Two novel spliced genes in human cytomegalovirus

Abstract

Two novel spliced genes (UL131A and UL128) flanking UL130 were predicted from sequence comparisons between human cytomegalovirus (HCMV) and its closest known relative, chimpanzee cytomegalovirus (CCMV), and the splicing patterns were confirmed by mRNA mapping experiments. Both genes were transcribed with late kinetics and shared a polyadenylation site. Comparisons with wild-type HCMV in infected human tissues showed that three of five isolates passaged in cell culture contained disruptions of UL128, one was frameshifted in UL131A and one exhibited a deletion affecting UL131A and UL130. CCMV and the Colburn strain of simian cytomegalovirus, which have been passaged in cell culture, also exhibit disruptions of UL128. These observations indicate that expression of either one of UL128 and UL131A is deleterious to growth of primate cytomegaloviruses in cell culture. Although the functions of these genes are unknown, sequence comparisons suggest that UL128 encodes a b-chemokine.The genome of the AD169 strain of human cytomegalovirus (HCMV; human herpesvirus 5) was characterized by Chee et al. (1990) as containing 189 putative protein-coding open reading frames (ORFs), some duplicated in an inverted repeat. An additional genome region was subsequently discovered in the Toledo strain (Cha et al., 1996). A recent comparison of these sequences with that of chimpanzee cytomegalovirus (CCMV) indicated that wild-type HCMV has 166-169 genes (Davison et al., 2003a, b). The present work concerns two of the eleven newly predicted genes in this redefined set.The upper part of Fig. 1(A) depicts the arrangement of ORFs UL131-UL128 as predicted by Chee et al. (1990), and the lower parts show alternative predictions based on comparisons between the AD169 and CCMV sequences. UL130 is unaltered, while spliced genes replace UL131 upstream and UL129 plus UL128 downstream. One of these genes is named UL131A because it occupies the same region as UL131 but does not share any encoded amino acid sequence, since the first exon is in a different reading frame from UL131. The other spliced gene retains the designation UL128 because it shares amino acid sequence with the original UL128 but not with UL129. Fig. 1(B, C) shows detailed alignments of the AD169 and CCMV sequences in these regions. Protein-coding regions were proposed from conservation of encoded amino acid sequences, and conceptually linked together via candidate splice donor and acceptor sites. This led to the hypothesis that UL131A and UL128 comprise two and three exons, respectively.In order to sustain this interpretation, it is necessary to propose that AD169 has a frameshift mutation (an additional residue making a tract of eight A residues) in UL131A exon 1 (Fig. 1B) and that CCMV has a frameshift mutation (an additional residue making a tract of eight C residues) in UL128 exon 1 (Fig. 1C). Davison et al. (2003a) confirmed the former lesion by resequencing the relevant AD169 region and comparing it with sequence obtained directly from clinical material. The proposed mutation in...

Identification and Characterization of U83A Viral Chemokine, a Broad and Potent β-Chemokine Agonist for Human CCRs with Unique Selectivity and Inhibition by Spliced Isoform

“…Sequencing studies of clinical material also suggested that this may be a polymorphic site (data not shown). Similar genomic variability has been observed in a homologous region in HCMV encoding a gene product with similarity to ␤-chemokines (17). The extended U83A now encoded a signal sequence comparable to that of U83B, with both start codons initiating at the same position, giving a similar predicted N terminus as shown by N-terminal sequencing (24).…”

“…This now encodes a predicted signal sequence much less likely to be used as described previously (23). Similarly, in fibroblast-passaged HCMV strains, deletions interrupt expression of a ␤-chemokine, suggesting that these chemokines may be detrimental or not necessary for in vitro cultivation in some cell types (17,19). Interestingly, as described earlier, the ␤-chemokine genes in HCMV and HHV-6 are genomic positional homologs, and in HCMV, this locus is associated with endothelial and DC tropism as well as capacity to transfer to lymphocytes, although its receptor specificity has not been characterized (18,19).…”

“…In human CMV (HCMV), for example, the UL146 chemokine is specific for ␣-chemokine receptors and can control dissemination of the virus in specifically chemoattracted neutrophils (16). There is another locus encoding a potential HCMV chemokine, UL131-128, and this also appears to affect endothelial, leukocyte, and dendritic cell (DC) tropism, in that passage in fibroblasts results in deletion or alteration at this locus (17)(18)(19). At least one transcript here encodes similarities to ␤-chemokines, which can chemoattract monocytes, and a similar function in murine CMV, vMCK, has been shown essential for virus dissemination (20 -22).…”

The susceptibility of primary cultured rhesus macaque kidney epithelial cells to rhesus cytomegalovirus strains

“…Notably, the epithelial/endothelial determinant locus (UL128-UL130-UL131A; hereafter UL128L) is especially susceptible to genetic alterations after only a few passages in cultured fibroblasts (Akter et al, 2003;Bradley et al, 2009;Cha et al, 1996;Cunningham et al, 2010;Hahn et al, 2004;Prichard et al, 2001;Ryckman et al, 2006Ryckman et al, , 2008Sinzger et al, 2008;Stanton et al, 2010). Proteins encoded by UL128L, together with glycoprotein H (gH) and L (gL), form a gH-anchored pentamer complex that mediates HCMV entry of EP and endothelial cells via receptor-dependent endocytosis.…”

Novel transcripts of human cytomegalovirus clinical strain found by cDNA library screening

“…The open reading frames (ORFs) of HCMV had been mapped on the genome by computer analysis and sequences compared between different HCMV strains and different CMV genomes (Chee et al, 1990;Rigoutsos et al, 2003;Dolan et al, 2004). To date, most HCMV genes have not been extensively characterized with respect to their expression patterns, although the coding potential and transcript structures of a few predicted genes had been identified by analysis of mRNA splicing, Northern blotting, identification of protein, and localization of the coding ORF (Jones and Muzithras, 1991;Adam et al, 1995;Wing and Huang, 1995;Atalay et al, 2002;Akter et al, 2003;Awasthi et al, 2004;Jenkins et al, 2004;Bego et al, 2005). Different transcripts expressed from a single piece of DNA sequence have been observed.…”