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Journal of Virology, February 2001, p. 1450-1458, Vol. 75, No. 3
0022-538X/01/$04.00+0   DOI: 10.1128/JVI.75.3.1450-1458.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.

Genetic Evidence of an Essential Role for Cytomegalovirus Small Capsid Protein in Viral Growth

Eva-Maria Borst, Sibylle Mathys, Markus Wagner, Walter Muranyi, and Martin Messerle*

Max von Pettenkofer-Institut für Hygiene und Medizinische Mikrobiologie, Lehrstuhl Virologie, Genzentrum, Ludwig-Maximilians-Universität München, D-81377 Munich, Germany

Received 21 August 2000/Accepted 6 November 2000

Many steps in the replication cycle of cytomegalovirus (CMV), like cell entry, capsid assembly, and egress of newly synthesized virions, have not been completely analyzed yet. In order to facilitate these studies, we decided to construct a recombinant CMV that incorporates the green fluorescent protein (GFP) into the nucleocapsid. A comparable herpes simplex virus type 1 (HSV-1) mutant has recently been generated by fusion of the GFP open reading frame (ORF) with the HSV-1 ORF encoding small capsid protein (SCP) VP26 (P. Desai and S. Person, J. Virol. 72:7563-7568, 1998). Recombinant CMV genomes expressing a fusion protein consisting of GFP and the SCP were constructed by the recently established bacterial artificial chromosome mutagenesis procedure. In transfected cells, the SCP-GFP fusion protein localized to distinct foci in the nucleus that may represent sites for capsid assembly (assemblons). However, no viable progeny was reconstituted from these mutant CMV genomes. CMV genomes with deletion of the SCP ORF also did not give rise to infectious virus. Rescue of the mutation by insertion of the SCP gene at an ectopic position in an SCP knockout genome indicates that, in contrast to the HSV-1 SCP, the CMV SCP is essential for viral growth. Expression of the SCP-GFP fusion protein together with the authentic SCP blocked the CMV infection cycle, suggesting that the SCP-GFP fusion protein exerts a dominant-negative effect on the assembly of new virions. The results of this study are discussed with regard to recently published data about the structure of the CMV virion and its differences from the HSV-1 virion.

* Corresponding author. Mailing address: Max von Pettenkofer-Institut, Genzentrum, Feodor-Lynen-Strasse 25, D-81377 Munich, Germany. Phone: 49 89 2180 6850. Fax: 49 89 2180 6898. E-mail: messerle{at}lmb.uni-muenchen.de.

Journal of Virology, February 2001, p. 1450-1458, Vol. 75, No. 3
0022-538X/01/$04.00+0   DOI: 10.1128/JVI.75.3.1450-1458.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.


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Copyright © 2001 by the American Society for Microbiology. All rights reserved.