Cell Type-Specific Biogenesis of Novel Vesicles Containing Viral Products in Human Cytomegalovirus Infection

Abstract

Human cytomegalovirus (HCMV) is a herpesvirus that, like all herpesvirus, establishes a lifelong infection. HCMV remains a significant cause of morbidity and mortality in immunocompromised individuals and HCMV seropositivity is associated with age-related pathology.

ABSTRACT

Human cytomegalovirus (HCMV), while highly restricted for the human species, infects a diverse array of cell types in the host. Patterns of infection are dictated by the cell type infected, but cell type-specific factors and how they impact tropism for specific cell types is poorly understood. Previous studies in primary endothelial cells showed that HCMV infection induces large multivesicular-like bodies (MVBs) that incorporate viral products, including dense bodies (DBs) and virions. Here, we define the nature of these large vesicles using a recombinant virus where UL32, encoding the pp150 tegument protein, is fused in frame with green fluorescent protein (GFP, TB40/E-UL32-GFP). In fibroblasts, UL32-GFP-positive vesicles were marked with classical markers of MVBs, including CD63 and lysobisphosphatidic acid (LBPA), both classical MVB markers, as well as clathrin and LAMP1. Unexpectedly, UL32-GFP-positive vesicles in primary human microvascular endothelial cells (HMVECs) were not labeled by CD63, and LBPA was completely lost from infected cells. We defined these UL32-positive vesicles in endothelial cells using markers for the cis -Golgi (GM130), the lysosome (LAMP1), and for autophagy (LC3B). These findings suggest that UL32-GFP-containing MVBs in fibroblasts are derived from the canonical endocytic pathway and take over the classical exosomal release pathway. In contrast, UL32-GFP-containing MVBs in HMVECs are derived from the early biosynthetic pathway and exploit a less-well-characterized early Golgi-LAMP1-associated noncanonical secretory autophagy pathway. These results reveal striking cell type-specific membrane trafficking differences in host pathways that are exploited by HCMV, which may reflect distinct pathways for virus egress.

IMPORTANCE Human cytomegalovirus (HCMV) is a herpesvirus that, like all herpesvirus, establishes a lifelong infection. HCMV remains a significant cause of morbidity and mortality in immunocompromised individuals and HCMV seropositivity is associated with age-related pathology. HCMV infects many cells in the human host and the biology underlying the different patterns of infection in different cell types is poorly understood. Endothelial cells are an important target of infection that contribute to hematogenous spread of the virus to tissues. Here, we define striking differences in the biogenesis of large vesicles that incorporate virions in fibroblasts and endothelial cells. In fibroblasts, HCMV is incorporated into canonical MVBs derived from an endocytic pathway, whereas HCMV matures through vesicles derived from the biosynthetic pathway in endothelial cells. This work defines basic biological differences between these cell types that may impact how progeny virus is trafficked out of infected cells.