Human Cytomegalovirus UL135 and UL136 Genes Are Required for Postentry Tropism in Endothelial Cells

Abstract

Endothelial cells (ECs) are a critical target of viruses, and infection of the endothelium represents a defining point in viral pathogenesis. Human cytomegalovirus (HCMV), the prototypical betaherpesvirus, encodes proteins specialized for entry into ECs and delivery of the genome to the nuclei of ECs. Virus strains competent to enter ECs replicate with differing efficiencies, suggesting that the virus encodes genes for postentry tropism in ECs. We previously reported a specific requirement for the UL133/8 locus of HCMV for replication in ECs. The UL133/8 locus harbors four genes: UL133, UL135, UL136, and UL138. In this study, we find that while UL133 and UL138 are dispensable for replication in ECs, both UL135 and UL136 are important. These genes are not required for virus entry or the expression of viral genes. The phenotypes associated with disruption of either gene reflect phenotypes observed for the UL133/8 NULL virus, which lacks the entire UL133/8 locus, but are largely distinct from one another. Viruses lacking UL135 fail to properly envelop capsids in the cytoplasm, produce fewer dense bodies (DB) than the wild-type (WT) virus, and are unable to incorporate viral products into multivesicular bodies (MVB). Viruses lacking UL136 also fail to properly envelop virions and produce larger dense bodies than the WT virus. Our results indicate roles for the UL135 and UL136 proteins in commandeering host membrane-trafficking pathways for virus maturation. UL135 and UL136 represent the first HCMV genes crucial for early-to late-stage tropism in ECs. H uman cytomegalovirus (HCMV) is a ubiquitous herpesvirus with 50 to 99% seroprevalence in the global population. Like all herpesviruses, HCMV persists for the lifetime of the host by way of latent infection (1-3). The persistence of HCMV is asymptomatic in immunocompetent individuals and is characterized by states of subclinical reactivation from latency and low-level virus shedding (1). However, in immunocompromised hosts, HCMV causes significant morbidity and mortality. Of particular concern are stem cell and solid-organ transplant patients, HIV/AIDS patients, and cancer patients undergoing chemotherapy or radiation regimens (4, 5). Additionally, HCMV is the leading cause of infectious disease-related birth defects in the United States, affecting 1 in 150 children born in the United States and most commonly resulting in mild to severe hearing loss (6). While CMV infection of seronegative women during pregnancy poses the most significant risk for severe sequelae in infants (microcephaly, cerebral palsy, and severe hearing loss or cognitive deficits), as many as 75% of congenital infections occur in infants whose mothers were seropositive at the time of conception, indicating that these infections result from reinfection or reactivation (7). Finally, the persistence of HCMV is increasingly associated with age-related pathologies, even when overt clinical symptoms are absent. Agerelated pathologies include vascular disease, immune dysfunction, and frailty (8-13)...

Inactivation of the Human Cytomegalovirus US20 Gene Hampers Productive Viral Replication in Endothelial Cells

“…So far, however, little is known about the functions exerted by viral tropism factors that act at a postentry stage and regulate, in a cell type-specific manner, subsequent stages of the virus replication cycle. In addition to the HCMV US20 and MCMV M45 (47) proteins, two other HCMV proteins were recently reported to contribute to HCMV tropism in endothelial cells by regulating post-immediate-early phases in the viral replication cycle (48,49,50). In these studies, the UL135 and UL136 proteins encoded within the UL133-to-UL138 locus of a clinical strain of the virus (48) were found to be required for efficient HCMV replication in primary endothelial cells (50).…”

“…In these studies, the UL135 and UL136 proteins encoded within the UL133-to-UL138 locus of a clinical strain of the virus (48) were found to be required for efficient HCMV replication in primary endothelial cells (50). The genes encoding those proteins were demonstrated to be dispensable for virus entry and normal expression of representative IE, E, and L proteins (49,50). However, the failure of UL135-and UL136-deficient viruses to produce infectious progeny in infected endothelial cells was related to defects in different stages of the final maturation of the virion, such as formation of the cVAC, the secondary envelopment, and egress (50).…”

“…The genes encoding those proteins were demonstrated to be dispensable for virus entry and normal expression of representative IE, E, and L proteins (49,50). However, the failure of UL135-and UL136-deficient viruses to produce infectious progeny in infected endothelial cells was related to defects in different stages of the final maturation of the virion, such as formation of the cVAC, the secondary envelopment, and egress (50). It was thus concluded that UL135 and UL136 function as determinants of viral tropism for endothelial cells by regulating the proper host membrane trafficking pathway required for an efficient viral maturation process in this specific cell type (50).…”

“…In addition to the HCMV US20 and MCMV M45 (47) proteins, two other HCMV proteins were recently reported to contribute to HCMV tropism in endothelial cells by regulating post-immediate-early phases in the viral replication cycle (48,49,50). In these studies, the UL135 and UL136 proteins encoded within the UL133-to-UL138 locus of a clinical strain of the virus (48) were found to be required for efficient HCMV replication in primary endothelial cells (50). The genes encoding those proteins were demonstrated to be dispensable for virus entry and normal expression of representative IE, E, and L proteins (49,50).…”

The pentameric complex of human Cytomegalovirus: cell tropism, virus dissemination, immune response and vaccine development

“…Firstly, HCMV UL135 and UL136 genes have been shown [66] to be indispensable for HCMV replication in ECs (post-entry tropism). Secondly, UL148, encoding an endoplasmic reticulum-resident glycoprotein, may regulate virion incorporation of gH/gL/gO and PC, thus modulating HCMV tropism [62].…”