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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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In1814 does not exit latency in vivo.(A) Mice latently infected with the indicated strains were subjected to                            HS, and whole TG were assayed for viral protein positive cells 22 hrs                            post stress. Each point in the scattergram represents the number of                            viral protein positive neurons detected in an individual TG. The                            horizontal bars are drawn at the mean. (B) A photomicrograph of a whole                            mounted ganglion processed for the detection of HSV-1 proteins 22 hr                            post HS shows a single neuron in which the latent genome has entered the                            lytic cycle. The brown precipitate evident in the cell body and axonal                            tract indicates the presence of viral proteins.
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ppat-1000352-g006: In1814 does not exit latency in vivo.(A) Mice latently infected with the indicated strains were subjected to HS, and whole TG were assayed for viral protein positive cells 22 hrs post stress. Each point in the scattergram represents the number of viral protein positive neurons detected in an individual TG. The horizontal bars are drawn at the mean. (B) A photomicrograph of a whole mounted ganglion processed for the detection of HSV-1 proteins 22 hr post HS shows a single neuron in which the latent genome has entered the lytic cycle. The brown precipitate evident in the cell body and axonal tract indicates the presence of viral proteins.

Mentions: Reactivation from latency is functionally defined by the detection of infectious virus. To expand our understanding of the process of reactivation, we have developed a strategy for quantifying at the single neuron level the number of neurons which exit latency as evidenced by detectable lytic viral protein expression [4],[78]. This method is the first to allow us to partition the process of reactivation into stages, and to begin the assignment of viral and host cell functions critical for either entry into the lytic cycle or for progression to infectious virus production [25],[28]. In addition, this approach obviates the inherent problem of detection of reactivated virus when a mutant with low plaquing efficiency (such as in1814) is employed. At 40 days pi, additional mice from the groups detailed above were induced to reactivate in vivo using HS. Latently infected control mice and treated mice (at 22 hrs post treatment) were euthanized, the ganglia were removed and processed for the detection of lytic viral proteins using WGIHC as detailed previously [4],[78]. This method can reliably detect a single neuron exiting latency among the 10's of thousands in the ganglion. In the uninduced groups of animals, lytic viral protein expressing neurons were not observed (0/10, 0/8, and 0/9, in1814, 1814R and 17syn+ infected ganglia, respectively). This was not unexpected as we have previously shown that the level of “spontaneous” reactivation of strain 17syn+ in the latently infected Swiss Webster mouse TG is very low [4],[78]. Consistent with the detection of infectious virus above, ganglia from mice latently infected with either 1814R or 17syn+ contained neurons expressing lytic viral proteins at 22 hrs post induction, a total of 60 and 55 neurons, respectively in the ganglia examined (Figure 6A and 6B). In contrast, no lytic viral protein expressing neurons were detected in in1814 ganglia post induction (0/40). These findings indicate that in vivo, the VP16 transactivating function is required for HSV to exit the latent state and produce detectable viral proteins.


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

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

In1814 does not exit latency in vivo.(A) Mice latently infected with the indicated strains were subjected to                            HS, and whole TG were assayed for viral protein positive cells 22 hrs                            post stress. Each point in the scattergram represents the number of                            viral protein positive neurons detected in an individual TG. The                            horizontal bars are drawn at the mean. (B) A photomicrograph of a whole                            mounted ganglion processed for the detection of HSV-1 proteins 22 hr                            post HS shows a single neuron in which the latent genome has entered the                            lytic cycle. The brown precipitate evident in the cell body and axonal                            tract indicates the presence of viral proteins.
© Copyright Policy
Related In: Results  -  Collection

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

ppat-1000352-g006: In1814 does not exit latency in vivo.(A) Mice latently infected with the indicated strains were subjected to HS, and whole TG were assayed for viral protein positive cells 22 hrs post stress. Each point in the scattergram represents the number of viral protein positive neurons detected in an individual TG. The horizontal bars are drawn at the mean. (B) A photomicrograph of a whole mounted ganglion processed for the detection of HSV-1 proteins 22 hr post HS shows a single neuron in which the latent genome has entered the lytic cycle. The brown precipitate evident in the cell body and axonal tract indicates the presence of viral proteins.
Mentions: Reactivation from latency is functionally defined by the detection of infectious virus. To expand our understanding of the process of reactivation, we have developed a strategy for quantifying at the single neuron level the number of neurons which exit latency as evidenced by detectable lytic viral protein expression [4],[78]. This method is the first to allow us to partition the process of reactivation into stages, and to begin the assignment of viral and host cell functions critical for either entry into the lytic cycle or for progression to infectious virus production [25],[28]. In addition, this approach obviates the inherent problem of detection of reactivated virus when a mutant with low plaquing efficiency (such as in1814) is employed. At 40 days pi, additional mice from the groups detailed above were induced to reactivate in vivo using HS. Latently infected control mice and treated mice (at 22 hrs post treatment) were euthanized, the ganglia were removed and processed for the detection of lytic viral proteins using WGIHC as detailed previously [4],[78]. This method can reliably detect a single neuron exiting latency among the 10's of thousands in the ganglion. In the uninduced groups of animals, lytic viral protein expressing neurons were not observed (0/10, 0/8, and 0/9, in1814, 1814R and 17syn+ infected ganglia, respectively). This was not unexpected as we have previously shown that the level of “spontaneous” reactivation of strain 17syn+ in the latently infected Swiss Webster mouse TG is very low [4],[78]. Consistent with the detection of infectious virus above, ganglia from mice latently infected with either 1814R or 17syn+ contained neurons expressing lytic viral proteins at 22 hrs post induction, a total of 60 and 55 neurons, respectively in the ganglia examined (Figure 6A and 6B). In contrast, no lytic viral protein expressing neurons were detected in in1814 ganglia post induction (0/40). These findings indicate that in vivo, the VP16 transactivating function is required for HSV to exit the latent state and produce detectable viral proteins.

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