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

Abstract

Human cytomegalovirus (HCMV), while highly restricted for the human species, infects an 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 the 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), lysosome (LAMP1), and autophagy (LC3B). These findings suggest that UL32-GFP containing MVBs in fibroblasts are derived from the canonical endocytic pathway and takeover classical exosomal release pathway. However, UL32-GFP containing MVBs in HMVECs are derived from the early biosynthetic pathway and exploit a less characterized early Golgi-LAMP1-associated non- canonical 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, that establishes a life-long infection. HCMV remains a significant cause of morbidity and mortality in the immunocompromised 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 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.

Intermittent Bulk Release of Human Cytomegalovirus

“…It is plausible to hypothesize that these virus populations might undergo different envelopment processes in the cell and are exocytosed with a different spatiotemporal profile (Scrivano et al, 2011). While this manuscript was in preparation, a study from the Wilson and Goodrum lab suggested that virus-containing MVBs in fibroblasts and endothelial cells are derived from different cellular membranes, which would add another potential HCMV egress pathway that could result in different virus populations; however, it is unclear if these pathways are functional in egress (Momtaz et al, 2021). Future work needs to focus on characterizing the particle populations that are exocytosed by these pathways with regards to their glycoprotein content and define their role in potentially divergent egress routes.…”

“…While the previous literature often called these virus-containing multivesicular structures "MVBs", we decided to dub them multi-viral bodies (MViB) as we could not untangle their descent clearly. A recent study from the Wilson and Goodrum labs suggested that virus-containing structures in HCMV fibroblasts and endothelial cells are derived from membranes of different cellular origins [26]. Importantly, a functional role for these "virus-containing MVBs" or "MViBs" in egress has lacked so far [14,[23][24][25].…”

“…It is plausible to hypothesize that these virus populations might undergo different envelopment processes in the cell and are exocytosed with a different spatio-temporal profile [4]. A recent study from the Wilson and Goodrum labs suggests that virus-containing MVBs in fibroblasts and endothelial cells are derived from different cellular membranes, which would add another potential HCMV egress pathway that could result in different virus populations; however, it is unclear if these pathways are functional in egress [26].…”

“…These large multivesicular structures, containing HCMV progeny, have been called endosomal cisternae 3 or multivesicular bodies (MVBs) in the literature, even though conclusive evidence regarding their biogenesis has been lacking [14,[23][24][25]. A recent study from the Wilson and Goodrum labs suggested that virus-containing MVBs in HCMV fibroblasts and endothelial cells are derived from membranes of different cellular origins [26].…”

The unconventional way out—Egress of HCMV through multiviral bodies

“…Although current data (Read et al, 2019;Schauflinger et al, 2013;Shaga Devan et al, 2021;Taisne et al, 2019) have mainly described that HCMV capsids individually bud into small vesicles in the AC (Figure 1a,b), several studies have described large vesicles filled with virus particles (Bughio et al, 2013;Fraile-Ramos et al, 2010;Momtaz et al, 2021;Schauflinger et al, 2011;Severi et al, 1988).…”