Nicholas T. Streck, Jillian Carmichael, Nicholas J. Buchkovich

Secondary envelopment of human cytomegalovirus (HCMV) occurs through a mechanism that is poorly understood. Many enveloped viruses utilize the endosomal sorting complexes required for transport (ESCRTs) for viral budding and envelopment. Although there are conflicting reports on the role of the ESCRT AAA-ATPase protein VPS4 in an HCMV infection, VPS4 may act in an envelopment role similar to its function during other viral infections. Because VPS4 is normally recruited by the ESCRT-III complex, we hypothesized that ESCRT-III subunits would also be required for HCMV infection. We investigated the role of ESCRT-III, the core ESCRT scission complex, during the late stages of infection. We show that inducible expression of dominant-negative ESCRT-III subunits during infection blocks endogenous ESCRT function, but does not inhibit virus production. We also show that HCMV forms enveloped intracellular and extracellular virions in the presence of dominant-negative ESCRT-III subunits, suggesting that ESCRT-III is not involved in the envelopment of HCMV. We also found that similar to ESCRT-III, inducible expression of a dominant-negative form of VPS4A did not inhibit the envelopment of virions or reduce virus titers. Thus, HCMV does not require the ESCRTs for secondary envelopment. However, we found that ESCRT-III subunits are required for efficient virus spread. This suggests a role for ESCRT-III during the spread of HCMV that is independent of viral envelopment.IMPORTANCEHuman cytomegalovirus (HCMV) is a prevalent opportunistic pathogen in the human population. HCMV infection in neonatal and immunocompromised patients can cause severe and possibly life-threatening complications in these at-risk patients. It is important to define mechanisms of the viral replication cycle to identify potential targets for new therapies. Secondary envelopment, or acquisition of the membrane envelope, of HCMV is a mechanism that needs further study. Using an inducible fibroblast system to carefully control for the toxicity associated with blocking ESCRT-III function, this study determines that the ESCRT proteins are not required for viral envelopment. However, the study does discover a non-envelopment role for the ESCRT-III complex in the efficient spread of the virus. Thus, this study advances our understanding of an important process essential for the replication of HCMV.