Analysis of the human cytomegalovirus genomic region from UL146 through UL147A reveals sequence hypervariability, genotypic stability, and overlapping transcripts.

BACKGROUND: Although the sequence of the human cytomegalovirus (HCMV) genome is generally conserved among unrelated clinical strains, some open reading frames (ORFs) are highly variable. UL146 and UL147, which encode CXC chemokine homologues are among these variable ORFs. RESULTS: The region of the HCMV genome from UL146 through UL147A was analyzed in clinical strains for sequence variability, genotypic stability, and transcriptional expression. The UL146 sequences in clinical strains from two geographically distant sites were assigned to 12 sequence groups that differ by over 60% at the amino acid level. The same groups were generated by sequences from the UL146-UL147 intergenic region and the UL147 ORF. In contrast to the high level of sequence variability among unrelated clinical strains, the sequences of UL146 through UL147A from isolates of the same strain were highly stable after repeated passage both in vitro and in vivo. Riboprobes homologous to these ORFs detected multiple overlapping transcripts differing in temporal expression. UL146 sequences are present only on the largest transcript, which also contains all of the downstream ORFs including UL148 and UL132. The sizes and hybridization patterns of the transcripts are consistent with a common 3'-terminus downstream of the UL132 ORF. Early-late expression of the transcripts associated with UL146 and UL147 is compatible with the potential role of CXC chemokines in pathogenesis associated with viral replication. CONCLUSION: Clinical isolates from two different geographic sites cluster in the same groups based on the hypervariability of the UL146, UL147, or the intergenic sequences, which provides strong evidence for linkage and no evidence for interstrain recombination within this region. The sequence of individual strains was absolutely stable in vitro and in vivo, which indicates that sequence drift is not a mechanism for the observed sequence hypervariability. There is also no evidence of transcriptional splicing, although multiple overlapping transcripts extending into the adjacent UL148 and UL132 open reading frames were detected using gene-specific probes.

Phenotyping of cytomegalovirus drug resistance mutations by using recombinant viruses incorporating a reporter gene.

A new recombinant phenotyping method was developed for the analysis of drug resistance mutations in human cytomegalovirus (CMV). CMV strain T2211 was derived from strain AD169 by inserting unique restriction sites and a secreted alkaline phosphatase (SEAP) reporter gene for rapid viral quantitation. Specific viral UL97 and pol gene mutations were transferred by recombination into T2211, and their drug resistance phenotypes (for ganciclovir, foscarnet, or cidofovir) were determined by the drug concentrations required to reduce supernatant SEAP activity by 50% (IC50). Changes in the IC50 conferred by the mutations tested (UL97 M460V, C592G, A594V, and L595S and pol del981-2) were similar to those previously reported in marker transfer and conventional plaque reduction assays. The combination of UL97 C592G and pol del981-2 conferred much higher ganciclovir resistance than either mutation alone. The UL97 polymorphism D605E had no measurable effect on ganciclovir susceptibility, alone or in combination with common UL97 resistance mutations. Transfer into strain T2211 facilitates the phenotyping of newly observed mutations, combinations of mutations, and clinical CMV sequences without an accompanying viral isolate.