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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

Construction and analyses of mutant US3 and UL13 alleles.(A) Schematic representation of the HSV-1 genome. Unique DNA sequences are represented by horizontal lines, and inverted repeats flanking the unique sequences are shown as open and black boxes with black and white arrows, respectively. US3 and UL13 regions where mutant alleles were generated are enlarged and position of the mCherry and GFP cassettes shown as red and green boxes, respectively. The approximate positions of primers designated a, b, c, d, and e, f, g, h used for PCR analyses are shown as short black arrows. (B) Southern blotting analyses of HSV-1 ΔUS3, ΔUL13, and ΔUL13/ΔU3 mutants. Viral DNA preparations from Vero cells infected with wild type KOS or mutant viruses were digested with AflII (top panel) and MluI (bottom panel) and subjected to Southern blotting. Membranes were probed with 32P-labeled UL13- and US3-specific oligonucleotide probes (S1 Table). Positions of DNA size standards are shown on the right. (C) PCR analyses of HSV-1 ΔUS3, ΔUL13, and ΔUL13/ΔUS3 mutants. Total DNA isolated from Vero cells infected with wild type HSV-1 KOS or mutant viruses were used as template for PCR reactions with 4 sets of primers. In each set, one primer is located outside of the region involved in homologous recombination and another primer is located within the mCherry or GFP cassettes. The PCR reactions were resolved on 1.2% agarose gels and visualized by ethidium bromide staining.
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pone.0131420.g001: Construction and analyses of mutant US3 and UL13 alleles.(A) Schematic representation of the HSV-1 genome. Unique DNA sequences are represented by horizontal lines, and inverted repeats flanking the unique sequences are shown as open and black boxes with black and white arrows, respectively. US3 and UL13 regions where mutant alleles were generated are enlarged and position of the mCherry and GFP cassettes shown as red and green boxes, respectively. The approximate positions of primers designated a, b, c, d, and e, f, g, h used for PCR analyses are shown as short black arrows. (B) Southern blotting analyses of HSV-1 ΔUS3, ΔUL13, and ΔUL13/ΔU3 mutants. Viral DNA preparations from Vero cells infected with wild type KOS or mutant viruses were digested with AflII (top panel) and MluI (bottom panel) and subjected to Southern blotting. Membranes were probed with 32P-labeled UL13- and US3-specific oligonucleotide probes (S1 Table). Positions of DNA size standards are shown on the right. (C) PCR analyses of HSV-1 ΔUS3, ΔUL13, and ΔUL13/ΔUS3 mutants. Total DNA isolated from Vero cells infected with wild type HSV-1 KOS or mutant viruses were used as template for PCR reactions with 4 sets of primers. In each set, one primer is located outside of the region involved in homologous recombination and another primer is located within the mCherry or GFP cassettes. The PCR reactions were resolved on 1.2% agarose gels and visualized by ethidium bromide staining.

Mentions: HSV-1 mutant viruses lacking portions of the UL13 or US3 genes were constructed by homologous recombination between infectious wild-type HSV-1 KOS DNA and plasmids carrying mCherry or GFP selection markers, respectively (Fig 1A). The infectious wild-type HSV-1 KOS DNA was prepared from a virus stock 12 passages removed from the original clinical isolate [86]. A plasmid bearing UL13 arms separated by a CMV promoter-mCherry reporter gene was recombined into HSV-1 KOS to yield a ΔUL13 virus in which codons 151–303 of the UL13 gene were deleted (Fig 1A). Likewise, a plasmid bearing US3 arms separated by a PGK promoter-GFP reporter gene was recombined into HSV-1 KOS to yield a ΔUS3 virus in which codons 107–356 of the US3 gene were deleted (Fig 1A). The HSV-1 ΔUL13/ΔUS3 mutant virus was constructed by homologous recombination between infectious HSV-1 ΔUL13 DNA and the plasmid containing the ΔUS3 allele and GFP selection marker (Fig 1A). The insertion of mCherry and GFP expression cassettes into the anticipated loci in the HSV-1 genome was independently verified by Southern blot analysis and PCR (Fig 1B and 1C). Southern blot analysis confirmed that each HSV-1 mutant virus carried an insertion of the expected size and restriction fragment length polymorphism within the UL13 and US3 loci (Fig 1B). Finally, PCR analysis confirmed the presence of the mCherry reporter gene in the UL13 locus of both the ΔUL13 and ΔUL13/ΔUS3 mutants, and reciprocally confirmed the presence of the GFP reporter gene in the US3 locus of the ΔUS3 and ΔUL13/ΔUS3 mutants (Fig 1C). Collectively, these data indicated that the intended mutations were successfully introduced into the HSV-1 genome.


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)

Construction and analyses of mutant US3 and UL13 alleles.(A) Schematic representation of the HSV-1 genome. Unique DNA sequences are represented by horizontal lines, and inverted repeats flanking the unique sequences are shown as open and black boxes with black and white arrows, respectively. US3 and UL13 regions where mutant alleles were generated are enlarged and position of the mCherry and GFP cassettes shown as red and green boxes, respectively. The approximate positions of primers designated a, b, c, d, and e, f, g, h used for PCR analyses are shown as short black arrows. (B) Southern blotting analyses of HSV-1 ΔUS3, ΔUL13, and ΔUL13/ΔU3 mutants. Viral DNA preparations from Vero cells infected with wild type KOS or mutant viruses were digested with AflII (top panel) and MluI (bottom panel) and subjected to Southern blotting. Membranes were probed with 32P-labeled UL13- and US3-specific oligonucleotide probes (S1 Table). Positions of DNA size standards are shown on the right. (C) PCR analyses of HSV-1 ΔUS3, ΔUL13, and ΔUL13/ΔUS3 mutants. Total DNA isolated from Vero cells infected with wild type HSV-1 KOS or mutant viruses were used as template for PCR reactions with 4 sets of primers. In each set, one primer is located outside of the region involved in homologous recombination and another primer is located within the mCherry or GFP cassettes. The PCR reactions were resolved on 1.2% agarose gels and visualized by ethidium bromide staining.
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pone.0131420.g001: Construction and analyses of mutant US3 and UL13 alleles.(A) Schematic representation of the HSV-1 genome. Unique DNA sequences are represented by horizontal lines, and inverted repeats flanking the unique sequences are shown as open and black boxes with black and white arrows, respectively. US3 and UL13 regions where mutant alleles were generated are enlarged and position of the mCherry and GFP cassettes shown as red and green boxes, respectively. The approximate positions of primers designated a, b, c, d, and e, f, g, h used for PCR analyses are shown as short black arrows. (B) Southern blotting analyses of HSV-1 ΔUS3, ΔUL13, and ΔUL13/ΔU3 mutants. Viral DNA preparations from Vero cells infected with wild type KOS or mutant viruses were digested with AflII (top panel) and MluI (bottom panel) and subjected to Southern blotting. Membranes were probed with 32P-labeled UL13- and US3-specific oligonucleotide probes (S1 Table). Positions of DNA size standards are shown on the right. (C) PCR analyses of HSV-1 ΔUS3, ΔUL13, and ΔUL13/ΔUS3 mutants. Total DNA isolated from Vero cells infected with wild type HSV-1 KOS or mutant viruses were used as template for PCR reactions with 4 sets of primers. In each set, one primer is located outside of the region involved in homologous recombination and another primer is located within the mCherry or GFP cassettes. The PCR reactions were resolved on 1.2% agarose gels and visualized by ethidium bromide staining.
Mentions: HSV-1 mutant viruses lacking portions of the UL13 or US3 genes were constructed by homologous recombination between infectious wild-type HSV-1 KOS DNA and plasmids carrying mCherry or GFP selection markers, respectively (Fig 1A). The infectious wild-type HSV-1 KOS DNA was prepared from a virus stock 12 passages removed from the original clinical isolate [86]. A plasmid bearing UL13 arms separated by a CMV promoter-mCherry reporter gene was recombined into HSV-1 KOS to yield a ΔUL13 virus in which codons 151–303 of the UL13 gene were deleted (Fig 1A). Likewise, a plasmid bearing US3 arms separated by a PGK promoter-GFP reporter gene was recombined into HSV-1 KOS to yield a ΔUS3 virus in which codons 107–356 of the US3 gene were deleted (Fig 1A). The HSV-1 ΔUL13/ΔUS3 mutant virus was constructed by homologous recombination between infectious HSV-1 ΔUL13 DNA and the plasmid containing the ΔUS3 allele and GFP selection marker (Fig 1A). The insertion of mCherry and GFP expression cassettes into the anticipated loci in the HSV-1 genome was independently verified by Southern blot analysis and PCR (Fig 1B and 1C). Southern blot analysis confirmed that each HSV-1 mutant virus carried an insertion of the expected size and restriction fragment length polymorphism within the UL13 and US3 loci (Fig 1B). Finally, PCR analysis confirmed the presence of the mCherry reporter gene in the UL13 locus of both the ΔUL13 and ΔUL13/ΔUS3 mutants, and reciprocally confirmed the presence of the GFP reporter gene in the US3 locus of the ΔUS3 and ΔUL13/ΔUS3 mutants (Fig 1C). Collectively, these data indicated that the intended mutations were successfully introduced into the HSV-1 genome.

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