Vaccinia Virus Core Protein VP8 Is Required for Virus Infectivity, but Not for Core Protein Processing or for INV and EEV Formation

Wilcock, Diane; Smith, Geoffrey L.

doi:10.1006/viro.1994.1346

Cited by 30 publications

(39 citation statements)

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“…L4R is a major core component of virus particles (7) that binds single-stranded DNA as well as RNA and stimulates the DNA helicase activity of I8R (38,39). When expression of L4R was repressed, noninfectious virus particles defective in transcription were produced (40,41). It was suggested that L4R may be involved in unwinding the DNA template for transcription.…”

Section: Resultsmentioning

confidence: 99%

Genome-wide analysis of vaccinia virus protein–protein interactions

McCraith

Holtzman²,

Moss³

et al. 2000

Proc. Natl. Acad. Sci. U.S.A.

265

205

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To detect interactions between proteins of vaccinia virus, we carried out a comprehensive two-hybrid analysis to assay every pairwise combination. We constructed an array of yeast transformants that contained each of the 266 predicted viral ORFs as Gal4 activation domain hybrid proteins. The array was individually mated to transformants containing each ORF as a Gal4 -DNAbinding domain hybrid, and diploids expressing the two-hybrid reporter gene were identified. Of the Ϸ70,000 combinations, we found 37 protein-protein interactions, including 28 that were previously unknown. In some cases, e.g., late transcription factors, both proteins were known to have related roles although there was no prior evidence of physical associations. For some other interactions, neither protein had a known role. In the majority of cases, however, one of the interacting proteins was known to be involved in DNA replication, transcription, virion structure, or host evasion, thereby providing a clue to the role of the other uncharacterized protein in a specific process.

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Section: Resultsmentioning

confidence: 99%

Genome-wide analysis of vaccinia virus protein–protein interactions

McCraith

Holtzman²,

Moss³

et al. 2000

Proc. Natl. Acad. Sci. U.S.A.

265

205

View full text Add to dashboard Cite

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“…L4 binds both RNA and DNA with high affinity, and it is highly abundant, being the sixth most abundant protein in the virions (9)(10)(11). Also, the absence of L4 from virions leads to formation of viral particles with fragile viral cores, from which the transcription enzymes are more easily extracted than from viral particles containing L4 (12). Interestingly, Sarov and Joklik (13) and Pedersen and colleagues (14), using biochemical and microscopy techniques, characterized intermediates of vaccinia virus that accumulate in the period immediately following virus entry and before the onset of virus DNA replication.…”

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confidence: 99%

“…L4 has no sequence homology with proteins outside the poxvirus family. L4 is synthesized late during infection and cleaved during maturation from IV to MV (12,15,16). Data showing that L4 stimulates the activity of the vaccinia virus I8 RNA helicase and that viral particles lacking L4 are inactive for core transcription even though they package all transcription factors suggest that L4 could participate in virus transcription (17,18).…”

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confidence: 99%

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Vaccinia Virus Mutations in the L4R Gene Encoding a Virion Structural Protein Produce Abnormal Mature Particles Lacking a Nucleocapsid

Jesus

Moussatché

Condit

2014

J Virol

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Electron micrographs from the 1960s revealed the presence of an S-shaped tubular structure in the center of the vaccinia virion core. Recently, we showed that packaging of virus transcription enzymes is necessary for the formation of the tubular structure, suggesting that the structure is equivalent to a nucleocapsid. Based on this study and on what is known about nucleocapsids of other viruses, we hypothesized that in addition to transcription enzymes, the tubular structure also contains the viral DNA and a structural protein as a scaffold. The vaccinia virion structural protein L4 stands out as the best candidate for the role of a nucleocapsid structural protein because it is abundant, it is localized in the center of the virion core, and it binds DNA. In order to gain more insight into the structure and relevance of the nucleocapsid, we analyzed thermosensitive and inducible mutants in the L4R gene. Using a cryo-fixation method for electron microscopy (high-pressure freezing followed by freeze-substitution) to preserve labile structures like the nucleocapsid, we were able to demonstrate that in the absence of functional L4, mature particles with defective internal structures are produced under nonpermissive conditions. These particles do not contain a nucleocapsid. In addition, the core wall of these virions is abnormal. This suggests that the nucleocapsid interacts with the core wall and that the nucleocapsid structure might be more complex than originally assumed. IMPORTANCEThe vaccinia virus nucleocapsid has been neglected since the 1960s due to a lack of electron microscopy techniques to preserve this labile structure. With the advent of cryo-fixation techniques, like high-pressure freezing/freeze-substitution, we are now able to consistently preserve and visualize the nucleocapsid. Because vaccinia virus early transcription is coupled to the viral core structure, detailing the structure of the nucleocapsid is indispensable for determining the mechanisms of vaccinia virus core-directed transcription. The present study represents our second attempt to understand the structure and biological significance of the nucleocapsid. We demonstrate the importance of the protein L4 for the formation of the nucleocapsid and reveal in addition that the nucleocapsid and the core wall may be associated, suggesting a higher level of complexity of the nucleocapsid than predicted. In addition, we prove the utility of high-pressure freezing in preserving the vaccinia virus nucleocapsid.

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“…Recombinant viruses have been generated in which the expression of specific core proteins is regulated by inducible promoters (29,45,48). The information obtained from such genetic studies has, however, been limited since in the absence of specific core proteins, assembly was either blocked at the IV stage (29,48) or generated IMVs that appeared morphologically indistinguishable from wild-type virus (45).…”

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confidence: 99%

Characterization of Vaccinia Virus Intracellular Cores: Implications for Viral Uncoating and Core Structure

Pedersen

Snijder

Schleich

et al. 2000

J Virol

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The entry of vaccinia virus (VV) into the host cell results in the delivery of the double-stranded DNA genomecontaining core into the cytoplasm. The core is disassembled, releasing the viral DNA in order to initiate VV cytoplasmic transcription and DNA replication. Core disassembly can be prevented using the VV early transcription inhibitor actinomycin D (actD), since early VV protein synthesis is required for core uncoating. In this study, VV intracellular cores were accumulated in the presence of actD and isolated from infected cells. The content of these cores was analyzed by negative staining EM and by Western blotting using a collection of antibodies to VV core and membrane proteins. By Western blot analyses, intracellular actD cores, as well as cores prepared by NP-40-dithiothreitol treatment of purified virions (NP-40/DTT cores), contained the core proteins p25 (encoded by L4R), 4a (A10L), 4b (A3L), and p39 (A4L) as well as small amounts of the VV membrane proteins p32 (D8L) and p35 (H3L). While NP-40/DTT cores contained the major putative DNA-binding protein p11 (F17R), actD cores entirely lacked this protein. Labeled cryosections of cells infected for different periods of time in the presence or absence of actD were subsequently used to follow the fate of VV core proteins by EM. These EM images confirmed that p11 was lost at the plasma membrane upon core penetration. The cores that accumulated in the presence of actD were labeled with antibodies to 4a, p39, p25, and DNA at all times examined. In the absence of the drug the cores gradually lost their electron-dense inner part, concomitant with the loss of p25 and DNA labeling. The remaining core shell still labeled with antibodies to p39 and 4a/4b, implying that these proteins are part of this structure. These combined data are discussed with respect to the structure of VV as well as core disassembly.

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Vaccinia Virus Core Protein VP8 Is Required for Virus Infectivity, but Not for Core Protein Processing or for INV and EEV Formation

Cited by 30 publications

References 0 publications

Genome-wide analysis of vaccinia virus protein–protein interactions

Genome-wide analysis of vaccinia virus protein–protein interactions

Vaccinia Virus Mutations in the L4R Gene Encoding a Virion Structural Protein Produce Abnormal Mature Particles Lacking a Nucleocapsid

Characterization of Vaccinia Virus Intracellular Cores: Implications for Viral Uncoating and Core Structure

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