A mutant of herpes simplex virus type 1 (HSV-1) in which glycoprotein H (gH) coding sequences were deleted and replaced by the Escherichia coli lacZ gene under the control of the human cytomegalovirus IE-1 gene promoter was constructed. The mutant was propagated in Vero cells which contained multiple copies of the HSV-1 gH gene under the control of the HSV-1 gD promoter and which therefore provide gH in trans following HSV-1 infection. Phenotypically gH-negative virions were obtained by a single growth cycle in Vero cells. These virions were noninfectious, as judged by plaque assay and by expression of I-galactosidase following high-multiplicity infection, but partial recovery of infectivity was achieved by using the fusogenic agent polyethylene glycol. Adsorption of gH-negative virions to cells blocked the adsorption of superinfecting wild-type virus, a result in contrast to that obtained with gD-negative virions (D. C. Johnson and M. W. Ligas, J. Virol. 62:4605-4612, 1988). The simplest conclusion is that gH is required for membrane fusion but not for receptor binding, a conclusion consistent with the conservation of gH in all herpesviruses.

A glycoprotein encoded by the ULl gene of herpes simplex virus type 1 (HSV-1) was detected in infected cells with antipeptide sera. The ULl gene has previously been implicated in virus-induced cell fusion (S. Little and P. A. Schaffer, Virology 112:686-697, 1981). Two protein species, a 30-kDa precursor form and a 40-kDa mature form of the glycoprotein, both of which were modified with N-linked oligosaccharides, were observed. This novel glycoprotein is the 10th HSV-1 glycoprotein to be described and was named glycoprotein L (gL). A complex was formed between gL and gH, a glycoprotein known to be essential for entry of HSV-1 into cells and for virus-induced cell fusion. Previously, it had been reported that gH expressed in the absence of other viral proteins was antigenically abnormal, not processed, and not expressed at the cell surface (U. A. Gompels and

This report describes the identification of a murine cytomegalovirus (MCMV) G protein-coupled receptor (GCR) homolog. This open reading frame (M33) is most closely related to, and collinear with, human cytomegalovirus UL33, and homologs are also present in human herpesvirus 6 and 7 (U12 for both viruses). Conserved counterparts in the sequenced alpha-or gammaherpesviruses have not been identified to date, suggesting that these genes encode proteins which are important for the biological characteristics of betaherpesviruses. We have detected transcripts for both UL33 and M33 as early as 3 or 4 h postinfection, and these reappear at late times. In addition, we have identified N-terminal splicing for both the UL33 and M33 RNA transcripts. For both open reading frames, splicing results in the introduction of amino acids which are highly conserved among known GCRs. To characterise the function of the M33 in the natural host, two independent MCMV recombinant viruses were prepared, each of which possesses an M33 open reading frame which has been disrupted with the ␤-galactosidase gene. While the recombinant M33 null viruses showed no phenotypic differences in replication from wild-type MCMV in primary mouse embryo fibroblasts in vitro, they showed severely restricted growth in the salivary glands of infected mice. These data suggest that M33 plays an important role in vivo, in particular in the dissemination to or replication in the salivary gland, and provide the first evidence for the function of a viral GCR homolog in vivo.

Chemokines are important mediators of the early inflammatory response to infection and modify a wide range of host immune responses. Functional homologs of cellular chemokines have been identified in a number of herpesviruses, suggesting that the subversion of the host chemokine response contributes to the pathogenesis of these viruses. Transcriptional and reverse transcription-PCR analyses demonstrated that the murine cytomegalovirus (MCMV) chemokine homolog, m131, was spliced at the 3′ end to the adjacent downstream open reading frame, m129, resulting in a predicted product of 31 kDa, which is significantly larger than most known chemokines. The in vivo impact of m131/129 was investigated by comparing the replication of MCMV mutants having m131/129 deleted (Δm131/129) with that of wild-type (wt) MCMV. Our studies demonstrate that both wt and Δm131/129 viruses replicated to equivalent levels during the first 2 to 3 days following in vivo infection. However, histological studies demonstrated that the early inflammatory response elicited by Δm131/129 was reduced compared with that of wt MCMV. Furthermore, the Δm131/129 mutants failed to establish a high-titer infection in the salivary glands. These results suggest that m131/129 possesses proinflammatory properties in vivo and is important for the dissemination of MCMV to or infection of the salivary gland. Notably, the Δm131/129 mutants were cleared more rapidly from the spleen and liver during acute infection compared with wt MCMV. The accelerated clearance of the mutants was dependent on NK cells and cells of the CD4+ CD8+ phenotype. These data suggest that m131/129 may also contribute to virus mechanisms of immune system evasion during early infection, possibly through the interference of NK cells and T cells.