Physical and molecular genetic analysis of the multistep proteolytic maturation pathway utilized by vaccinia virus P4a protein

Whitehead, Stephen S.; Bersani, Neil A.; Hruby, Dennis E.

doi:10.1099/0022-1317-76-3-717

Cited by 9 publications

(5 citation statements)

References 16 publications

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“…Core protein 4a is processed from a precursor core protein, p4a, which is encoded by the viral A10L gene (71). p4a protein is 102 kDa in size and is cleaved at sites between amino acids (aa) 614 and 615 and aa 697 and 698 to generate core protein 4a and p25, respectively (66,68). Both p4a and 4a proteins interact with A4L protein during virion morphogenesis (57).…”

Section: Discussionmentioning

confidence: 99%

Molecular Chaperone Hsp90 Is Important for Vaccinia Virus Growth in Cells

Hung

Chung

Chang

2002

J Virol

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Molecular chaperones assist protein folding, and some chaperones are induced by heat, nutrient depletion, or pathogen invasion. This study investigates the role played by Hsp90 in the life cycle of vaccinia virus. The titer of vaccinia intracellular mature virions (IMV) was reduced by 2 orders of magnitude in RK13 cells treated with geldanamycin (GA), which blocks the ATPase activity of Hsp90. GA does not affect expression from the viral early promoter, but treatment with GA delays DNA replication and intermediate gene transcription and reduces expression from the viral late promoter. Vaccinia virus infection does not induce Hsp90 expression; however, intracellular distribution of Hsp90 is altered in virus-infected cells. Hsp90 is restricted to the cytoplasm of mock-infected cells; in contrast, Hsp90 is transiently associated with virosomes in virus-infected cells although it is not incorporated into IMV. In addition, Hsp90 interacts with viral core protein 4a, the mature form of the A10L gene product, in virus-infected cells. In conclusion, these results suggest that a cellular chaperone protein, Hsp90, is important for vaccinia virus growth in cultured cells and that viral core protein 4a associates with Hsp90-containing complexes in the infected cells.

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Section: Discussionmentioning

confidence: 99%

Molecular Chaperone Hsp90 Is Important for Vaccinia Virus Growth in Cells

Hung

Chung

Chang

2002

J Virol

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“…Protein processing plays an important role during the vaccinia virus life cycle (68,69,75,76). For example, p4a and p4b are major precursors of vaccinia virus core proteins that are proteolytically processed to become mature core proteins 4a and 4b (68,69).…”

Section: Resultsmentioning

confidence: 99%

Vaccinia Virus J1R Protein: a Viral Membrane Protein That Is Essential for Virion Morphogenesis

Chiu

Chang

2002

J Virol

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“…P4a is synthesized at late times in the viral infection as a 102-kDa protein, which is posttranslationally cleaved to two smaller polypeptides. The processing occurs at two locations: cleavage at the N-terminal Ala-Gly-Ser site between amino acids 614 and 615 and cleavage at the C-terminal Ala-Gly-Thr site between amino acids 697 and 698 lead to release of 62-kDa (4a) and 23-kDa polypeptides, respectively (55,56,59). These cleavage sites are distinct but related to the consensus Ala-Gly-Ala motif for the proteolytic maturation of other VV structural proteins (58).…”

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confidence: 99%

The Major Core Protein P4a (A10L Gene) of Vaccinia Virus Is Essential for Correct Assembly of Viral DNA into the Nucleoprotein Complex To Form Immature Viral Particles

Heljasvaara¹,

Rodrı́guez²,

Risco

et al. 2001

J Virol

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The vaccinia virus (VV) A10L gene codes for a major core protein, P4a. This polypeptide is synthesized at late times during viral infection and is proteolytically cleaved during virion assembly. To investigate the role of P4a in the virus life cycle and morphogenesis, we have generated an inducer-dependent conditional mutant (VVindA10L) in which expression of the A10L gene is under the control of the Escherichia coli lacI operator/ repressor system. Repression of the A10L gene severely impairs virus growth, as observed by both the inability of the virus to form plaques and the 2-log reduction of viral yields. This defect can be partially overcome by addition of the inducer isopropyl-␤-D-thiogalactopyranoside (IPTG). Synthesis of viral proteins other than P4a occurred, although early shutoff of host protein synthesis and expression of viral late polypeptides are clearly delayed, both in the absence and in the presence of IPTG, compared with cells infected with the parental virus. Viral DNA replication and concatemer resolution appeared to proceed normally in the absence of the A10L gene product. In cells infected with VVindA10L in the absence of the inducer virion assembly is blocked, as defined by electron microscopy. Numerous spherical immature viral particles that appear devoid of dense viroplasmic material together with highly electron-dense regular structures are abundant in VVindA10L-infected cells. These regularly spaced structures can be specifically labeled with anti-DNA antibodies as well as with a DNase-gold conjugate, indicating that they contain DNA. Some images suggest that these DNA structures enter into spherical immature viral particles. In this regard, although it has not been firmly established, it has been suggested that DNA uptake occurs after formation of spherical immature particles. Overall, our results showed that P4a and/or its cleaved products are essential for the correct assembly of the nucleoprotein complex within immature viral particles.Vaccinia virus (VV), the prototype member of the Poxviridae family, is a large DNA virus whose replication and assembly occur entirely in the cytoplasm of the host cell, in particular areas termed viral factories or virosomes (33). The linear double-stranded DNA genome has a capacity to encode over 200 polypeptides (22), of which approximately 100 are incorporated into virus particles (14). The viral genes can be divided into three classes according to their temporally regulated expression. The early genes are transcribed prior to DNA replication, while the intermediate and late genes are transcribed only during or after replication of the viral genome (33).Detailed information about VV morphogenesis has been obtained from studies by conventional electron microscopy (6, 7, 9, 21, 31, 32). Virus assembly begins within cytoplasmic viral factories with the formation of crescent-shaped membranes whose origin remains controversial. These membranes subsequently enclose granular material from the virosomes, forming spherical particles known as immature vir...

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Physical and molecular genetic analysis of the multistep proteolytic maturation pathway utilized by vaccinia virus P4a protein

Cited by 9 publications

References 16 publications

Molecular Chaperone Hsp90 Is Important for Vaccinia Virus Growth in Cells

Molecular Chaperone Hsp90 Is Important for Vaccinia Virus Growth in Cells

Vaccinia Virus J1R Protein: a Viral Membrane Protein That Is Essential for Virion Morphogenesis

The Major Core Protein P4a (A10L Gene) of Vaccinia Virus Is Essential for Correct Assembly of Viral DNA into the Nucleoprotein Complex To Form Immature Viral Particles

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