Abstract
The herpes simplex virus type 1 (HSV-1) immediate early protein, ICP4, participates in the regulation of viral gene expression by both activating and repressing RNA polII transcription. We used affinity purification of ICP4 expressed in infected cells followed by mass spectrometry and western blot analysis to determine the composition of cellular complexes associated with ICP4 throughout infection. ICP4 was associated with TFIID complexes containing a distinct set of TAFs. These complexes were most abundant early, but were detected throughout infection, whereas Mediator was found in ICP4 containing complexes later in infection, indicating a temporal pattern for the utilization of these complexes for the transcription of the viral genome. The form of Mediator copurifying with ICP4 was enriched for the kinase domain and also lacked the activator-specific component, Med26, suggesting that Mediator-ICP4 interactions may be involved in repression of viral transcription. The N-terminal 774 amino acids of ICP4, which retains partial function, were sufficient to form complexes with TFIID and Mediator, although these interactions were not as strong as with full-length ICP4. Additionally, components involved in transcription elongation, chromatin remodeling, and mRNA processing were isolated with ICP4. Together our data indicate that ICP4 plays a more integrated role in mediating HSV transcription, possibly affecting multiple steps in transcription and gene expression.
Highlights
The genome of Herpes Simplex Virus Type 1 (HSV-1) is transcribed by RNA polymerase II (RNA polII) [1]
To examine the temporal characteristics of Infected Cell Polypeptide 4 (ICP4) mediated interactions, KOS and TAPwtICP4 infected samples were collected at 3, 6, and 12 hours post infection as these time points traverse the entire cascade of HSV gene expression. n208 and Tandem Affinity Purification (TAP)-n208 samples were collected at 6h post infection
The studies presented aimed to isolate transcription complexes associated with ICP4 throughout infection to provide a better understanding of the mechanisms underlying expression of the HSV-1 genome
Summary
The genome of Herpes Simplex Virus Type 1 (HSV-1) is transcribed by RNA polymerase II (RNA polII) [1]. Immediate early (IE) genes are transcribed in the absence of prior protein synthesis, largely due to VP16 present in the infecting virion subsequently acting on the promoters of IE genes to activate their transcription [4,5,6]. Functional IE proteins are required for the transcription of early (E) genes [3], the products of which are involved in viral DNA synthesis. The syntheses of IE and E proteins along with viral DNA replication are prerequisites for efficient late (L) transcription, the protein products of which mostly comprise the virion structure or are required for its assembly. The IE protein, Infected Cell Polypeptide 4 (ICP4), is an activator and repressor of transcription. Positioned binding sites in several viral genes mediate transcriptional repression by ICP4
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