Herpes simplex virus type 1 glycoprotein K is not essential for infectious virus production in actively replicating cells but is required for efficient envelopment and translocation of infectious virions from the cytoplasm to the extracellular space

Abstract

We characterized the glycoprotein K (gK)-null herpes simplex virus type 1 [HSV-1] (KOS) ⌬gK and compared it to the gK-null virus HSV-1 F-gK␤ (L. Hutchinson et al., J. Virol. 69:5401-5413, 1995). ⌬gK and F-gK␤ mutant viruses produced small plaques on Vero cell monolayers at 48 h postinfection. F-gK␤ caused extensive fusion of 143TK cells that was sensitive to melittin, a specific inhibitor of gK-induced cell fusion, while ⌬gK virus did not fuse 143TK cells. A recombinant plasmid containing the truncated gK gene specified by F-gK␤ failed to rescue the ICP27-null virus KOS (d27-1), while a plasmid with the ⌬gK deletion rescued the d27-1 virus efficiently. ⌬gK virus yield was approximately 100,000-fold lower in stationary cells than in actively replicating Vero cells. The plaquing efficiencies of ⌬gK and F-gK␤ virus stocks on VK302 cells were similar, while the plaquing efficiency of F-gK␤ virus stocks on Vero cells was reduced nearly 10,000-fold in comparison to that of ⌬gK virus. Mutant ⌬gK and F-gK␤ infectious virions accumulated within Vero and HEp-2 cells but failed to translocate to extracellular spaces. ⌬gK capsids accumulated in the nuclei of Vero but not HEp-2 cells. Enveloped ⌬gK virions were visualized in the cytoplasms of both Vero and HEp-2 cells, and viral capsids were found in the cytoplasm of HEp-2 cells within vesicles. Glycoproteins B, C, D, and H were expressed on the surface of ⌬gK-infected Vero cells in amounts similar to those for KOS-infected Vero cells.These results indicate that gK is involved in nucleocapsid envelopment, and more importantly in the translocation of infectious virions from the cytoplasm to the extracellular spaces, and that actively replicating cells can partially compensate for the envelopment but not for the cellular egress deficiency of the ⌬gK virus.

Pseudorabies Virus Glycoprotein K Requires the UL20 Gene Product for Processing

“…Most strikingly, in cells infected with gK-negative PrV, numerous intracellular nucleocapsids were found close to the plasma membrane, and fusion events were observed which might represent entry stages of released virions. Since HSV-1 UL20 and gK-null mutants exhibited different phenotypes on different cells, indicating partial complementation by cellular factors (2,16), we cannot exclude that the differences in phenotypes between HSV-1 and PrV are, at least partially, due to the difference in cell systems. However, from our data, we deduce that UL20 function precedes gK function, which would imply that a UL20-gK double mutant should exhibit essentially a UL20 phenotype.…”

“…Although in both HSV-1 and PrV, the UL20 and gK proteins are involved in virion maturation and egress, there are differences in the phenotypes displayed by the respective mutants. In HSV-1, deletion of UL20 resulted in accumulation of enveloped virions in the perinuclear space (2) and disruption of the Golgi apparatus with concomitant alteration of glycoprotein maturation (1), whereas in the absence of gK, enveloped virions accumulated in the cytoplasm (16). In PrV, deletion of UL20 resulted in accumulation of enveloped virions within intracytoplasmic vesicles (10).…”

“…HSV-1 and PrV gKs have been implicated in virus morphogenesis and egress. In cells infected with HSV-1 F-gK␤, which contains a lacZ expression cassette within the UL53 ORF numerous nonenveloped capsids were found in the cytoplasm (14), whereas in cells infected with a gK-deletion mutant of strain KOS, enveloped virions appeared to accumulate in the cytoplasm (16). These findings pointed to an important role for HSV-1 gK in nucleocapsid envelopment and efficient transport of enveloped virions to the extracellular space.…”

“…Only small plaques or foci of infected cells were observed after infection of noncomplementing cells with gK mutants of HSV-1 and PrV (14,25), and productive viral replication was prevented by deletion of VZV gK (32). Generally, the yield of gK mutants from noncomplementing cells is decreased compared to that of wild-type viruses, although final titers may depend on the cell type and particular virus mutant (14,16).…”

Herpes simplex virus type 1 morphogenesis and virus‐cell interactions: significance of cytoskeleton and methodological aspects

“…The plaque size was used as a parameter of the cell‐to‐cell spread of HSV‐1 (IV), which reflects the virulence. The plaque size is sensitive to the virus strain (79), the passage number of the virus isolate (90) and host cells (IV), the temperature (91), the gK gene and the plaquing cell type (92).…”