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De novo synthesis of VP16 coordinates the exit from HSV latency in vivo.

Thompson RL, Preston CM, Sawtell NM - PLoS Pathog. (2009)

Bottom Line: TG neurons latently infected with the VP16TF mutant in1814 do not express detectable viral proteins following stress, whereas viruses with mutations in the other major viral transcription regulators ICP0 and ICP4 do exit the latent state.These findings support the novel hypothesis that de novo expression of VP16 regulates entry into the lytic program in neurons at all phases of the viral life cycle.HSV reactivation from latency conforms to a model in which stochastic derepression of the VP16 promoter and expression of VP16 initiates entry into the lytic cycle.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Genetics, Microbiology, and Biochemistry, University of Cincinnati School of Medicine, Cincinnati, Ohio, USA.

ABSTRACT
The mechanism controlling the exit from herpes simplex virus latency (HSV) is of central importance to recurrent disease and transmission of infection, yet interactions between host and viral functions that govern this process remain unclear. The cascade of HSV gene transcription is initiated by the multifunctional virion protein VP16, which is expressed late in the viral replication cycle. Currently, it is widely accepted that VP16 transactivating function is not involved in the exit from latency. Utilizing the mouse ocular model of HSV pathogenesis together with genetically engineered viral mutants and assays to quantify latency and the exit from latency at the single neuron level, we show that in vivo (i) the VP16 promoter confers distinct regulation critical for viral replication in the trigeminal ganglion (TG) during the acute phase of infection and (ii) the transactivation function of VP16 (VP16TF) is uniquely required for the exit from latency. TG neurons latently infected with the VP16TF mutant in1814 do not express detectable viral proteins following stress, whereas viruses with mutations in the other major viral transcription regulators ICP0 and ICP4 do exit the latent state. Analysis of a VP16 promoter/reporter mutant in the background of in1814 demonstrates that the VP16 promoter is activated in latently infected neurons following stress in the absence of other viral proteins. These findings support the novel hypothesis that de novo expression of VP16 regulates entry into the lytic program in neurons at all phases of the viral life cycle. HSV reactivation from latency conforms to a model in which stochastic derepression of the VP16 promoter and expression of VP16 initiates entry into the lytic cycle.

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Replication kinetics of the VP16 transactivation mutants in1814 and                            17VP16Δ422.(A) A schematic of the experimental design employed to biologically                            characterize the VP16 mutants is shown. (B) A diagram of the genomic                            structures of in1814 and 17VP16Δ422 is shown. (C,D) Mice were                            infected as detailed in Methods                            and, at the indicated times pi, tissues from three mice from each group                            were assayed for infectious virus. Solid lines represent titers obtained                            by standard plaque assay, whereas dashed lines represent the same                            samples titrated in the presence of 3 mM HMBA, as described in Methods. The area under the curve                            (AUC) was calculated for each curve, and the fold increase in AUC in the                            presence of HMBA is indicated below the graphs. Genomically restored                            isolates of both in1814 (1814R) and 17VP16Δ422                            (17VP16Δ422R) were analyzed and found to be not different from                            the parental strain, 17syn+ (not shown).
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ppat-1000352-g003: Replication kinetics of the VP16 transactivation mutants in1814 and 17VP16Δ422.(A) A schematic of the experimental design employed to biologically characterize the VP16 mutants is shown. (B) A diagram of the genomic structures of in1814 and 17VP16Δ422 is shown. (C,D) Mice were infected as detailed in Methods and, at the indicated times pi, tissues from three mice from each group were assayed for infectious virus. Solid lines represent titers obtained by standard plaque assay, whereas dashed lines represent the same samples titrated in the presence of 3 mM HMBA, as described in Methods. The area under the curve (AUC) was calculated for each curve, and the fold increase in AUC in the presence of HMBA is indicated below the graphs. Genomically restored isolates of both in1814 (1814R) and 17VP16Δ422 (17VP16Δ422R) were analyzed and found to be not different from the parental strain, 17syn+ (not shown).

Mentions: VP16 is an essential multifunctional protein. However, mutations which impair the transactivation function of VP16 have been generated and characterized in vitro and in vivo. Mutant in1814 contains a 12 bp insertion that disrupts a domain required for the VP16 induced complex formation and thus the transactivation function of the protein [40],[70]. The carboxy-terminal acidic activation domain has been deleted in two mutants, V422 [41] and RP5 [30], both built in HSV-1 strain KOS. While these three mutants are phenotypically similar in vitro, important differences have been reported in their in vivo phenotypes. Despite the impaired replication reported for both in1814 and RP5 in mouse eyes and TG, in1814 established latent infections efficiently and reactivated in explant assays [16],[71],[72]. RP5 failed to accomplish either of these outcomes [30]. The in vivo phenotypic differences between HSV strains 17syn+ and KOS is a confounding issue [69],[73]. Therefore, mutant 17VP16Δ422 was constructed as detailed in methods. We utilized mutants in1814 and 17VP16Δ422 to evaluate the role of VP16 transactivation on reactivation in vivo as outlined in Figure 3A.


De novo synthesis of VP16 coordinates the exit from HSV latency in vivo.

Thompson RL, Preston CM, Sawtell NM - PLoS Pathog. (2009)

Replication kinetics of the VP16 transactivation mutants in1814 and                            17VP16Δ422.(A) A schematic of the experimental design employed to biologically                            characterize the VP16 mutants is shown. (B) A diagram of the genomic                            structures of in1814 and 17VP16Δ422 is shown. (C,D) Mice were                            infected as detailed in Methods                            and, at the indicated times pi, tissues from three mice from each group                            were assayed for infectious virus. Solid lines represent titers obtained                            by standard plaque assay, whereas dashed lines represent the same                            samples titrated in the presence of 3 mM HMBA, as described in Methods. The area under the curve                            (AUC) was calculated for each curve, and the fold increase in AUC in the                            presence of HMBA is indicated below the graphs. Genomically restored                            isolates of both in1814 (1814R) and 17VP16Δ422                            (17VP16Δ422R) were analyzed and found to be not different from                            the parental strain, 17syn+ (not shown).
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2654966&req=5

ppat-1000352-g003: Replication kinetics of the VP16 transactivation mutants in1814 and 17VP16Δ422.(A) A schematic of the experimental design employed to biologically characterize the VP16 mutants is shown. (B) A diagram of the genomic structures of in1814 and 17VP16Δ422 is shown. (C,D) Mice were infected as detailed in Methods and, at the indicated times pi, tissues from three mice from each group were assayed for infectious virus. Solid lines represent titers obtained by standard plaque assay, whereas dashed lines represent the same samples titrated in the presence of 3 mM HMBA, as described in Methods. The area under the curve (AUC) was calculated for each curve, and the fold increase in AUC in the presence of HMBA is indicated below the graphs. Genomically restored isolates of both in1814 (1814R) and 17VP16Δ422 (17VP16Δ422R) were analyzed and found to be not different from the parental strain, 17syn+ (not shown).
Mentions: VP16 is an essential multifunctional protein. However, mutations which impair the transactivation function of VP16 have been generated and characterized in vitro and in vivo. Mutant in1814 contains a 12 bp insertion that disrupts a domain required for the VP16 induced complex formation and thus the transactivation function of the protein [40],[70]. The carboxy-terminal acidic activation domain has been deleted in two mutants, V422 [41] and RP5 [30], both built in HSV-1 strain KOS. While these three mutants are phenotypically similar in vitro, important differences have been reported in their in vivo phenotypes. Despite the impaired replication reported for both in1814 and RP5 in mouse eyes and TG, in1814 established latent infections efficiently and reactivated in explant assays [16],[71],[72]. RP5 failed to accomplish either of these outcomes [30]. The in vivo phenotypic differences between HSV strains 17syn+ and KOS is a confounding issue [69],[73]. Therefore, mutant 17VP16Δ422 was constructed as detailed in methods. We utilized mutants in1814 and 17VP16Δ422 to evaluate the role of VP16 transactivation on reactivation in vivo as outlined in Figure 3A.

Bottom Line: TG neurons latently infected with the VP16TF mutant in1814 do not express detectable viral proteins following stress, whereas viruses with mutations in the other major viral transcription regulators ICP0 and ICP4 do exit the latent state.These findings support the novel hypothesis that de novo expression of VP16 regulates entry into the lytic program in neurons at all phases of the viral life cycle.HSV reactivation from latency conforms to a model in which stochastic derepression of the VP16 promoter and expression of VP16 initiates entry into the lytic cycle.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Genetics, Microbiology, and Biochemistry, University of Cincinnati School of Medicine, Cincinnati, Ohio, USA.

ABSTRACT
The mechanism controlling the exit from herpes simplex virus latency (HSV) is of central importance to recurrent disease and transmission of infection, yet interactions between host and viral functions that govern this process remain unclear. The cascade of HSV gene transcription is initiated by the multifunctional virion protein VP16, which is expressed late in the viral replication cycle. Currently, it is widely accepted that VP16 transactivating function is not involved in the exit from latency. Utilizing the mouse ocular model of HSV pathogenesis together with genetically engineered viral mutants and assays to quantify latency and the exit from latency at the single neuron level, we show that in vivo (i) the VP16 promoter confers distinct regulation critical for viral replication in the trigeminal ganglion (TG) during the acute phase of infection and (ii) the transactivation function of VP16 (VP16TF) is uniquely required for the exit from latency. TG neurons latently infected with the VP16TF mutant in1814 do not express detectable viral proteins following stress, whereas viruses with mutations in the other major viral transcription regulators ICP0 and ICP4 do exit the latent state. Analysis of a VP16 promoter/reporter mutant in the background of in1814 demonstrates that the VP16 promoter is activated in latently infected neurons following stress in the absence of other viral proteins. These findings support the novel hypothesis that de novo expression of VP16 regulates entry into the lytic program in neurons at all phases of the viral life cycle. HSV reactivation from latency conforms to a model in which stochastic derepression of the VP16 promoter and expression of VP16 initiates entry into the lytic cycle.

Show MeSH
Related in: MedlinePlus