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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

Expression of viral proteins in Vero cells infected with HSV-1 KOS or ΔUS3 and ΔUL13 mutants.(A) Vero cells were inoculated in duplicates as described in Fig 3. At 14 hours post-inoculation, whole-cell lysates were prepared and resolved by SDS-PAGE followed by immunoblot analyses with respective antibodies. Each lane contains an amount of lysate equal to 105 cells. Levels of β-actin served as a loading control. (B) The relative intensity of signals was quantified by densitometry using ImageJ [106]. Results are shown for proteins whose levels of expression changed substantially as percent change in signal intensity relative to the wild type (HSV-1 KOS) (set to 100) ± SD of the duplicate infections.
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pone.0131420.g004: Expression of viral proteins in Vero cells infected with HSV-1 KOS or ΔUS3 and ΔUL13 mutants.(A) Vero cells were inoculated in duplicates as described in Fig 3. At 14 hours post-inoculation, whole-cell lysates were prepared and resolved by SDS-PAGE followed by immunoblot analyses with respective antibodies. Each lane contains an amount of lysate equal to 105 cells. Levels of β-actin served as a loading control. (B) The relative intensity of signals was quantified by densitometry using ImageJ [106]. Results are shown for proteins whose levels of expression changed substantially as percent change in signal intensity relative to the wild type (HSV-1 KOS) (set to 100) ± SD of the duplicate infections.

Mentions: To verify the effects of loss of the US3 or UL13 kinases on early and late stages of HSV-1 replication, expression levels of several viral genes were compared. Vero cells were inoculated with 2.5 pfu per cell of HSV-1 KOS, or HSV-1 kinase mutants. At 14 hours post-inoculation, whole-cell lysates were prepared and subjected to SDS-PAGE and immunoblotting analyses, and expression levels of several HSV-1 proteins were quantified by densitometry. Uninfected (UI) Vero cells were included as a negative control (Fig 4). The steady-state levels of the ICP0 protein (expressed from an immediate-early gene) were nearly identical across all viruses (Fig 4A). Likewise, the steady-state levels of another immediate-early protein, ICP4, were equal in cells inoculated with all tested viruses (not shown). Among the proteins expressed from HSV-1 late genes, the steady-state levels of glycoproteins B, E, and G were not significantly affected by the loss of US3 and UL13 protein kinases. In contrast, the steady-state levels of glycoprotein C (gC) were reduced more significantly: the levels of precursor gC (pr-gC) decreased 2-fold during the infection with the ΔUS3 mutant, 10-fold during the infection with the ΔUL13 mutant, and were essentially undetectable during the infection with the ΔUL13/ΔUS3 mutant (Fig 4B). Consequently, the steady-state levels of mature gC were reduced by 3- and 5-fold for the ΔUL13 and ΔUL13/ΔUS3 mutants, respectively. Likewise, during the infection with the ΔUL13/ΔUS3 mutant, the steady-state levels of glycoprotein D (gD) were reduced such that the levels of precursor gD (pr-gD) were 7-fold lower and mature gD 2-fold lower relative to the HSV-1 KOS (Fig 4B). These results correlate with the observation of a small-plaque phenotype exhibited by the UL13-deficient HSV-1 mutants (Fig 2) and corroborate the viral DNA accumulation data (Fig 3), suggesting that the HSV-1 ΔUL13 mutants are deficient in their ability to spread from one host cell to another and that the activity of viral protein kinases is crucial at the late phase of viral infection. Notably, two of the tested viral proteins, ICP0 and gE, are reported substrates of the UL13 protein kinase [38–40], yet the loss of UL13 kinase had no effect on the steady-state levels of these proteins (Fig 4A).


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)

Expression of viral proteins in Vero cells infected with HSV-1 KOS or ΔUS3 and ΔUL13 mutants.(A) Vero cells were inoculated in duplicates as described in Fig 3. At 14 hours post-inoculation, whole-cell lysates were prepared and resolved by SDS-PAGE followed by immunoblot analyses with respective antibodies. Each lane contains an amount of lysate equal to 105 cells. Levels of β-actin served as a loading control. (B) The relative intensity of signals was quantified by densitometry using ImageJ [106]. Results are shown for proteins whose levels of expression changed substantially as percent change in signal intensity relative to the wild type (HSV-1 KOS) (set to 100) ± SD of the duplicate infections.
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pone.0131420.g004: Expression of viral proteins in Vero cells infected with HSV-1 KOS or ΔUS3 and ΔUL13 mutants.(A) Vero cells were inoculated in duplicates as described in Fig 3. At 14 hours post-inoculation, whole-cell lysates were prepared and resolved by SDS-PAGE followed by immunoblot analyses with respective antibodies. Each lane contains an amount of lysate equal to 105 cells. Levels of β-actin served as a loading control. (B) The relative intensity of signals was quantified by densitometry using ImageJ [106]. Results are shown for proteins whose levels of expression changed substantially as percent change in signal intensity relative to the wild type (HSV-1 KOS) (set to 100) ± SD of the duplicate infections.
Mentions: To verify the effects of loss of the US3 or UL13 kinases on early and late stages of HSV-1 replication, expression levels of several viral genes were compared. Vero cells were inoculated with 2.5 pfu per cell of HSV-1 KOS, or HSV-1 kinase mutants. At 14 hours post-inoculation, whole-cell lysates were prepared and subjected to SDS-PAGE and immunoblotting analyses, and expression levels of several HSV-1 proteins were quantified by densitometry. Uninfected (UI) Vero cells were included as a negative control (Fig 4). The steady-state levels of the ICP0 protein (expressed from an immediate-early gene) were nearly identical across all viruses (Fig 4A). Likewise, the steady-state levels of another immediate-early protein, ICP4, were equal in cells inoculated with all tested viruses (not shown). Among the proteins expressed from HSV-1 late genes, the steady-state levels of glycoproteins B, E, and G were not significantly affected by the loss of US3 and UL13 protein kinases. In contrast, the steady-state levels of glycoprotein C (gC) were reduced more significantly: the levels of precursor gC (pr-gC) decreased 2-fold during the infection with the ΔUS3 mutant, 10-fold during the infection with the ΔUL13 mutant, and were essentially undetectable during the infection with the ΔUL13/ΔUS3 mutant (Fig 4B). Consequently, the steady-state levels of mature gC were reduced by 3- and 5-fold for the ΔUL13 and ΔUL13/ΔUS3 mutants, respectively. Likewise, during the infection with the ΔUL13/ΔUS3 mutant, the steady-state levels of glycoprotein D (gD) were reduced such that the levels of precursor gD (pr-gD) were 7-fold lower and mature gD 2-fold lower relative to the HSV-1 KOS (Fig 4B). These results correlate with the observation of a small-plaque phenotype exhibited by the UL13-deficient HSV-1 mutants (Fig 2) and corroborate the viral DNA accumulation data (Fig 3), suggesting that the HSV-1 ΔUL13 mutants are deficient in their ability to spread from one host cell to another and that the activity of viral protein kinases is crucial at the late phase of viral infection. Notably, two of the tested viral proteins, ICP0 and gE, are reported substrates of the UL13 protein kinase [38–40], yet the loss of UL13 kinase had no effect on the steady-state levels of these proteins (Fig 4A).

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