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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

EM analysis of Vero cells infected with ΔUS3 and ΔUL13 HSV-1 mutants.Monolayers of Vero cells on Thermonox coverslips were inoculated as described in Fig 3. At 16 hours post-inoculation, the monolayers were fixed and processed for EM analyses. (A) Separate images of nucleus, cytoplasm, and plasma membranes/extracellular space were recorded for 20 individual cells per each virus. The number of nucleocapsids and virions were manually counted in each image in a blinded fashion. Results are presented as the mean number of virions per compartment ± sem (n = 20; ***—p<0.001). (B) Representative images of the plasma membranes/extracellular space for cells infected with different viruses (bars represent 500 nm).
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pone.0131420.g007: EM analysis of Vero cells infected with ΔUS3 and ΔUL13 HSV-1 mutants.Monolayers of Vero cells on Thermonox coverslips were inoculated as described in Fig 3. At 16 hours post-inoculation, the monolayers were fixed and processed for EM analyses. (A) Separate images of nucleus, cytoplasm, and plasma membranes/extracellular space were recorded for 20 individual cells per each virus. The number of nucleocapsids and virions were manually counted in each image in a blinded fashion. Results are presented as the mean number of virions per compartment ± sem (n = 20; ***—p<0.001). (B) Representative images of the plasma membranes/extracellular space for cells infected with different viruses (bars represent 500 nm).

Mentions: Studies of CHPKs of human β- and γ- herpesviruses suggest that lack of their function interferes with nuclear egress as evidenced by accumulation of nucleocapsids in the nuclei of infected cells [8, 9, 11]. Likewise, the US3-deficient HSV-1 mutants exhibited a similar phenotype in electron microscopy studies [24, 33]. However, the UL13- HSV-1 mutants have never been analyzed by the electron microscopy. To address this knowledge gap, Vero cells were inoculated with HSV-1 KOS, or HSV-1 ΔUL13/ΔUS3, ΔUS3, and ΔUL13 mutants at MOI of 2.5 pfu per cell. At 16 hours post-inoculation the cells were fixed and processed for analysis by transmission electron microscopy (TEM). Separate images of nucleus, cytoplasm and plasma membranes/extracellular space were recorded for 20 individual cells for each virus. The number of nucleocapsids and virions were manually enumerated in each image in a blinded manner (Fig 7A). The number of nucleocapsids in the nuclei of cells infected with the ΔUS3 mutant was significantly higher than in the nuclei of the cells infected with the HSV-1 KOS, ΔUL13, or ΔUS3/ΔUL13 mutants (79±16 vs 24±5, 6±2, and 6±1 nucleocapsids, respectively) (Fig 7A, p<0.001). Importantly, a portion of the nucleocapsids was trapped in invaginations of the nuclear membrane, as described in previous studies [24, 33]. However, this increased accumulation of nucleocapsids had no effect on virion yields in the extracellular space and infectivity of ΔUS3 mutant virions (Figs 5–7) corroborating a recent study by Wild et al. [95]. The HSV-1 ΔUL13 mutant yields differed only slightly from the wild type HSV-1 KOS: the yield of nucleocapsids in the nuclei was slightly lower and the yield of virions in the cytoplasm was slightly higher. Neither of these differences was statistically significant. Most importantly, the virion yields on the membrane or extracellular space were significantly lower in cells infected with the HSV-1 ΔUL13/ΔUS3 mutant than the cells infected with the HSV-1 KOS, or HSV-1 ΔUS3 and ΔUL13 mutants (1±0.3 vs 32±7, 28±5, and 17±5, respectively) (Fig 7A, p<0.001). These data are consistent with the micro-plaque phenotype (Fig 2) and with the apparent defect in virion release (Figs 5 and 6) during infection with the ΔUL13 and ΔUL13/ΔUS3 mutants. Notably, only cells infected with the HSV-1 ΔUL13/ΔUS3 mutant often released large number(s) of membrane-bound vesicles (S1 Fig). Since one of the functions ascribed to the US3 kinase is inhibition of apoptosis [54, 66–77], it is important to emphasize that (a) release of these vesicles was not observed in cells infected with the HSV-1 ΔUS3 mutant, (b) release of the vesicles was not accompanied by changes in chromatin structure and nuclear architecture characteristic of apoptosis, and (c) previous studies indicated that the apoptotic phenotype in HSV-1-infected cells is typically observed at later times (24 hours post-inoculation or later), whereas the experiments described here were terminated at 16 hours post-inoculation. These data suggest that the membrane-bound vesicles released during the infection with the HSV-1 ΔUL13/ΔUS3 mutant are not consistent with the apoptotic phenotype; however, these vesicles might contain HSV-1 antigens detected in the 25–35% fractions of the sucrose gradients by ELISA (Fig 6A).


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)

EM analysis of Vero cells infected with ΔUS3 and ΔUL13 HSV-1 mutants.Monolayers of Vero cells on Thermonox coverslips were inoculated as described in Fig 3. At 16 hours post-inoculation, the monolayers were fixed and processed for EM analyses. (A) Separate images of nucleus, cytoplasm, and plasma membranes/extracellular space were recorded for 20 individual cells per each virus. The number of nucleocapsids and virions were manually counted in each image in a blinded fashion. Results are presented as the mean number of virions per compartment ± sem (n = 20; ***—p<0.001). (B) Representative images of the plasma membranes/extracellular space for cells infected with different viruses (bars represent 500 nm).
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pone.0131420.g007: EM analysis of Vero cells infected with ΔUS3 and ΔUL13 HSV-1 mutants.Monolayers of Vero cells on Thermonox coverslips were inoculated as described in Fig 3. At 16 hours post-inoculation, the monolayers were fixed and processed for EM analyses. (A) Separate images of nucleus, cytoplasm, and plasma membranes/extracellular space were recorded for 20 individual cells per each virus. The number of nucleocapsids and virions were manually counted in each image in a blinded fashion. Results are presented as the mean number of virions per compartment ± sem (n = 20; ***—p<0.001). (B) Representative images of the plasma membranes/extracellular space for cells infected with different viruses (bars represent 500 nm).
Mentions: Studies of CHPKs of human β- and γ- herpesviruses suggest that lack of their function interferes with nuclear egress as evidenced by accumulation of nucleocapsids in the nuclei of infected cells [8, 9, 11]. Likewise, the US3-deficient HSV-1 mutants exhibited a similar phenotype in electron microscopy studies [24, 33]. However, the UL13- HSV-1 mutants have never been analyzed by the electron microscopy. To address this knowledge gap, Vero cells were inoculated with HSV-1 KOS, or HSV-1 ΔUL13/ΔUS3, ΔUS3, and ΔUL13 mutants at MOI of 2.5 pfu per cell. At 16 hours post-inoculation the cells were fixed and processed for analysis by transmission electron microscopy (TEM). Separate images of nucleus, cytoplasm and plasma membranes/extracellular space were recorded for 20 individual cells for each virus. The number of nucleocapsids and virions were manually enumerated in each image in a blinded manner (Fig 7A). The number of nucleocapsids in the nuclei of cells infected with the ΔUS3 mutant was significantly higher than in the nuclei of the cells infected with the HSV-1 KOS, ΔUL13, or ΔUS3/ΔUL13 mutants (79±16 vs 24±5, 6±2, and 6±1 nucleocapsids, respectively) (Fig 7A, p<0.001). Importantly, a portion of the nucleocapsids was trapped in invaginations of the nuclear membrane, as described in previous studies [24, 33]. However, this increased accumulation of nucleocapsids had no effect on virion yields in the extracellular space and infectivity of ΔUS3 mutant virions (Figs 5–7) corroborating a recent study by Wild et al. [95]. The HSV-1 ΔUL13 mutant yields differed only slightly from the wild type HSV-1 KOS: the yield of nucleocapsids in the nuclei was slightly lower and the yield of virions in the cytoplasm was slightly higher. Neither of these differences was statistically significant. Most importantly, the virion yields on the membrane or extracellular space were significantly lower in cells infected with the HSV-1 ΔUL13/ΔUS3 mutant than the cells infected with the HSV-1 KOS, or HSV-1 ΔUS3 and ΔUL13 mutants (1±0.3 vs 32±7, 28±5, and 17±5, respectively) (Fig 7A, p<0.001). These data are consistent with the micro-plaque phenotype (Fig 2) and with the apparent defect in virion release (Figs 5 and 6) during infection with the ΔUL13 and ΔUL13/ΔUS3 mutants. Notably, only cells infected with the HSV-1 ΔUL13/ΔUS3 mutant often released large number(s) of membrane-bound vesicles (S1 Fig). Since one of the functions ascribed to the US3 kinase is inhibition of apoptosis [54, 66–77], it is important to emphasize that (a) release of these vesicles was not observed in cells infected with the HSV-1 ΔUS3 mutant, (b) release of the vesicles was not accompanied by changes in chromatin structure and nuclear architecture characteristic of apoptosis, and (c) previous studies indicated that the apoptotic phenotype in HSV-1-infected cells is typically observed at later times (24 hours post-inoculation or later), whereas the experiments described here were terminated at 16 hours post-inoculation. These data suggest that the membrane-bound vesicles released during the infection with the HSV-1 ΔUL13/ΔUS3 mutant are not consistent with the apoptotic phenotype; however, these vesicles might contain HSV-1 antigens detected in the 25–35% fractions of the sucrose gradients by ELISA (Fig 6A).

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