Analysis of two hypervariable human cytomegalovirus genes, UL146 and UL139

Abstract Human cytomegalovirus (HCMV) is a highly host-specific, ubiquitous herpesvirus that results in asymptomatic infection for the majority of those infected. However, it produces serious clinical disease in neonates and immunocompromised individuals such as transplant recipients and AIDS patients. The majority of the 236 kbp genome is highly conserved, but there are a number of highly variable regions, coding and non-coding, scattered throughout the genome. Numerous studies have been published investigating the genotypes of hypervariable genes, most focussed on potential associations between genotype and clinical disease or tropism. In general, no convincing connections between genotype and disease have been found. The present study investigated two hypervariable HCMV genes, UL146 and UL139, in a large number of clinical samples (179) from a number of locations worldwide in Europe, Africa, Asia and Australia. A total of 14 UL146 genotypes (G1-G14) were detected, which agrees with previous findings based on many fewer samples. For UL139, eight genotypes were detected, three of them (G5, G7 and G8) novel. The genotypes of both genes appear to have evolved under constraint rather than positive selection. Possible bias in the geographical distribution of the UL146 and UL139 genotypes was investigated. In general, all genotypes were found in all areas and any variation from the expected distribution was probably a result of small sample numbers from certain regions, specifically Asia and Australia. This general finding is in agreement with that of a previously published study on gene UL73. No evidence for linkage disequilibrium between UL146 and UL139 genotypes was found. This is in accordance with a previously published study of linkage disequilibrium among six other genes (UL55, UL74, UL75, UL115, US9 and US28), and is consistent with the theory that recombination has played a role in HCMV evolution. The absence of linkage between highly variable genes complicates attempts to examine associations between genotype and disease, as many combinations of genotypes are possible. Investigation of transcriptional expression of UL146 and UL139 from HCMV strain Merlin in fibroblast cell culture revealed that UL146 is expressed with late kinetics and UL139 with early-late kinetics. Northern blot and RACE data suggested that UL146 is 3’-coterminally expressed with UL147, UL147A, UL148 and UL132, and that UL139 is 3’-coterminally expressed with UL140 and UL141. To determine whether the high degree of sequence divergence corresponds to structural divergence, the UL146 genotypes were homology modelled on the related human chemokines IL-8, gro-a and IF9S. All 14 genotypes were predicted to be structurally very similar, which suggests they may also be functionally similar. However, small differences between the structures of human chemokines are known to result in slightly differing binding affinities for cellular receptors, and therefore even small differences between UL146 genotypes could conceivably confer functional differences. UL139 has been predicted to encode a type 1 membrane glycoprotein. No information has been published regarding UL139 function, although a short region of similarity with the cellular signal transducer CD24 has been noted previously, tentatively suggesting an immunomodulatory role. Preliminary experiments to characterise UL139 were performed utilising recombinant adenovirus vectors expressing tagged UL139 variants from three genotypes (G1, G5 and G7). The tagged UL139 variants expressed proteins that were considerably larger in mass than predicted from amino acid sequences. This extra mass may be attributable to glycosylation as well as other forms of post translational modification. Mixed infections of HCMV strains in immunocompromised individuals, such as transplant recipients, have been associated with enhanced pathogenesis and increased risk of transplant rejection. The presence of mixed infections also further complicates attempts to establish connections between genotype and disease outcome. In the analysis of UL146 and UL139 genotypes, multiple genotypes were detected in 14% of samples and in 29% when repeated experimental results were included, and even these values may be underestimations. The utility of a QPCR-based assay using genotype-specific primers was assessed as a means of more accurately determining the occurrence of mixed infections, and showed promise. Passage of HCMV strains in cell culture has been shown to result in various mutations. AD169, a commonly used laboratory strain, lacks 15 kbp sequence that includes UL146 and UL139. An alternative stock of AD169 (AD169varUC) was obtained that was thought to contain most or all of the deleted region and, indeed, both UL146 and UL139 were detected. Further sequencing confirmed that this stock is derived from AD169 and revealed that it contains all but 3.2 kbp of the 15 kbp absent from commonly used AD169 stocks. The 3.2 kbp deletion affects UL144, UL142, UL141 and UL140. This propensity of HCMV to undergo mutation during cell culture highlights the importance of studying characterised strains that are as close to wild type virus as possible.