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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

Viral DNA accumulation during the infection with HSV-1 KOS or HSV-1 kinase mutants.(A) Vero cells were inoculated with wild type HSV-1 KOS or HSV-1 ΔUS3, ΔUL13, and ΔUL13/ΔUS3 mutants at an MOI of 2.5 pfu per cell. At indicated time points, lysates were prepared and blotted onto a nylon membrane. The accumulation of viral DNA was assayed by hybridization to a 32P-labeled US6-specific oligonucleotide probe. ACV-treated KOS served as a negative control. (B) Relative intensity of hybridization signals was quantified by phosphorimager analysis. Results are shown as fold change in hybridization signal intensity relative to the background ± standard deviation (SD) of two duplicate infections.
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pone.0131420.g003: Viral DNA accumulation during the infection with HSV-1 KOS or HSV-1 kinase mutants.(A) Vero cells were inoculated with wild type HSV-1 KOS or HSV-1 ΔUS3, ΔUL13, and ΔUL13/ΔUS3 mutants at an MOI of 2.5 pfu per cell. At indicated time points, lysates were prepared and blotted onto a nylon membrane. The accumulation of viral DNA was assayed by hybridization to a 32P-labeled US6-specific oligonucleotide probe. ACV-treated KOS served as a negative control. (B) Relative intensity of hybridization signals was quantified by phosphorimager analysis. Results are shown as fold change in hybridization signal intensity relative to the background ± standard deviation (SD) of two duplicate infections.

Mentions: To differentiate between these two possibilities, HSV-1 DNA accumulation was compared in Vero cells inoculated with 2.5 pfu per cell of either HSV-1 KOS, or HSV-1 ΔUL13, ΔUS3, and ΔUL13/ΔUS3 mutants. Uninfected (UI) Vero cells were included as a negative control, and an additional group of Vero cells were inoculated with HSV-1 KOS in the presence of 300 μM acyclovir (ACV) served as a positive-control for inhibition of viral DNA synthesis. DNA samples were isolated at 3-hour intervals between 9 and 24 hours post inoculation, immobilized on a nylon membrane, and hybridized to an HSV-1-specific probe (Fig 3A). PhosphorImager analysis of the dot-blot indicated that viral DNA yields and the kinetics of viral DNA accumulation were similar in cells inoculated with HSV-1 KOS, or HSV-1 ΔUL13 and ΔUL13/ΔUS3 mutants (Fig 3A). In cells inoculated with HSV-1 ΔUS3 mutant, viral DNA accumulated to ~ 6-fold higher levels at 9 hours post-inoculation, but was similar to other tested viruses at later time points. Viral DNA accumulation in cells inoculated with all tested viruses peaked between 18 and 24 hours post-inoculation and reached levels that were 170 to 260-fold higher than uninfected Vero cells (Fig 3B). In contrast, HSV-1 KOS-infected cells treated with ACV exhibited levels of viral DNA accumulation that did not significantly differ from the uninfected Vero cells (Fig 3B).


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)

Viral DNA accumulation during the infection with HSV-1 KOS or HSV-1 kinase mutants.(A) Vero cells were inoculated with wild type HSV-1 KOS or HSV-1 ΔUS3, ΔUL13, and ΔUL13/ΔUS3 mutants at an MOI of 2.5 pfu per cell. At indicated time points, lysates were prepared and blotted onto a nylon membrane. The accumulation of viral DNA was assayed by hybridization to a 32P-labeled US6-specific oligonucleotide probe. ACV-treated KOS served as a negative control. (B) Relative intensity of hybridization signals was quantified by phosphorimager analysis. Results are shown as fold change in hybridization signal intensity relative to the background ± standard deviation (SD) of two duplicate infections.
© Copyright Policy
Related In: Results  -  Collection

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

pone.0131420.g003: Viral DNA accumulation during the infection with HSV-1 KOS or HSV-1 kinase mutants.(A) Vero cells were inoculated with wild type HSV-1 KOS or HSV-1 ΔUS3, ΔUL13, and ΔUL13/ΔUS3 mutants at an MOI of 2.5 pfu per cell. At indicated time points, lysates were prepared and blotted onto a nylon membrane. The accumulation of viral DNA was assayed by hybridization to a 32P-labeled US6-specific oligonucleotide probe. ACV-treated KOS served as a negative control. (B) Relative intensity of hybridization signals was quantified by phosphorimager analysis. Results are shown as fold change in hybridization signal intensity relative to the background ± standard deviation (SD) of two duplicate infections.
Mentions: To differentiate between these two possibilities, HSV-1 DNA accumulation was compared in Vero cells inoculated with 2.5 pfu per cell of either HSV-1 KOS, or HSV-1 ΔUL13, ΔUS3, and ΔUL13/ΔUS3 mutants. Uninfected (UI) Vero cells were included as a negative control, and an additional group of Vero cells were inoculated with HSV-1 KOS in the presence of 300 μM acyclovir (ACV) served as a positive-control for inhibition of viral DNA synthesis. DNA samples were isolated at 3-hour intervals between 9 and 24 hours post inoculation, immobilized on a nylon membrane, and hybridized to an HSV-1-specific probe (Fig 3A). PhosphorImager analysis of the dot-blot indicated that viral DNA yields and the kinetics of viral DNA accumulation were similar in cells inoculated with HSV-1 KOS, or HSV-1 ΔUL13 and ΔUL13/ΔUS3 mutants (Fig 3A). In cells inoculated with HSV-1 ΔUS3 mutant, viral DNA accumulated to ~ 6-fold higher levels at 9 hours post-inoculation, but was similar to other tested viruses at later time points. Viral DNA accumulation in cells inoculated with all tested viruses peaked between 18 and 24 hours post-inoculation and reached levels that were 170 to 260-fold higher than uninfected Vero cells (Fig 3B). In contrast, HSV-1 KOS-infected cells treated with ACV exhibited levels of viral DNA accumulation that did not significantly differ from the uninfected Vero cells (Fig 3B).

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