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

Abstract

The human pathogen Herpes Simplex Virus 1 (HSV-1) produces a lifelong infection in the majority of the world's 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 in clusters at the corners, edges, and cell-cell contacts. We show that this clustering effect is the result of individual particles undergoing exocytosis at preferential sites. We also show that the viral membrane proteins gE, gI, and US9, which have important functions in intracellular transport in neurons, have a subtle effect on 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.

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

“…To visualize HSV-1 exocytosis, we infected Vero cells with HSV-1 IH01, which expresses gM-pHluorin. We previously showed that gM-pHluorin is incorporated into virus particles, and exhibits pH-dependent green fluorescence [19]. In live cells, the lumen of secretory vesicles is acidic (pH of 5.2-5.7) [31, 32], which quenches the green fluorescence of gM-pHluorin (Figure 5A).…”

“…HSV-1 Viruses. HSV-1 IH01 was constructed and propagated as previously described [19]. HSV-1 OK14 was obtained from Lynn Enquist (Princeton University) [17] from whole-genome sequenced archival stocks.…”

“…Previously, we constructed a recombinant virus, HSV-1 IH01 expressing a pH-sensitive variant of GFP fused to the extravirion loop of glycoprotein M (gM-pHluorin) [19]. In fixed and immunostained cells, cellular pH equilibrates to that of the extracellular buffer, so gM-pHluorin exhibits consistent green fluorescence.…”

“…In live cells, the lumen of secretory vesicles is acidic (pH of 5.2-5.7) [31,32], which quenches the green fluorescence of gM-pHluorin (Figure 5A). At the moment of exocytosis, the pHluorin moiety is exposed to the extracellular pH and its fluorescence is dequenched [31,11,12,19], allowing us to identify virus particle exocytosis events using TIRF microscopy (Figure 5A). Vero cells were transduced with a non-replicating adenovirus vector expressing mCherry-Rab6a, coinfected with HSV-1 IH01, and imaged by live-cell TIRF microscopy beginning at 6 hpi.…”

“…In cells that were positive for both HSV-1 gM-pHluorin and mCherry-Rab6a, both proteins accumulated in the same locations in the cell periphery (Figure 5, Cell 3, and Figure 5C). 2 and 4), and clusters of colocalized gM-pHluorin and mRFP-VP26 capsids [19], a subset of these clustered particles represent complete, mature virions.…”