Limits...
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

HSV-1 ΔUL13 mutants exhibit ‘small-plaque’ phenotype.(A) Vero cells and (C) US3+- and UL13+- cells were inoculated with 300 pfu of HSV-1 KOS or HSV-1 kinase mutants. At 48 (A) and 60 (C) hours post-inoculation, the monolayers were fixed and labeled with FITC-conjugated anti-HSV-1 antibody. Representative plaques are shown at magnification of 100×. (B) and (D) Ten plaques in each infection were photographed, and infected cells were enumerated. Results are presented as the mean number of infected cells per plaque ± sem (n = 10 plaques; ***—p<0.001).
© Copyright Policy
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4482649&req=5

pone.0131420.g002: HSV-1 ΔUL13 mutants exhibit ‘small-plaque’ phenotype.(A) Vero cells and (C) US3+- and UL13+- cells were inoculated with 300 pfu of HSV-1 KOS or HSV-1 kinase mutants. At 48 (A) and 60 (C) hours post-inoculation, the monolayers were fixed and labeled with FITC-conjugated anti-HSV-1 antibody. Representative plaques are shown at magnification of 100×. (B) and (D) Ten plaques in each infection were photographed, and infected cells were enumerated. Results are presented as the mean number of infected cells per plaque ± sem (n = 10 plaques; ***—p<0.001).

Mentions: To formally determine if the HSV-1 ΔUL13/ΔUS3 mutant exhibited greater defect in viral spread relative to the HSV-1 ΔUL13 or ΔUS3 mutants, monolayers of Vero cells were inoculated with 300 pfu of either HSV-1 KOS, or HSV-1 ΔUL13, ΔUS3, and ΔUL13/ΔUS3 mutants and overlaid with medium containing 0.5% methylcellulose. At 48 hours post-inoculation, the monolayers were fixed and immunofluorescently labeled with FITC-conjugated polyclonal HSV-1 antibody. Ten plaques were randomly selected per culture, photographed (Fig 2A), and the number of infected cells per plaque enumerated (Fig 2B). Plaques formed by wild-type HSV-1 KOS contained 202 ± 15 viral antigen-positive cells, and loss of US3 function alone (ΔUS3) did not significantly alter plaque size (232 ± 21) (Fig 2B). Loss of UL13 function alone (ΔUL13) reduced the number of infected cells per plaque by ~50% (106 ± 10) relative to HSV-1 KOS (Fig 2B; p<0.0001). Consistent with our observations during plaque purification, loss of both UL13 and US3 kinases further reduced HSV-1 plaque size to 20% of wild-type HSV-1. Specifically, plaques formed by the HSV-1 ΔUL13/ΔUS3 mutant contained 47 ± 5 viral antigen-positive cells, and this reduction was significant relative to HSV-1 KOS (p<0.0001), the HSV-1 ΔUS3 mutant (p<0.0001), and the HSV-1 ΔUL13 mutant (Fig 2B; p<0.05).


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)

HSV-1 ΔUL13 mutants exhibit ‘small-plaque’ phenotype.(A) Vero cells and (C) US3+- and UL13+- cells were inoculated with 300 pfu of HSV-1 KOS or HSV-1 kinase mutants. At 48 (A) and 60 (C) hours post-inoculation, the monolayers were fixed and labeled with FITC-conjugated anti-HSV-1 antibody. Representative plaques are shown at magnification of 100×. (B) and (D) Ten plaques in each infection were photographed, and infected cells were enumerated. Results are presented as the mean number of infected cells per plaque ± sem (n = 10 plaques; ***—p<0.001).
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4482649&req=5

pone.0131420.g002: HSV-1 ΔUL13 mutants exhibit ‘small-plaque’ phenotype.(A) Vero cells and (C) US3+- and UL13+- cells were inoculated with 300 pfu of HSV-1 KOS or HSV-1 kinase mutants. At 48 (A) and 60 (C) hours post-inoculation, the monolayers were fixed and labeled with FITC-conjugated anti-HSV-1 antibody. Representative plaques are shown at magnification of 100×. (B) and (D) Ten plaques in each infection were photographed, and infected cells were enumerated. Results are presented as the mean number of infected cells per plaque ± sem (n = 10 plaques; ***—p<0.001).
Mentions: To formally determine if the HSV-1 ΔUL13/ΔUS3 mutant exhibited greater defect in viral spread relative to the HSV-1 ΔUL13 or ΔUS3 mutants, monolayers of Vero cells were inoculated with 300 pfu of either HSV-1 KOS, or HSV-1 ΔUL13, ΔUS3, and ΔUL13/ΔUS3 mutants and overlaid with medium containing 0.5% methylcellulose. At 48 hours post-inoculation, the monolayers were fixed and immunofluorescently labeled with FITC-conjugated polyclonal HSV-1 antibody. Ten plaques were randomly selected per culture, photographed (Fig 2A), and the number of infected cells per plaque enumerated (Fig 2B). Plaques formed by wild-type HSV-1 KOS contained 202 ± 15 viral antigen-positive cells, and loss of US3 function alone (ΔUS3) did not significantly alter plaque size (232 ± 21) (Fig 2B). Loss of UL13 function alone (ΔUL13) reduced the number of infected cells per plaque by ~50% (106 ± 10) relative to HSV-1 KOS (Fig 2B; p<0.0001). Consistent with our observations during plaque purification, loss of both UL13 and US3 kinases further reduced HSV-1 plaque size to 20% of wild-type HSV-1. Specifically, plaques formed by the HSV-1 ΔUL13/ΔUS3 mutant contained 47 ± 5 viral antigen-positive cells, and this reduction was significant relative to HSV-1 KOS (p<0.0001), the HSV-1 ΔUS3 mutant (p<0.0001), and the HSV-1 ΔUL13 mutant (Fig 2B; p<0.05).

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