Bergeman, M. H. | Search bioRxiv

bioRxiv2023

Herpes Simplex Virus 1 (HSV-1) Uses the Rab6 Post-Golgi Secretory Pathway For Viral Egress

Herpes Simplex Virus 1 (HSV-1) is an alpha herpesvirus that infects a majority of the world population. The mechanisms and cellular host factors involved in the intracellular transport and exocytosis of HSV-1 particles are not fully understood. To elucidate these late steps in the replication cycle, we developed a live-cell fluorescence microscopy assay of HSV-1 virion intracellular trafficking and exocytosis. This method allows us to track individual virus particles, and identify the precise moment and location of particle exocytosis using a pH-sensitive reporter. We show that HSV-1 uses the host Rab6 post-Golgi secretory pathway during egress. The small GTPase, Rab6, binds to nascent secretory vesicles at the trans-Golgi network and regulates vesicle trafficking and exocytosis at the plasma membrane. HSV-1 particles colocalize with Rab6a in the region of the Golgi, cotraffic with Rab6a to the cell periphery, and undergo exocytosis from Rab6a vesicles. Consistent with previous reports, we find that HSV-1 particles accumulate at preferential egress sites in infected cells. The Rab6a secretory pathway mediates this preferential/polarized egress, since Rab6a vesicles accumulate near the plasma membrane similarly in uninfected cells. These data suggest that, following particle envelopment, HSV-1 egress follows a pre-existing cellular secretory pathway to exit infected cells rather than novel, virus-induced mechanisms. IMPORTANCEHSV-1 infects a majority of people. It establishes a life-long latent infection, and occasionally reactivates, typically causing characteristic oral or genital lesions. Rarely in healthy natural hosts, but more commonly in zoonotic infections and in elderly, newborn, or immunocompromised patients, HSV-1 can cause severe herpes encephalitis. The precise cellular mechanisms used by HSV-1 remain an important area of research. In particular, the egress pathways that newly-assembled virus particles use to exit from infected cells are unclear. In this study, we used fluorescence microscopy to visualize individual virus particles exiting from cells, and found that HSV-1 particles use the pre-existing cellular secretory pathway, regulated by the cellular protein, Rab6.

microbiology↗

bioRxiv2023

Live-cell Fluorescence Microscopy of HSV-1 Cellular Egress by Exocytosis

1.1The human pathogen Herpes Simplex Virus 1 (HSV-1) produces a lifelong infection in the majority of the worlds population. While the generalities of alpha herpesvirus assembly and egress pathways are known, the precise molecular and spatiotemporal details remain unclear. In order to study this aspect of HSV-1 infection, we engineered a recombinant HSV-1 strain expressing a pH-sensitive reporter, gM-pHluorin. Using a variety of fluorescent microscopy modalities, we can detect individual virus particles undergoing intracellular transport and exocytosis at the plasma membrane. We show that particles exit from epithelial cells individually, not bulk release of many particles at once, as has been reported for other viruses. In multiple cell types, HSV-1 particles accumulate over time at the cell periphery and cell-cell contacts. We show that this accumulation effect is the result of individual particles undergoing exocytosis at preferential sites and that these egress sites can contribute to cell-cell spread. We also show that the viral membrane proteins gE, gI, and US9, which have important functions in intracellular transport in neurons, are not required for preferential egress and clustering in non-neuronal cells. Importantly, by comparing HSV-1 to a related alpha herpesvirus, pseudorabies virus, we show that this preferential exocytosis and clustering effect is cell type-dependent, not virus dependent. This preferential egress and clustering appears to be the result of the arrangement of the microtubule cytoskeleton, as virus particles co-accumulate at the same cell protrusions as an exogenous plus end-directed kinesin motor. ImportanceAlpha herpesviruses produce lifelong infections of their human and animal hosts. The majority of people in the world are infected with Herpes Simplex Virus 1 (HSV-1), which typically causes recurrent oral or genital lesions. However, HSV-1 can also spread to the central nervous system, causing severe encephalitis, and might also contribute to the development of neurodegenerative diseases. Many of the steps of how these viruses infect and replicate inside host cells are known in depth, but the final step, exiting from the infected cell, is not fully understood. In this study, we engineered a novel variant of HSV-1 that allows us to visualize how individual virus particles exit from infected cells. With this imaging assay, we investigated preferential egress site formation in certain cell types and their contribution to cell-cell spread of HSV-1.

microbiology↗