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Journal of Virology, June 2001, p. 5692-5696, Vol. 75, No. 12
0022-538X/01/$04.00+0   DOI: 10.1128/JVI.75.12.5692-5696.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.

Virus Reconstituted from Infectious Bacterial Artificial Chromosome (BAC)-Cloned Murine Gammaherpesvirus 68 Acquires Wild-Type Properties In Vivo Only after Excision of BAC Vector Sequences

Heiko Adler,* Martin Messerle, and Ulrich H. Koszinowski

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

Received 12 January 2001/Accepted 14 March 2001

We studied the in vivo biological properties of viruses reconstituted from the genome of murine gammaherpesvirus 68 (MHV-68) cloned as an infectious bacterial artificial chromosome (BAC). Recombinant virus Rgamma HV68A98.01, containing BAC vector sequences, is attenuated in vivo as determined by (i) viral titers in the lungs during the acute phase of infection, (ii) the extent of splenomegaly, and (iii) the number of latently infected spleen cells reactivating virus in an ex vivo reactivation assay. Since the BAC vector sequences were flanked by loxP sites, passaging the virus in fibroblasts expressing Cre recombinase resulted in the generation of recombinant virus Rgamma HV68A98.02, with biological properties comparable to those of wild-type MHV-68. On the basis of these data we conclude (i) that excision of BAC vector sequences from cloned MHV-68 genomes is critical for reconstitution of the wild-type phenotypic properties of this virus and (ii) that the BAC-cloned MHV-68 genome is suitable for the construction of mutants and mutant libraries whose phenotypes can be reliably assessed in vivo.

* Corresponding author. Mailing address: Max von Pettenkofer-Institut für Hygiene und Medizinische Mikrobiologie, Lehrstuhl Virologie, Genzentrum, Ludwig-Maximilians-Universität München, Feodor-Lynen-Strasse 25, D-81377 Munich, Germany. Phone: 49-89-2180-6858. Fax: 49-89-2180-6899. E-mail: adler{at}lmb.uni-muenchen.de.

Journal of Virology, June 2001, p. 5692-5696, Vol. 75, No. 12
0022-538X/01/$04.00+0   DOI: 10.1128/JVI.75.12.5692-5696.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.


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