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Transient reversal of episome silencing precedes VP16-dependent transcription during reactivation of latent HSV-1 in neurons.

Kim JY, Mandarino A, Chao MV, Mohr I, Wilson AC - PLoS Pathog. (2012)

Bottom Line: This allows synthesis of viral regulatory proteins including VP16, which accumulate in the cytoplasm of the host neuron.During the second phase, VP16 and its cellular cofactor HCF-1, which is also predominantly cytoplasmic, concentrate in the nucleus where they assemble an activator complex on viral promoters.Thus regulated localization of de novo synthesized VP16 is likely to be a critical determinant of HSV-1 reactivation in sympathetic neurons.

View Article: PubMed Central - PubMed

Affiliation: Department of Microbiology, New York University School of Medicine, New York, New York, USA.

ABSTRACT
Herpes simplex virus type-1 (HSV-1) establishes latency in peripheral neurons, creating a permanent source of recurrent infections. The latent genome is assembled into chromatin and lytic cycle genes are silenced. Processes that orchestrate reentry into productive replication (reactivation) remain poorly understood. We have used latently infected cultures of primary superior cervical ganglion (SCG) sympathetic neurons to profile viral gene expression following a defined reactivation stimulus. Lytic genes are transcribed in two distinct phases, differing in their reliance on protein synthesis, viral DNA replication and the essential initiator protein VP16. The first phase does not require viral proteins and has the appearance of a transient, widespread de-repression of the previously silent lytic genes. This allows synthesis of viral regulatory proteins including VP16, which accumulate in the cytoplasm of the host neuron. During the second phase, VP16 and its cellular cofactor HCF-1, which is also predominantly cytoplasmic, concentrate in the nucleus where they assemble an activator complex on viral promoters. The transactivation function supplied by VP16 promotes increased viral lytic gene transcription leading to the onset of genome amplification and the production of infectious viral particles. Thus regulated localization of de novo synthesized VP16 is likely to be a critical determinant of HSV-1 reactivation in sympathetic neurons.

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Related in: MedlinePlus

Transactivation function of VP16 is required during Phase II.(A) Structure of VP16 showing the 12-bp insertion (in1814) between the structured N-terminal domain and the C-terminal activation domain (AD) that disrupts VP16-induced complex assembly [32]. (B) SCG neurons were infected with mutant (in1814) or marker rescue (in1814R) viruses (MOI = 1) in the presence of 100 µM acyclovir and maintained for 7 days before measuring the relative amounts of viral genomic DNA by qPCR. (C) Reactivation was induced with 20 µM LY294002 in media lacking ACV and maintained for 7 days before harvest and plaque assay to detect infectious virus. (D) Comparison of viral transcript levels during reactivation by in1814 (‘M’) and in1814R (‘R’) at 15 and 20 h post-induction (Phase I) and at 72 h post-induction (Phase II). For each time point, transcript levels from the in1814 (‘M’) sample were set to 1 and the value for the corresponding transcript from in1814R (‘R’) plotted as the fold difference. (E) Depletion of VP16 using RNA interference. Latently infected neuron cultures were infected with a lentivirus expressing a VP16-specific short-hairpin RNA [shRNA] (KD) or with a control lentivirus (Con). ShRNAs were allowed to accumulate for 5 days before reactivation was induced with LY294002 and allowed to proceed for 5 days in media lacking ACV. Lysates were prepared and probed by immunoblotting to detect VP16 and the loading control, Rho-GDI. (F) Quantitation of infectious virus by plaque assay. (G) Comparison of viral transcript levels in the absence of VP16. Values from the control culture are plotted relative to the corresponding value from the VP16 shRNA (KD) culture.
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ppat-1002540-g004: Transactivation function of VP16 is required during Phase II.(A) Structure of VP16 showing the 12-bp insertion (in1814) between the structured N-terminal domain and the C-terminal activation domain (AD) that disrupts VP16-induced complex assembly [32]. (B) SCG neurons were infected with mutant (in1814) or marker rescue (in1814R) viruses (MOI = 1) in the presence of 100 µM acyclovir and maintained for 7 days before measuring the relative amounts of viral genomic DNA by qPCR. (C) Reactivation was induced with 20 µM LY294002 in media lacking ACV and maintained for 7 days before harvest and plaque assay to detect infectious virus. (D) Comparison of viral transcript levels during reactivation by in1814 (‘M’) and in1814R (‘R’) at 15 and 20 h post-induction (Phase I) and at 72 h post-induction (Phase II). For each time point, transcript levels from the in1814 (‘M’) sample were set to 1 and the value for the corresponding transcript from in1814R (‘R’) plotted as the fold difference. (E) Depletion of VP16 using RNA interference. Latently infected neuron cultures were infected with a lentivirus expressing a VP16-specific short-hairpin RNA [shRNA] (KD) or with a control lentivirus (Con). ShRNAs were allowed to accumulate for 5 days before reactivation was induced with LY294002 and allowed to proceed for 5 days in media lacking ACV. Lysates were prepared and probed by immunoblotting to detect VP16 and the loading control, Rho-GDI. (F) Quantitation of infectious virus by plaque assay. (G) Comparison of viral transcript levels in the absence of VP16. Values from the control culture are plotted relative to the corresponding value from the VP16 shRNA (KD) culture.

Mentions: VP16 is essential for HSV-1 replication, functioning in the assembly of the tegument layer and as a potent transactivator of the IE genes, which in turn drive the expression of the E genes [30], [31]. Recombinant viruses have been characterized that carry mutations in VP16 that preserve the essential tegument forming function but selectively impair the transactivation function, either by preventing the formation of the VP16-induced complex on DNA response elements found in each IE promoter [32] or by removing the C-terminal transactivation domain [33], [34]. Previous work has shown these mutants can still establish latency in animal models, and depending on the reactivation assay used, can display severe defects in reactivation [35], [36]. To examine the contribution of VP16 to LY294002-induced reactivation in the SCG system, we performed infection studies using in1814, a derivative of strain 17syn+ that contains a 12-bp insertion at codon 397 (Figure 4A) [32]. Neuron cultures were infected in the presence of ACV with equal titers (MOI = 1) of in1814 or a repaired version (in1814R) that behaves similar to wild type strain 17syn+ and allowed to establish latency. To obtain equivalent titers each virus stock was grown on complementing human U2OS cells and then plaque assayed on rat embryo fibroblasts in the presence of 5 mM hexamethylene bisacetamide (HMBA), a differentiation agent that overcomes the requirement for transactivation by VP16 during productive replication at low multiplicity [37]. The in1814 and in1814R viruses established latency at a similar frequency as judged by quantitation of viral genomic DNA after six days in the presence of ACV (Figure 4B). Slightly more genomes were detected with in1814, however, this difference was not statistically significant (P>0.29). The ability of in1814 to establish latency mirrors its behavior in several latency models [33], [35], [36], [38], [39]. Next, latently infected cultures were induced with LY294002 and the yield of infectious virus was determined by plaque assay on Vero cells in the presence of HMBA (Figure 4C). In spite of the similar numbers of genome at the time of induction, we observed a profound difference in the yield of infectious virus. Indeed, over multiple experiments we were unable to detect a single infectious particle for in1814. This indicates that loss of the transactivation function of VP16 has a highly deleterious effect on the ability of HSV-1 to reactivate successfully in cultured neurons following a specific stimulus. This agrees with previous studies using the murine hyperthermal stress model for reactivation [36].


Transient reversal of episome silencing precedes VP16-dependent transcription during reactivation of latent HSV-1 in neurons.

Kim JY, Mandarino A, Chao MV, Mohr I, Wilson AC - PLoS Pathog. (2012)

Transactivation function of VP16 is required during Phase II.(A) Structure of VP16 showing the 12-bp insertion (in1814) between the structured N-terminal domain and the C-terminal activation domain (AD) that disrupts VP16-induced complex assembly [32]. (B) SCG neurons were infected with mutant (in1814) or marker rescue (in1814R) viruses (MOI = 1) in the presence of 100 µM acyclovir and maintained for 7 days before measuring the relative amounts of viral genomic DNA by qPCR. (C) Reactivation was induced with 20 µM LY294002 in media lacking ACV and maintained for 7 days before harvest and plaque assay to detect infectious virus. (D) Comparison of viral transcript levels during reactivation by in1814 (‘M’) and in1814R (‘R’) at 15 and 20 h post-induction (Phase I) and at 72 h post-induction (Phase II). For each time point, transcript levels from the in1814 (‘M’) sample were set to 1 and the value for the corresponding transcript from in1814R (‘R’) plotted as the fold difference. (E) Depletion of VP16 using RNA interference. Latently infected neuron cultures were infected with a lentivirus expressing a VP16-specific short-hairpin RNA [shRNA] (KD) or with a control lentivirus (Con). ShRNAs were allowed to accumulate for 5 days before reactivation was induced with LY294002 and allowed to proceed for 5 days in media lacking ACV. Lysates were prepared and probed by immunoblotting to detect VP16 and the loading control, Rho-GDI. (F) Quantitation of infectious virus by plaque assay. (G) Comparison of viral transcript levels in the absence of VP16. Values from the control culture are plotted relative to the corresponding value from the VP16 shRNA (KD) culture.
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Related In: Results  -  Collection

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ppat-1002540-g004: Transactivation function of VP16 is required during Phase II.(A) Structure of VP16 showing the 12-bp insertion (in1814) between the structured N-terminal domain and the C-terminal activation domain (AD) that disrupts VP16-induced complex assembly [32]. (B) SCG neurons were infected with mutant (in1814) or marker rescue (in1814R) viruses (MOI = 1) in the presence of 100 µM acyclovir and maintained for 7 days before measuring the relative amounts of viral genomic DNA by qPCR. (C) Reactivation was induced with 20 µM LY294002 in media lacking ACV and maintained for 7 days before harvest and plaque assay to detect infectious virus. (D) Comparison of viral transcript levels during reactivation by in1814 (‘M’) and in1814R (‘R’) at 15 and 20 h post-induction (Phase I) and at 72 h post-induction (Phase II). For each time point, transcript levels from the in1814 (‘M’) sample were set to 1 and the value for the corresponding transcript from in1814R (‘R’) plotted as the fold difference. (E) Depletion of VP16 using RNA interference. Latently infected neuron cultures were infected with a lentivirus expressing a VP16-specific short-hairpin RNA [shRNA] (KD) or with a control lentivirus (Con). ShRNAs were allowed to accumulate for 5 days before reactivation was induced with LY294002 and allowed to proceed for 5 days in media lacking ACV. Lysates were prepared and probed by immunoblotting to detect VP16 and the loading control, Rho-GDI. (F) Quantitation of infectious virus by plaque assay. (G) Comparison of viral transcript levels in the absence of VP16. Values from the control culture are plotted relative to the corresponding value from the VP16 shRNA (KD) culture.
Mentions: VP16 is essential for HSV-1 replication, functioning in the assembly of the tegument layer and as a potent transactivator of the IE genes, which in turn drive the expression of the E genes [30], [31]. Recombinant viruses have been characterized that carry mutations in VP16 that preserve the essential tegument forming function but selectively impair the transactivation function, either by preventing the formation of the VP16-induced complex on DNA response elements found in each IE promoter [32] or by removing the C-terminal transactivation domain [33], [34]. Previous work has shown these mutants can still establish latency in animal models, and depending on the reactivation assay used, can display severe defects in reactivation [35], [36]. To examine the contribution of VP16 to LY294002-induced reactivation in the SCG system, we performed infection studies using in1814, a derivative of strain 17syn+ that contains a 12-bp insertion at codon 397 (Figure 4A) [32]. Neuron cultures were infected in the presence of ACV with equal titers (MOI = 1) of in1814 or a repaired version (in1814R) that behaves similar to wild type strain 17syn+ and allowed to establish latency. To obtain equivalent titers each virus stock was grown on complementing human U2OS cells and then plaque assayed on rat embryo fibroblasts in the presence of 5 mM hexamethylene bisacetamide (HMBA), a differentiation agent that overcomes the requirement for transactivation by VP16 during productive replication at low multiplicity [37]. The in1814 and in1814R viruses established latency at a similar frequency as judged by quantitation of viral genomic DNA after six days in the presence of ACV (Figure 4B). Slightly more genomes were detected with in1814, however, this difference was not statistically significant (P>0.29). The ability of in1814 to establish latency mirrors its behavior in several latency models [33], [35], [36], [38], [39]. Next, latently infected cultures were induced with LY294002 and the yield of infectious virus was determined by plaque assay on Vero cells in the presence of HMBA (Figure 4C). In spite of the similar numbers of genome at the time of induction, we observed a profound difference in the yield of infectious virus. Indeed, over multiple experiments we were unable to detect a single infectious particle for in1814. This indicates that loss of the transactivation function of VP16 has a highly deleterious effect on the ability of HSV-1 to reactivate successfully in cultured neurons following a specific stimulus. This agrees with previous studies using the murine hyperthermal stress model for reactivation [36].

Bottom Line: This allows synthesis of viral regulatory proteins including VP16, which accumulate in the cytoplasm of the host neuron.During the second phase, VP16 and its cellular cofactor HCF-1, which is also predominantly cytoplasmic, concentrate in the nucleus where they assemble an activator complex on viral promoters.Thus regulated localization of de novo synthesized VP16 is likely to be a critical determinant of HSV-1 reactivation in sympathetic neurons.

View Article: PubMed Central - PubMed

Affiliation: Department of Microbiology, New York University School of Medicine, New York, New York, USA.

ABSTRACT
Herpes simplex virus type-1 (HSV-1) establishes latency in peripheral neurons, creating a permanent source of recurrent infections. The latent genome is assembled into chromatin and lytic cycle genes are silenced. Processes that orchestrate reentry into productive replication (reactivation) remain poorly understood. We have used latently infected cultures of primary superior cervical ganglion (SCG) sympathetic neurons to profile viral gene expression following a defined reactivation stimulus. Lytic genes are transcribed in two distinct phases, differing in their reliance on protein synthesis, viral DNA replication and the essential initiator protein VP16. The first phase does not require viral proteins and has the appearance of a transient, widespread de-repression of the previously silent lytic genes. This allows synthesis of viral regulatory proteins including VP16, which accumulate in the cytoplasm of the host neuron. During the second phase, VP16 and its cellular cofactor HCF-1, which is also predominantly cytoplasmic, concentrate in the nucleus where they assemble an activator complex on viral promoters. The transactivation function supplied by VP16 promotes increased viral lytic gene transcription leading to the onset of genome amplification and the production of infectious viral particles. Thus regulated localization of de novo synthesized VP16 is likely to be a critical determinant of HSV-1 reactivation in sympathetic neurons.

Show MeSH
Related in: MedlinePlus