Rapid Genetic Engineering of Human Cytomegalovirus by Using a Lambda Phage Linear Recombination System: Demonstration that pp28 (UL99) Is Essential for Production of Infectious Virus

Abstract

A highly efficient lambda phage recombination system previously utilized for studies of bacterial artificial chromosome (BAC)-maintained mouse chromosomal DNA was adapted for the study of the role of human cytomegalovirus (HCMV)-encoded pp28 (UL99) in virus replication. Incorporating a two-step mutagenesis strategy with blue/white selection in Escherichia coli containing a HCMV AD169 BAC, we have shown that we can rapidly introduce point mutations into the HCMV BAC using linear PCR fragments. All manipulations were carried out in bacteria, which greatly accelerated the introduction and analysis of mutations in the viral genome. Our results indicated that HCMV pp28 was essential for the production of infectious virus and that introduction of a single base change that resulted in loss of the myristylation site on pp28 was also associated with the lack of production of infectious virus. Although the block in the viral morphogenesis cannot be determined from these studies, the latter finding suggested that authentic intracellular localization of pp28, not only the expression of the protein, is required for virus assembly.The role of individual herpesvirus genes in virus replication and the pathogenesis of virus-induced disease have been best understood from studies using viral genetics. The propagation of infectious clones of herpesviruses as bacterial artificial chromosomes (BACs) has revolutionized the experimental manipulation of viral genomes. Importantly, this technology has allowed application of tools of prokaryotic molecular biology to the study of viral genetics, thereby facilitating the genetic analysis of many herpesviruses (4-6, 16, 23, 24, 27). Previously, mutagenic approaches based on homologous recombination in eukaryotic cells were limited to genetic manipulation of only the most rapidly replicating and promiscuous herpesviruses, such as herpes simplex virus (HSV) and pseudorabies virus (PRV) (19). In this study, we have adapted a recombination strategy originally developed for mutagenesis of BACs containing mouse chromosomes to mutagenize the human cytomegalovirus (HCMV) genome (8,10,25,26). This methodology takes advantage of a lambda phage recombination system (RED locus) expressed from a temperature-sensitive promoter that enables the use of linear single-stranded DNA to target genes of interest carried on BACs maintained in Escherichia coli (8,26). We have used this recombination system together with a blue/white lacZ-based selection approach to define the importance of HCMV tegument protein pp28 (UL99) for HCMV replication. The product of the HCMV UL99 open reading frame (ORF) is a 190-amino-acid (aa) phosphorylated tegument protein, which is modified by myristylation (17,18,21). Homologues of pp28 are found in all herpesviruses. Studies with the HSV homologue UL11 have indicated that this protein is also myristylated, traffics in the cytoplasm, and is essential for wild-type levels of virus replication in vitro (2, 3, 11-13). Furthermore, more recent studies of the PRV UL11 * Corresponding aut...

Role of tegument proteins in herpesvirus assembly and egress

“…In HCMV, the pUL11 homolog pUL99 has been found to interact with the pUL16 homolog pUL94 (Liu et al, 2009). Consistent with the previously described model of capsid transport and budding, herpesviruses lacking pUL16, pUL21 or pUL11 (or their homologs) have defects in virus egress, resulting in decreased amounts of extracellular viruses produced and the accumulation of non-enveloped capsids in the cytoplasm (MacLean et al, 1989(MacLean et al, , 1992Baines and Roizman, 1992;Baines et al, 1994;Kopp et al, 2003;Schimmer and Neubauer, 2003;Silva et al, 2003;Britt et al, 2004;Jones and Lee, 2004;Silva et al, 2005;Seo and Britt, 2006;Guo et al, 2009).…”

Murine cytomegalovirus encodes a miR-27 inhibitor disguised as a target

“…The MCMV-24 mutant was constructed using the adapted protocol described in Ref. 42, with modifications described in SI Materials and Methods. For in situ hybridization analyses, LNA-incorporated miRNA probes were purchased from Exiqon and the m169 probe and antisense control probe were generated using the DIG RNA labeling kit (Roche).…”

Functional analysis of human cytomegalovirus pUS28 mutants in infected cells

“…In order to investigate the pUS28 domains involved in signalling at physiological levels in the context of an HCMV infection, HCMV FIX-BAC containing the genome of the clinical isolate VR1814 was used to create sitedirected mutants of US28 by lambda red-mediated recombination (Borst et al, 1999;Britt et al, 2004;Datsenko & Wanner, 2000;Hahn et al, 2002;Oppenheim et al, 2004;Wagner & Koszinowski, 2004). Using this methodology, we constructed DUS28, FLAG-US28/WT, FLAG-US28/R129A and FLAG-US28/DN recombinant viruses.…”