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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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Mutant in1814 establishes latency as efficiently as genomically wild                            type isolates.Groups of mice were infected with strain 17Syn+, in1814, and the                            genomically rescued variant 1814R, as described in Methods. At 40 days pi, the ganglia of 3 mice per                            group were processed for single neuron PCR. Individual neurons were                            examined for the presence of the viral genome and the number of viral                            genomes present in positive neurons was determined using a quantitative                            PCR assay as detailed in Methods.                            (A) Shown is the percentage of neurons positive for the viral genome.                            The number of neurons positive for the viral genome over the number                            tested is shown in the histograms. (B) Each point on the scattergram                            represents the number of viral genomes present in an individual neuron.                            The horizontal bars are drawn at the mean value of genome copies per                            positive neuron.
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ppat-1000352-g004: Mutant in1814 establishes latency as efficiently as genomically wild type isolates.Groups of mice were infected with strain 17Syn+, in1814, and the genomically rescued variant 1814R, as described in Methods. At 40 days pi, the ganglia of 3 mice per group were processed for single neuron PCR. Individual neurons were examined for the presence of the viral genome and the number of viral genomes present in positive neurons was determined using a quantitative PCR assay as detailed in Methods. (A) Shown is the percentage of neurons positive for the viral genome. The number of neurons positive for the viral genome over the number tested is shown in the histograms. (B) Each point on the scattergram represents the number of viral genomes present in an individual neuron. The horizontal bars are drawn at the mean value of genome copies per positive neuron.

Mentions: In preliminary studies, 17VP16Δ422 was determined to establish latent infections, but at very low levels compared to 17syn+ (not shown). Thus it becomes impractical to study in vivo reactivation with this mutant because the efficiency of reactivation in vivo following HS is directly correlated with the number of latently infected neurons in the ganglion (r2 = 0.99) [56],[76]. Likewise quantification of the number of latently infected neurons in in1814 infected TG compared to the parental strain (17syn+) and rescue (1814R) is critical for interpreting the outcome of experiments to quantify viral reactivation. The number of latently infected neurons and the number of viral genomes within individual infected neurons in TG from 3 mice from each group was quantified using a single neuron PCR assay termed CXA [25],[28][66][77]. In this assay, ganglia stabilized by fixation are enzymatically dissociated and individual neurons from enriched neuronal fractions are harvested and analyzed by QPCR, providing information on both the frequency of latently infected neurons and the number of viral genome copies in the individual neurons analyzed. As anticipated from the results of preliminary experiments, similar numbers of latently infected neurons were observed in in1814, 1814R, and 17syn+ infected ganglia, 28%, 25% and 26%, respectively (Figure 4A). The number of viral genomes detected within individual latently infected neurons is shown in the scattergram (Figure 4B). No significant difference among the viral genome copy number profiles was observed (mean copy number = 56.5, 51.8 and 44.8, respectively p = 0.94; ANOVA).


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

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

Mutant in1814 establishes latency as efficiently as genomically wild                            type isolates.Groups of mice were infected with strain 17Syn+, in1814, and the                            genomically rescued variant 1814R, as described in Methods. At 40 days pi, the ganglia of 3 mice per                            group were processed for single neuron PCR. Individual neurons were                            examined for the presence of the viral genome and the number of viral                            genomes present in positive neurons was determined using a quantitative                            PCR assay as detailed in Methods.                            (A) Shown is the percentage of neurons positive for the viral genome.                            The number of neurons positive for the viral genome over the number                            tested is shown in the histograms. (B) Each point on the scattergram                            represents the number of viral genomes present in an individual neuron.                            The horizontal bars are drawn at the mean value of genome copies per                            positive neuron.
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Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2654966&req=5

ppat-1000352-g004: Mutant in1814 establishes latency as efficiently as genomically wild type isolates.Groups of mice were infected with strain 17Syn+, in1814, and the genomically rescued variant 1814R, as described in Methods. At 40 days pi, the ganglia of 3 mice per group were processed for single neuron PCR. Individual neurons were examined for the presence of the viral genome and the number of viral genomes present in positive neurons was determined using a quantitative PCR assay as detailed in Methods. (A) Shown is the percentage of neurons positive for the viral genome. The number of neurons positive for the viral genome over the number tested is shown in the histograms. (B) Each point on the scattergram represents the number of viral genomes present in an individual neuron. The horizontal bars are drawn at the mean value of genome copies per positive neuron.
Mentions: In preliminary studies, 17VP16Δ422 was determined to establish latent infections, but at very low levels compared to 17syn+ (not shown). Thus it becomes impractical to study in vivo reactivation with this mutant because the efficiency of reactivation in vivo following HS is directly correlated with the number of latently infected neurons in the ganglion (r2 = 0.99) [56],[76]. Likewise quantification of the number of latently infected neurons in in1814 infected TG compared to the parental strain (17syn+) and rescue (1814R) is critical for interpreting the outcome of experiments to quantify viral reactivation. The number of latently infected neurons and the number of viral genomes within individual infected neurons in TG from 3 mice from each group was quantified using a single neuron PCR assay termed CXA [25],[28][66][77]. In this assay, ganglia stabilized by fixation are enzymatically dissociated and individual neurons from enriched neuronal fractions are harvested and analyzed by QPCR, providing information on both the frequency of latently infected neurons and the number of viral genome copies in the individual neurons analyzed. As anticipated from the results of preliminary experiments, similar numbers of latently infected neurons were observed in in1814, 1814R, and 17syn+ infected ganglia, 28%, 25% and 26%, respectively (Figure 4A). The number of viral genomes detected within individual latently infected neurons is shown in the scattergram (Figure 4B). No significant difference among the viral genome copy number profiles was observed (mean copy number = 56.5, 51.8 and 44.8, respectively p = 0.94; ANOVA).

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