Use of recombinant virus to assess human cytomegalovirus early and late promoters in the context of the viral genome.

We have developed a system to study human cytomegalovirus (HCMV) cis-acting promoter elements within the context of the viral genome. A recombinant HCMV (RV134) containing a marker gene (beta-glucuronidase) was used to insert HCMV promoter-chloramphenicol acetyltransferase gene constructs into the viral genome between open reading frames US9 and US10. Using this system, we have studied the promoters for the early DNA polymerase gene (UL54), the early-late lower matrix phosphoprotein gene (pp65, UL83), and the true late 28-kDa structural phosphoprotein gene (pp28, UL99). Transient-expression assays demonstrated that the pp65 and pp28 promoters are activated earlier and to higher levels than typically observed with the endogenous gene. In contrast, insertion of these promoters into the viral genome resulted in kinetics which mimicked that of the endogenous genes. In addition, we have also tested a variant of the pp28 promoter (d24/26CAT) which is deleted from -609 to -41. This promoter behaved similarly to the wild-type pp28 promoter, indicating that sequences from -40 to +106 are sufficient for conferring true late kinetics. Taken together, these data demonstrate that the viral genome affords a level of regulation on HCMV gene expression that has been previously unrealized. Therefore, these experiments provide a model system for the analysis of cis-acting promoter regulatory elements in the context of the viral genome.

Proteolytic activity of human cytomegalovirus UL80 protease cleavage site mutants.

The human cytomegalovirus UL80 open reading frame encodes protease and assembly protein from its N- and C-terminal regions, respectively. We reported previously that a 30-kDa protease is derived by autoproteolytic processing of a polyprotein which is the translation product of the entire UL80 open reading frame (E. Z. Baum, G. A. Bebernitz, J. D. Hulmes, V. P. Muzithras, T. R. Jones, and Y. Gluzman, J. Virol. 67:497-506, 1993). Three autoproteolytic cleavage sites within the UL80 polyprotein were characterized; site 143 is within the protease domain and inactivates the protease. In this article, we report (i) expression analyses of UL80 in infected cells, including the processing kinetics of the UL80 polyprotein; (ii) the existence of an additional cleavage site (site 209) within the protease domain of the UL80 polyprotein; and (iii) the effect of mutagenesis at each of the cleavage sites upon proteolytic activity and steady-state levels of the UL80 processing products. During the course of infection, UL80 polyprotein processing begins at cleavage site 643 and follows at sites 256 and 143. Cleavage at site 643 and/or 256 within the polyprotein is not a prerequisite for efficient protease activity, since all three proteases (85-, 80-, and 30-kDa proteins) were equally active in cleaving the assembly protein precursor to its mature form. Inhibition of cleavage at site 143 resulted in a three- to sixfold increase in the steady-state level of the 30-kDa protease, supporting the hypothesis that cleavage at this site may represent a mechanism by which cytomegalovirus regulates the level of active protease.

Expression and analysis of the human cytomegalovirus UL80-encoded protease: identification of autoproteolytic sites.

The 45-kDa assembly protein of human cytomegalovirus is encoded by the C-terminal portion of the UL80 open reading frame (ORF). For herpes simplex virus, packaging of DNA is accompanied by cleavage of its assembly protein precursor at a site near its C terminus, by a protease encoded by the N-terminal region of the same ORF (F. Liu and B. Roizman, J. Virol. 65:5149-5156, 1991). By analogy with herpes simplex virus, we investigated whether a protease is contained within the N-terminal portion of the human cytomegalovirus UL80 ORF. The entire UL80 ORF was expressed in Escherichia coli, under the control of the phage T7 promoter. UL80 should encode a protein of 85 kDa. Instead, the wild-type construct produces a set of proteins with molecular masses of 50, 30, 16, 13, and 5 kDa. In contrast, when mutant UL80 is deleted of the first 14 amino acids, it produces only an 85-kDa protein. These results suggest that the UL80 polyprotein undergoes autoproteolysis. We demonstrate by deletional analysis and by N-terminal sequencing that the 30-kDa protein is the protease and that it originates from the N terminus of UL80. The UL80 polyprotein is cleaved at the following three sites: (i) at the C terminus of the assembly protein domain, (ii) between the 30- and 50-kDa proteins, and (iii) within the 30-kDa protease itself, which yields the 16- and 13-kDa proteins and may be a mechanism to inactivate the protease.

A cluster of dispensable genes within the human cytomegalovirus genome short component: IRS1, US1 through US5, and the US6 family.

By insertional mutagenesis, human cytomegalovirus recombinants deleted of each of the US6 glycoprotein family genes were isolated. A recombinant lacking IRS1, US1 through US5, and most of the US6 family was also isolated. The growth kinetics of these mutants were similar to that of the wild type. A dispensable cluster of genes was identified.

Replacement mutagenesis of the human cytomegalovirus genome: US10 and US11 gene products are nonessential.

The US6 gene family, located within the unique short region (US) of the human cytomegalovirus (HCMV) genome, contains six open reading frames (US6 through US11) which may encode glycoproteins, such as gcII (D. Gretch, B. Kari, R. Gehrz, and M. Stinski, J. Virol. 62:1956-1962, 1988). By homologous recombination, several different recombinant HCMV were created which contain a marker gene, beta-glucuronidase, inserted within this gene family. It was demonstrated that beta-glucuronidase has utility as a marker gene for the identification of recombinants in this herpesvirus system, without the occurrence of deletions in other regions of the viral genome. DNA and RNA blot analyses attested to the fidelity of the recombination. Immunoprecipitation experiments using monospecific polyclonal antisera indicated that the US10 and/or US11 gene products were not expressed in the recombinants, as predicted. These results, along with single-cycle growth analyses, indicated that the US10 and US11 gene products are nonessential for virus replication and growth in tissue culture. HCMV recombinants expressing beta-glucuronidase seemed to be genetically stable.

Fine mapping of transcripts expressed from the US6 gene family of human cytomegalovirus strain AD169.

By sequence analysis (K. Weston and B. G. Barrell, J. Mol. Biol. 192:177-208, 1986), the human cytomegalovirus (HCMV) strain AD169 HindIII X DNA fragment contains six open reading frames (US6 through US11; called the US6 family) which may encode glycoproteins. Sense transcripts from the US6 family were mapped. The kinetics of appearance of steady-state cytoplasmic RNA was different for each transcription unit. The 1.5-kb US11-US10 and the 1.7-kb US9-US8 transcripts belonged to the early kinetic class. The former reached peak abundance by 8 h postinfection, while the latter peaked at 24 h postinfection. These RNAs greatly decreased in abundance by 48 to 72 h after infection, unlike transcripts from other HCMV early transcription units reported previously. US6 and US7 messages were most abundant at late times postinfection. US6 transcripts utilized different initiation sites at early or late times postinfection. There was evidence for both spliced and unspliced messages from this family. In a transient expression assay, chimeric plasmids containing the regions upstream of the mapped transcription initiation sites were active in promoting indicator gene expression in HCMV-infected, but not uninfected, human foreskin fibroblast cells.