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Journal of Virology, July 2001, p. 5778-5795, Vol. 75, No. 13
0022-538X/01/$04.00+0   DOI: 10.1128/JVI.75.13.5778-5795.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.

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

Ritva Heljasvaara,1 Dolores Rodríguez,1 Cristina Risco,2 José L. Carrascosa,2 Mariano Esteban,1,* and Juan Ramón Rodríguez1

Departments of Molecular and Cellular Biology1 and Macromolecular Structure,2 Centro Nacional de Biotecnología, Consejo Superior de Investigaciones Cientifícas, Campus Universidad Autónoma, Cantoblanco, 28049 Madrid, Spain

Received 28 July 2000/Accepted 2 April 2001

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-beta -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.

* Corresponding author. Mailing address: Department of Molecular and Cellular Biology, Centro Nacional de Biotecnología, Consejo Superior de Investigaciones Científicas, Campus Universidad Autónoma, Cantoblanco, 28049 Madrid, Spain. Phone: 34-91-585-4503. Fax: 34-91-5854506. E-mail: mesteban{at}cnb.uam.es.

Journal of Virology, July 2001, p. 5778-5795, Vol. 75, No. 13
0022-538X/01/$04.00+0   DOI: 10.1128/JVI.75.13.5778-5795.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.


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