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

Abstract

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...

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

“…Several other vaccinia virus mutants that are also defective at IV formation during morphogenesis were identified, such as A10L, A14L, and A30L (20,48,63,64). The phenotypes of these mutants are not identical to that of the J1R mutant.…”

“…However, the viral membrane structures observed in these cells were more fragmented and tubular-vesicular, indicating an additional role for A14L in crescent formation (48,64). Mutations in A10L produced DNA bundles that were not incorporated into IMV, resulting in empty spherical viral particles similar to those observed with the J1R mutant (20). However, the organization of electron-dense viroplasm structures into regularly spaced bands that accumulated in the cytoplasm of cells was observed only in cells infected with the A10L mutant virus and not with the J1R mutant virus.…”

“…J1R protein is not required for crescent formation; instead, it plays a role in DNA packaging in IV formation. Also shown are several other viral proteins (A10L, A14L, A17L, A30L, and D13L) in which a mutation interferes with virion morphogenesis before or during the formation of IV (20,45,47,48,59,63,79,82). CEV, cell-associated virions; EEV, extracellular enveloped viruses.…”

Membrane remodelling during vaccinia virus morphogenesis

“…It is well established that VACV DNA is associated with nucleoproteins in the cytoplasm of the infected cells from early times after infection (Dahl and Kates, 1970;Polisky and Kates, 1972;Soloski and Holowczak, 1981;Heljasvaara et al, 2001). The fact that certain areas of the condensed DNA crystalloid inside IVNs show fibres arranged with a spacing of approx.…”

Cidofovir Inhibits Genome Encapsidation and Affects Morphogenesis during the Replication of Vaccinia Virus

“…Abnormal IV, including dense spherical particles, were consistently observed, resembling aberrant forms described during an infection with VACV mutants defective in the expression of I7, D6, A32, A4, A10, J1, I6, G7, and A13. Some of these mutants are deficient in the proper formation of IV (3,6,7,17,22,23,25,27,47,50,53). Importantly, DNA crystalloids were also detected in cells infected in the presence of CDV, and the ratio of IVN to total IV decreased.…”