Syncytial mutations in the herpes simplex virus type 1 gK (UL53) gene occur in two distinct domains

The initial goal of this study was to reexamine the requirement of UL21 for herpes simplex virus 1 (HSV-1) replication. Previous studies suggested that UL21 is dispensable for replication in cell cultures, but a recent report on HSV-2 challenges those findings. As was done for the HSV-2 study, a UL21-null virus was made and propagated on complementing cells to discourage selection of compensating mutations. This HSV-1 mutant was able to replicate in noncomplementing cells, even at a low multiplicity of infection (MOI), though a reduction in titer was observed. Also, increased proportions of empty capsids were observed in the cytoplasm, suggesting a role for UL21 in preventing their exit from the nucleus. Surprisingly, passage of the null mutant resulted in rapid outgrowth of syncytial (Syn) variants. This was unexpected because UL21 has been shown to be required for the Syn phenotype. However, earlier experiments made use of only the A855V syncytial mutant of glycoprotein B (gB), and the Syn phenotype can also be produced by substitutions in glycoprotein K (gK), UL20, and UL24. Sequencing of the syncytial variants revealed mutations in the gK locus, but UL21 was shown to be dispensable for UL20 and UL24 To test whether UL21 is needed only for the A855V mutant, additional gB derivatives were examined in the context of the null virus, and all produced lytic rather than syncytial sites of infection. Thus, UL21 is required only for the gB phenotype. This is the first example of a differential requirement for a viral protein across the four loci. UL21 is conserved among alphaherpesviruses, but its role is poorly understood. This study shows that HSV-1 can replicate without UL21, although the virus titers are greatly reduced. The null virus had greater proportions of empty (DNA-less) capsids in the cytoplasm of infected cells, suggesting that UL21 may play a role in retaining them in the nucleus. This is consistent with reports showing UL21 to be capsid associated and localized to the nuclei of infected cells. UL21 also appears to be needed for viral membrane activities. It was found to be required for virus-mediated cell fusion, but only for mutants that harbor syncytial mutations in gB (not variants of gK, UL20, or UL24). The machinery needed for syncytial formation is similar to that needed for direct spread of the virus through cell junctions, and these studies show that UL21 is required for cell-to-cell spread even in the absence of syncytial mutations.

Section: Figmentioning

confidence: 53%

The UL21 Tegument Protein of Herpes Simplex Virus 1 Is Differentially Required for the Syncytial Phenotype

Sarfo

Starkey

Mellinger

et al. 2017

“…Furthermore, it was not possible to detect this truncated gK in F-gK␤-infected cells (28). Interestingly, this 112-amino-acid sequence contains the Syn1 locus that demarcates the putative fusion domain of gK (24).…”

Section: Discussionmentioning

confidence: 96%

“…Syncytial mutations that cause extensive virus-induced cell fusion can arise in at least four different regions of the viral genome, including the UL20 gene (6,35); the UL24 gene (30,49); the UL27 gene, encoding glycoprotein B (gB) (13,41); and the UL53 gene, coding for glycoprotein K (gK) (8,22,42,48). Syncytial mutations in UL53 (gK) gene are more frequently isolated than syncytial mutations in any other genes (8,9,22,24,(42)(43)(44)48). Studies with anti-gK serum raised against peptides that represented immunogenic portions of gK detected the glycoprotein in the nuclear and perinuclear space, but not in the plasma membrane, of infected cells.…”

mentioning

confidence: 99%

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

Jayachandra

Baghian

Kousoulas

1997

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.

“…7). Characteristically, this model predicts that all syncytial mutations are on the external portion of gK located within either domain I or domain III, suggesting that these domains may cooperate in virus-induced cell fusion (11,30).…”

Section: Discussionmentioning

confidence: 98%

Genetic Analysis of the Role of Herpes Simplex Virus Type 1 Glycoprotein K in Infectious Virus Production and Egress

Foster

Kousoulas

1999

Herpes simplex virus type 1 (KOS)ΔgK is a mutant virus which lacks glycoprotein K (gK) and exhibits defects in virion egress (S. Jayachandra, A. Baghian, and K. G. Kousoulas, J. Virol. 69:5401–5413, 1997). To further understand the role of gK in virus egress, we constructed recombinant viruses, ΔgKhpd-1, -2, -3, and -4, that specified gK amino-terminal portions of 139, 239, 268, and 326 amino acids, respectively, corresponding to truncations immediately after each of the four putative membrane-spanning domains of gK. ΔgKhpd-1 and ΔgKhpd-2 viruses produced lower yields and smaller plaques than ΔgK. Numerous ΔgKhpd-1 capsids accumulated predominately within large double-membrane vesicles of which the inner membrane appeared to be derived from viral envelopes while the outer membrane appeared to originate from the outer nuclear membrane. The mutant virus ΔgKhpd-3 produced higher yields and larger plaques than the ΔgK virus. The mutant virus ΔgKhpd-4 produced yields and plaques similar to those of the wild-type virus strain KOS, indicating that deletion of the carboxy-terminal 12 amino acids did not adversely affect virus replication and egress. Comparisons of the gK primary sequences specified by alphaherpesviruses revealed the presence of a cysteine-rich motif (CXXCC), located within domain III in the lumen side of gK, and a tyrosine-based motif, YTKΦ (where Φ is any bulky hydrophobic amino acid), located between the second and third hydrophobic domains (domain II) in the cytoplasmic side of gK. The mutant virus gK/Y183S, which was constructed to specify gK with a single-amino-acid change (Y to S) within the YTKΦ motif, replicated less efficiently than the ΔgK virus. The mutant virus gK/C304S-C307S, which was constructed to specify two serine instead of cysteine residues within the cysteine-rich motif (CXXCC changed to SXXSC) of gK domain III, replicated more efficiently than the ΔgK virus. Our data suggests that gK contains domains in its amino-terminal portion that promote aberrant nucleocapsid envelopment and/or membrane fusion between different virion envelopes and contains domains within its domains II and III that function in virus replication and egress.