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The UL13 and US3 Protein Kinases of Herpes Simplex Virus 1 Cooperate to Promote the Assembly and Release of Mature, Infectious Virions.

Gershburg S, Geltz J, Peterson KE, Halford WP, Gershburg E - PLoS ONE (2015)

Bottom Line: Loss of US3 function alone had largely negligible effect on viral DNA accumulation, gene expression, virion release, and spread.Loss of UL13 function alone also had no appreciable effects on viral DNA levels.These data show that the UL13 kinase plays an important role in the late phase of HSV-1 infection, likely by affecting virion assembly and/or release.

View Article: PubMed Central - PubMed

Affiliation: Department of Medical Microbiology, Immunology and Cell Biology, Southern Illinois University School of Medicine, Springfield, IL 62794-9626, United States of America.

ABSTRACT
Herpes simplex virus type 1 (HSV-1) encodes two bona fide serine/threonine protein kinases, the US3 and UL13 gene products. HSV-1 ΔUS3 mutants replicate with wild-type efficiency in cultured cells, and HSV-1 ΔUL13 mutants exhibit <10-fold reduction in infectious viral titers. Given these modest phenotypes, it remains unclear how the US3 and UL13 protein kinases contribute to HSV-1 replication. In the current study, we designed a panel of HSV-1 mutants, in which portions of UL13 and US3 genes were replaced by expression cassettes encoding mCherry protein or green fluorescent protein (GFP), respectively, and analyzed DNA replication, protein expression, and spread of these mutants in several cell types. Loss of US3 function alone had largely negligible effect on viral DNA accumulation, gene expression, virion release, and spread. Loss of UL13 function alone also had no appreciable effects on viral DNA levels. However, loss of UL13 function did result in a measurable decrease in the steady-state levels of two viral glycoproteins (gC and gD), release of total and infectious virions, and viral spread. Disruption of both genes did not affect the accumulation of viral DNA, but resulted in further reduction in gC and gD steady-state levels, and attenuation of viral spread and infectious virion release. These data show that the UL13 kinase plays an important role in the late phase of HSV-1 infection, likely by affecting virion assembly and/or release. Moreover, the data suggest that the combined activities of the US3 and UL13 protein kinases are critical to the efficient assembly and release of infectious virions from HSV-1-infected cells.

No MeSH data available.


Related in: MedlinePlus

The yields of extracellular virus are significantly reduced in Vero cells infected with HSV-1 ΔUL13 mutants.(A) Vero cells were inoculated with wild type HSV-1 KOS or HSV-1 kinase mutants at MOIs of 2.5 pfu per cell. Viral yields were determined by plaque assay in culture supernatants (extracellular) and cell lysates (intracellular) at 1, 3, 6, 9, 12, 15, 18, and 24 hours post-inoculation. (B) Vero cells were inoculated with wild type HSV-1 KOS or HSV-1 kinase mutants at MOIs of 2.5 pfu per cell. Viral yields were determined by plaque assay in culture supernatants (extracellular) collected at 36 hours post-inoculation. (C) Vero and UL13+- cells were inoculated with the HSV-1 ΔUL13/ΔUS3 mutant at an MOI of 0.1 pfu per cell. Viral yields were determined by plaque assay in culture supernatants (extracellular). For all panels, results are presented as the mean virus yield ± sem (n = 6; ***—p<0.001).
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pone.0131420.g005: The yields of extracellular virus are significantly reduced in Vero cells infected with HSV-1 ΔUL13 mutants.(A) Vero cells were inoculated with wild type HSV-1 KOS or HSV-1 kinase mutants at MOIs of 2.5 pfu per cell. Viral yields were determined by plaque assay in culture supernatants (extracellular) and cell lysates (intracellular) at 1, 3, 6, 9, 12, 15, 18, and 24 hours post-inoculation. (B) Vero cells were inoculated with wild type HSV-1 KOS or HSV-1 kinase mutants at MOIs of 2.5 pfu per cell. Viral yields were determined by plaque assay in culture supernatants (extracellular) collected at 36 hours post-inoculation. (C) Vero and UL13+- cells were inoculated with the HSV-1 ΔUL13/ΔUS3 mutant at an MOI of 0.1 pfu per cell. Viral yields were determined by plaque assay in culture supernatants (extracellular). For all panels, results are presented as the mean virus yield ± sem (n = 6; ***—p<0.001).

Mentions: To examine whether the activity of UL13 and US3 kinases contribute to virion morphogenesis or release of infectious virions, titers of intracellular and extracellular infectious virions were compared in Vero cells. The cells were inoculated with 2.5 pfu per cell of HSV-1 KOS, or HSV-1 ΔUL13/ΔUS3, ΔUS3, and ΔUL13 mutants. The titers of intracellular infectious virus in cell monolayers and extracellular infectious virus in culture supernatants were measured at 1, 3, 6, 9, 12, 15, 18, and 24 hours post-inoculation by plaque assay (Fig 5A). Loss of US3 kinase alone (ΔUS3) had only a negligible effect (2-fold decrease) on titers of the intracellular and extracellular infectious virions relative to the wild-type HSV-1 KOS (Fig 5A). Loss of UL13 kinase alone (ΔUL13) yielded an 8-fold decrease in titers of the intracellular virus relative to KOS by 24 hours post-inoculation, but decreased the titers of the extracellular virus by 20-fold relative to KOS (Fig 5A). Moreover, loss of both US3 and UL13 kinases (ΔUL13/ΔUS3) yielded a 13-fold decrease in titers of the intracellular virus relative to KOS and a 72-fold decrease in titers of the extracellular virus relative to KOS (Fig 5A). The titers of the ΔUL13 mutant viruses started to lag between 9 hours post-inoculation for the intracellular virions to 12 hours post-inoculation for the extracellular virions (Fig 5A). The titers of the extracellular infectious virions decrease even more severely when Vero cells were inoculated with a low MOI of 0.1 pfu per cell. Measured at 36 hours post-inoculation, the titers of the ΔUL13 mutants decreased 27-fold (for the HSV-1 ΔUL13 mutant) and 171-fold (for the HSV-1 ΔUL13/ΔUS3 mutant) relative to the wild-type HSV-1 KOS (Fig 5B).


The UL13 and US3 Protein Kinases of Herpes Simplex Virus 1 Cooperate to Promote the Assembly and Release of Mature, Infectious Virions.

Gershburg S, Geltz J, Peterson KE, Halford WP, Gershburg E - PLoS ONE (2015)

The yields of extracellular virus are significantly reduced in Vero cells infected with HSV-1 ΔUL13 mutants.(A) Vero cells were inoculated with wild type HSV-1 KOS or HSV-1 kinase mutants at MOIs of 2.5 pfu per cell. Viral yields were determined by plaque assay in culture supernatants (extracellular) and cell lysates (intracellular) at 1, 3, 6, 9, 12, 15, 18, and 24 hours post-inoculation. (B) Vero cells were inoculated with wild type HSV-1 KOS or HSV-1 kinase mutants at MOIs of 2.5 pfu per cell. Viral yields were determined by plaque assay in culture supernatants (extracellular) collected at 36 hours post-inoculation. (C) Vero and UL13+- cells were inoculated with the HSV-1 ΔUL13/ΔUS3 mutant at an MOI of 0.1 pfu per cell. Viral yields were determined by plaque assay in culture supernatants (extracellular). For all panels, results are presented as the mean virus yield ± sem (n = 6; ***—p<0.001).
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pone.0131420.g005: The yields of extracellular virus are significantly reduced in Vero cells infected with HSV-1 ΔUL13 mutants.(A) Vero cells were inoculated with wild type HSV-1 KOS or HSV-1 kinase mutants at MOIs of 2.5 pfu per cell. Viral yields were determined by plaque assay in culture supernatants (extracellular) and cell lysates (intracellular) at 1, 3, 6, 9, 12, 15, 18, and 24 hours post-inoculation. (B) Vero cells were inoculated with wild type HSV-1 KOS or HSV-1 kinase mutants at MOIs of 2.5 pfu per cell. Viral yields were determined by plaque assay in culture supernatants (extracellular) collected at 36 hours post-inoculation. (C) Vero and UL13+- cells were inoculated with the HSV-1 ΔUL13/ΔUS3 mutant at an MOI of 0.1 pfu per cell. Viral yields were determined by plaque assay in culture supernatants (extracellular). For all panels, results are presented as the mean virus yield ± sem (n = 6; ***—p<0.001).
Mentions: To examine whether the activity of UL13 and US3 kinases contribute to virion morphogenesis or release of infectious virions, titers of intracellular and extracellular infectious virions were compared in Vero cells. The cells were inoculated with 2.5 pfu per cell of HSV-1 KOS, or HSV-1 ΔUL13/ΔUS3, ΔUS3, and ΔUL13 mutants. The titers of intracellular infectious virus in cell monolayers and extracellular infectious virus in culture supernatants were measured at 1, 3, 6, 9, 12, 15, 18, and 24 hours post-inoculation by plaque assay (Fig 5A). Loss of US3 kinase alone (ΔUS3) had only a negligible effect (2-fold decrease) on titers of the intracellular and extracellular infectious virions relative to the wild-type HSV-1 KOS (Fig 5A). Loss of UL13 kinase alone (ΔUL13) yielded an 8-fold decrease in titers of the intracellular virus relative to KOS by 24 hours post-inoculation, but decreased the titers of the extracellular virus by 20-fold relative to KOS (Fig 5A). Moreover, loss of both US3 and UL13 kinases (ΔUL13/ΔUS3) yielded a 13-fold decrease in titers of the intracellular virus relative to KOS and a 72-fold decrease in titers of the extracellular virus relative to KOS (Fig 5A). The titers of the ΔUL13 mutant viruses started to lag between 9 hours post-inoculation for the intracellular virions to 12 hours post-inoculation for the extracellular virions (Fig 5A). The titers of the extracellular infectious virions decrease even more severely when Vero cells were inoculated with a low MOI of 0.1 pfu per cell. Measured at 36 hours post-inoculation, the titers of the ΔUL13 mutants decreased 27-fold (for the HSV-1 ΔUL13 mutant) and 171-fold (for the HSV-1 ΔUL13/ΔUS3 mutant) relative to the wild-type HSV-1 KOS (Fig 5B).

Bottom Line: Loss of US3 function alone had largely negligible effect on viral DNA accumulation, gene expression, virion release, and spread.Loss of UL13 function alone also had no appreciable effects on viral DNA levels.These data show that the UL13 kinase plays an important role in the late phase of HSV-1 infection, likely by affecting virion assembly and/or release.

View Article: PubMed Central - PubMed

Affiliation: Department of Medical Microbiology, Immunology and Cell Biology, Southern Illinois University School of Medicine, Springfield, IL 62794-9626, United States of America.

ABSTRACT
Herpes simplex virus type 1 (HSV-1) encodes two bona fide serine/threonine protein kinases, the US3 and UL13 gene products. HSV-1 ΔUS3 mutants replicate with wild-type efficiency in cultured cells, and HSV-1 ΔUL13 mutants exhibit <10-fold reduction in infectious viral titers. Given these modest phenotypes, it remains unclear how the US3 and UL13 protein kinases contribute to HSV-1 replication. In the current study, we designed a panel of HSV-1 mutants, in which portions of UL13 and US3 genes were replaced by expression cassettes encoding mCherry protein or green fluorescent protein (GFP), respectively, and analyzed DNA replication, protein expression, and spread of these mutants in several cell types. Loss of US3 function alone had largely negligible effect on viral DNA accumulation, gene expression, virion release, and spread. Loss of UL13 function alone also had no appreciable effects on viral DNA levels. However, loss of UL13 function did result in a measurable decrease in the steady-state levels of two viral glycoproteins (gC and gD), release of total and infectious virions, and viral spread. Disruption of both genes did not affect the accumulation of viral DNA, but resulted in further reduction in gC and gD steady-state levels, and attenuation of viral spread and infectious virion release. These data show that the UL13 kinase plays an important role in the late phase of HSV-1 infection, likely by affecting virion assembly and/or release. Moreover, the data suggest that the combined activities of the US3 and UL13 protein kinases are critical to the efficient assembly and release of infectious virions from HSV-1-infected cells.

No MeSH data available.


Related in: MedlinePlus