomics and Virus-Host Protein Interactions

Vliet

Mohamed

Zhang

et al. 2009

Microbiol Mol Biol Rev

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SUMMARY Studies of the functional proteins encoded by the poxvirus genome provide information about the composition of the virus as well as individual virus-virus protein and virus-host protein interactions, which provides insight into viral pathogenesis and drug discovery. Widely used proteomic techniques to identify and characterize specific protein-protein interactions include yeast two-hybrid studies and coimmunoprecipitations. Recently, various mass spectrometry techniques have been employed to identify viral protein components of larger complexes. These methods, combined with structural studies, can provide new information about the putative functions of viral proteins as well as insights into virus-host interaction dynamics. For viral proteins of unknown function, identification of either viral or host binding partners provides clues about their putative function. In this review, we discuss poxvirus proteomics, including the use of proteomic methodologies to identify viral components and virus-host protein interactions. High-throughput global protein expression studies using protein chip technology as well as new methods for validating putative protein-protein interactions are also discussed.

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“…The origin and function of ORFan genes are still a matter of controversy, with opinions ranging from considering them pieces of junk DNA (1,8,40,44) to seeing them as quickly evolving sequences encoding normally expressed functional proteins (38, 39). Recent clinical evidence raised the possibility that mimivirus might be a human pathogen causing pneumonia (4, 24, 34), as suspected when it was first isolated from a cooling tower following an outbreak of pneumonia (23).Mass spectrometry-based analysis has recently emerged as a technique of choice to identify more comprehensively the set of viral proteins associated with viral particles (19,29,49). We now present the application of this technique to the largest known, and presumably most complex, viral particle, Acanthamoeba polyphaga mimivirus.…”

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confidence: 99%

Mimivirus Giant Particles Incorporate a Large Fraction of Anonymous and Unique Gene Products

Renesto

Abergel²,

Decloquement

et al. 2006

J Virol

117

137

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Acanthamoeba polyphaga mimivirus is the largest known virus in both particle size and genome complexity. Its 1.2-Mb genome encodes 911 proteins, among which only 298 have predicted functions. The composition of purified isolated virions was analyzed by using a combined electrophoresis/mass spectrometry approach allowing the identification of 114 proteins. Besides the expected major structural components, the viral particle packages 12 proteins unambiguously associated with transcriptional machinery, 3 proteins associated with DNA repair, and 2 topoisomerases. Other main functional categories represented in the virion include oxidative pathways and protein modification. More than half of the identified virion-associated proteins correspond to anonymous genes of unknown function, including 45 "ORFans." As demonstrated by both Western blotting and immunogold staining, some of these "ORFans," which lack any convincing similarity in the sequence databases, are endowed with antigenic properties. Thus, anonymous and unique genes constituting the majority of the mimivirus gene complement encode bona fide proteins that are likely to participate in well-integrated processes.Acanthamoeba polyphaga mimivirus (mimivirus) is the largest virus isolated so far (23). Based on its highly specific characteristics, this double-stranded-DNA icosahedral virus (47) is the first member of the new Mimiviridae family (33, 43). Computational annotation of its 1.2-Mb genome (33) revealed many atypical features, including the presence of key translation enzymes, a full complement of DNA repair pathway components, and the unique presence of three different topoisomerases (of types IA, IB, and II) (2, 33). Another unique characteristic of mimivirus is the presence of nearly identical promoter sequence motifs upstream of half of its 911 proteinencoding genes (42), which are presumably associated with proteins expressed during the early or late-early phase. Only 23% of the predicted coding genes exhibit convincing homology to proteins of known function, and 39% of them do not exhibit a clear (E values, Ͻ10 Ϫ5 ) sequence database match (33). Such coding regions without sequence similarity to other genes in databases are considered orphan open reading frames (ORFs) and termed "ORFans" (12). The origin and function of ORFan genes are still a matter of controversy, with opinions ranging from considering them pieces of junk DNA (1,8,40,44) to seeing them as quickly evolving sequences encoding normally expressed functional proteins (38, 39). Recent clinical evidence raised the possibility that mimivirus might be a human pathogen causing pneumonia (4, 24, 34), as suspected when it was first isolated from a cooling tower following an outbreak of pneumonia (23).Mass spectrometry-based analysis has recently emerged as a technique of choice to identify more comprehensively the set of viral proteins associated with viral particles (19,29,49). We now present the application of this technique to the largest known, and presumably most complex, viral particle,...

show abstract

Characterization of the major capsid proteins of myxoma virus particles using MALDI-TOF mass spectrometry

Cited by 22 publications

References 25 publications

Vaccinia Virus A6L Encodes a Virion Core Protein Required for Formation of Mature Virion

Vaccinia Virus A6L Encodes a Virion Core Protein Required for Formation of Mature Virion

Poxvirus Proteomics and Virus-Host Protein Interactions

Mimivirus Giant Particles Incorporate a Large Fraction of Anonymous and Unique Gene Products

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