The role of distinct gO genotypes in mixed HCMV infections

A large number of human cytomegalovirus (HCMV) strains circulate in the human population distinguishable by heterogeneous sequences within the HCMV genome for which a number of distinct genotypes have been defined. Infections with more than one HCMV strain are common. To date, it is far from being understood how genetically different HCMV strains within a mixture interfere with each other and nothing is known about the function of the distinct HCMV genotypes in mixed HCMV infection dynamics. First clinical evidence that mixed HCMV infections might alter the infection dynamics has been provided by the findings that immunocompromised patients with mixed infections show higher plasma HCMV DNA loads over a longer period of time compared to those with single infections. Studies in the mouse model have shown that functional trans-complementation between distinct variants of murine CMV alters virus fitness and that profound within-host interaction between co-infecting murine CMV strains results in significant changes in the infection dynamics. HCMV infects a broad spectrum of cell types in vivo important for HCMV dissemination and lifelong persistence. In this regard, glycoprotein O (gO) is of particular interest. It plays a critical role in HCMV cell tropism by regulating the incorporation of two different glycoprotein complexes, gH/gL/gO and gH/gL/pUL128-131A, into the virus envelope. Remarkably, among these glycoproteins, only gO shows high sequence diversity between clinical HCMV strains. It exhibits eight gO genotypes circulating widespread in the human population with mixed gO genotype infections being frequently observed. We hypothesize that the distinct gO genotypes and mixtures thereof differ in their infectivity for different cell types and thereby might affect the HCMV infection dynamics. This research proposal aims at elucidating potential differences among the gO genotypes and mixtures thereof by phenotypic characterization of a set of gO mutant strains using different cell types as representative of cell types that are infected in vivo. In the first part of the project, we will generate bacterial artificial chromosome (BAC) derived gO mutant HCMV strains differing only in the gO genotype sequence. For this purpose, the parental strains vBAC-luc and vBAC-gfp which express either the luciferase protein or green fluorescent protein as indicator of infection will be used. Both of the parental strains derive from TB40-BAC4 known to be able to infect the cell types using in this study. In the second part of the project, we propose to apply a broad phenotypic characterization by the use of four different cell types. As HCMV remains a major cause of viral disease, especially for congenitally infected children and immunocompromised transplant patients the implications of this project are to gain insights into the pathogenic potential of mixed HCMV strain infections with the goal to provide worthful information for novel therapeutic approaches.

More than half of all adults worldwide are infected with human cytomegalovirus (HCMV), yet hardly noticed since infections usually occur without any symptoms. Patients with a strongly compromised immune system, however, may become severely ill, with symptoms ranging from mild to fatal. Moreover, HCMV infections of the unborn child may cause irrevocable harm leading to hearing loss or mental disabilities in newborns.Numerous different HCMV strains circulate in humans, and individuals can be infected with more than one HCMV strain, either synchroneously or serially aquired. HCMV possesses a lipid membrane in which various glycoprotein complexes are embedded. One of these complexes consists of three individual glyoproteins, gH, gL, and gO. It is assumed that this trimeric gH/gL/gO complex plays a key role in HCMV entry into various cell types. Circulating HCMV strains may differ in their gO sequences, and currently 8 different so-called gO genotypes are identified. It is still unclear whether or how gO heterogeneity affects HCMV infection and growth.In this project, we have investigated how efficiently distinct HCMV strains, which differ solely in the gO genotype sequence, can infect and replicate in various human cell types, either as a single strain or a mixture of strains, and how single point mutations of the gO sequence influence the stability and formation of the gH/gL/gO trimeric complex.Our study provides evidence that dependent on the gO genotype significantly more or less cells are infected and correspondingly more or less virus is released from infected cells into cell-culture supernatant. Interestingly, the respective growth properties of the distinct strains behave similar in single and mixed HCMV infections. We were also able to show that the distinct gO genotypes display structural differences and this appears to shape the overall three-dimensional structure of the trimeric complex. Taken together, these findings suggest that gO sequence diversity influences the interactions of the virus with the host cell.Our work has thus generated novel insights into the role of HCMV strain diversity on infectivity. A better understanding of the biological consequences of HCMV strain diversity in the human host can be very helpful for the development of novel antiviral agents against HCMV.