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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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VP16 is expressed during Phase I but is localized to the cytoplasm.(A) Detection of VP16 in latently infected neurons by indirect immunofluorescence microscopy using an α-VP16 rabbit polyclonal antibody (red). Individual nuclei were visualized with DAPI (blue). Three representative fields are shown from cover slips collected at 20 h post-induction (Phase I) and single fields from 48 and 96 h (Phase II). An arrowhead is used to indicate the cell body of neurons with a predominantly cytoplasmic VP16 signal, whereas those displaying a strong nuclear VP16 signal are indicated with an asterisk. Neurons that do not express VP16 are left unmarked but are evident from the DAPI stained nuclei. (B) Quantitation of the immunofluorescence analysis. Numbers of VP16 positive neurons are expressed as a percentage of all neurons scored. Data is compiled from three independent reactivation experiments. Between 500 and 3,000 neurons were scored per cover slip. (C) Percentage of VP16 positive neurons with a predominantly nuclear rather than cytoplasmic α-VP16 signal.
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ppat-1002540-g006: VP16 is expressed during Phase I but is localized to the cytoplasm.(A) Detection of VP16 in latently infected neurons by indirect immunofluorescence microscopy using an α-VP16 rabbit polyclonal antibody (red). Individual nuclei were visualized with DAPI (blue). Three representative fields are shown from cover slips collected at 20 h post-induction (Phase I) and single fields from 48 and 96 h (Phase II). An arrowhead is used to indicate the cell body of neurons with a predominantly cytoplasmic VP16 signal, whereas those displaying a strong nuclear VP16 signal are indicated with an asterisk. Neurons that do not express VP16 are left unmarked but are evident from the DAPI stained nuclei. (B) Quantitation of the immunofluorescence analysis. Numbers of VP16 positive neurons are expressed as a percentage of all neurons scored. Data is compiled from three independent reactivation experiments. Between 500 and 3,000 neurons were scored per cover slip. (C) Percentage of VP16 positive neurons with a predominantly nuclear rather than cytoplasmic α-VP16 signal.

Mentions: Although the transactivation function of VP16 is dispensable for Phase I, VP16 mRNA is readily detected (Figures 2–4). This prompted us to ask when the VP16 protein is synthesized. To do this we performed indirect immunofluorescence microscopy on latently infected neurons undergoing reactivation (Figure 6). Using a rabbit polyclonal antibody against VP16, we detected a strong signal in approximately 5% of the neurons at the onset of reactivation (0 h, see discussion), which rose to more than 20% of the culture at 15 and 20 h, which corresponds to Phase I (Figure 6A and 6B). The signal corresponds to VP16 because the antibody recognizes a single species of the correct size on immunoblots (Figure 4E), is absent from the remaining 80% or more neurons in the cultures (Figure 6A) and can be suppressed or eliminated by coinfection with the VP16 shRNA lentivirus (data not shown).


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)

VP16 is expressed during Phase I but is localized to the cytoplasm.(A) Detection of VP16 in latently infected neurons by indirect immunofluorescence microscopy using an α-VP16 rabbit polyclonal antibody (red). Individual nuclei were visualized with DAPI (blue). Three representative fields are shown from cover slips collected at 20 h post-induction (Phase I) and single fields from 48 and 96 h (Phase II). An arrowhead is used to indicate the cell body of neurons with a predominantly cytoplasmic VP16 signal, whereas those displaying a strong nuclear VP16 signal are indicated with an asterisk. Neurons that do not express VP16 are left unmarked but are evident from the DAPI stained nuclei. (B) Quantitation of the immunofluorescence analysis. Numbers of VP16 positive neurons are expressed as a percentage of all neurons scored. Data is compiled from three independent reactivation experiments. Between 500 and 3,000 neurons were scored per cover slip. (C) Percentage of VP16 positive neurons with a predominantly nuclear rather than cytoplasmic α-VP16 signal.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3285597&req=5

ppat-1002540-g006: VP16 is expressed during Phase I but is localized to the cytoplasm.(A) Detection of VP16 in latently infected neurons by indirect immunofluorescence microscopy using an α-VP16 rabbit polyclonal antibody (red). Individual nuclei were visualized with DAPI (blue). Three representative fields are shown from cover slips collected at 20 h post-induction (Phase I) and single fields from 48 and 96 h (Phase II). An arrowhead is used to indicate the cell body of neurons with a predominantly cytoplasmic VP16 signal, whereas those displaying a strong nuclear VP16 signal are indicated with an asterisk. Neurons that do not express VP16 are left unmarked but are evident from the DAPI stained nuclei. (B) Quantitation of the immunofluorescence analysis. Numbers of VP16 positive neurons are expressed as a percentage of all neurons scored. Data is compiled from three independent reactivation experiments. Between 500 and 3,000 neurons were scored per cover slip. (C) Percentage of VP16 positive neurons with a predominantly nuclear rather than cytoplasmic α-VP16 signal.
Mentions: Although the transactivation function of VP16 is dispensable for Phase I, VP16 mRNA is readily detected (Figures 2–4). This prompted us to ask when the VP16 protein is synthesized. To do this we performed indirect immunofluorescence microscopy on latently infected neurons undergoing reactivation (Figure 6). Using a rabbit polyclonal antibody against VP16, we detected a strong signal in approximately 5% of the neurons at the onset of reactivation (0 h, see discussion), which rose to more than 20% of the culture at 15 and 20 h, which corresponds to Phase I (Figure 6A and 6B). The signal corresponds to VP16 because the antibody recognizes a single species of the correct size on immunoblots (Figure 4E), is absent from the remaining 80% or more neurons in the cultures (Figure 6A) and can be suppressed or eliminated by coinfection with the VP16 shRNA lentivirus (data not shown).

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