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Activation of adenoviral gene expression by protein IX is not required for efficient virus replication.

ABSTRACT: The adenovirus (Ad) protein IX (pIX) is a minor component of the Ad capsid and is in part responsible for virion stability; virions lacking pIX are heat labile and lose their infectivity if the DNA content is greater than approximately 35 kb. More recently, pIX has been identified as a transcriptional activator and, in transient-transfection assays, was shown to enhance expression from the E1A, E4, and major late Ad promoters by as much as 70-fold. In this study, we examined the role of pIX's ability to activate transcription during Ad replication. In transient-transfection assays, pIX had a minimal effect on expression from the E1A promoter, increasing expression by only 1.4-fold. We used helper-dependent Ad vectors, which had all Ad protein coding sequences deleted with the exception of E1A and which had capsids that either contained or lacked pIX, to show that pIX derived from decapsidation of the infecting virion does not influence expression of E1A. Similarly, expression of pIX from the Ad genome did not alter the expression levels of E1A. Viruses that had pIX deleted showed a threefold reduction in virus yield and expression of late genes compared to those of a similar virus which encoded pIX. This phenotype could not be rescued by growing the virus in cells which constitutively express pIX. Our results indicate that, although pIX can affect transcription from a variety of viral promoters, it does not appear to play a significant role in activation of Ad promoters during normal Ad replication.

SUBMITTER: Sargent KL

PROVIDER: S-EPMC400331 | biostudies-other | 2004 May

REPOSITORIES: biostudies-other

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Project description:UnlabelledThe negative-sense RNA genome of influenza A virus is transcribed and replicated by the viral RNA-dependent RNA polymerase (RdRP). The viral RdRP is an important host range determinant, indicating that its function is affected by interactions with cellular factors. However, the identities and the roles of most of these factors remain unknown. Here, we employed affinity purification followed by mass spectrometry to identify cellular proteins that interact with the influenza A virus RdRP in infected human cells. We purified RdRPs using a recombinant influenza virus in which the PB2 subunit of the RdRP is fused to a Strep-tag. When this tagged subunit was purified from infected cells, copurifying proteins included the other RdRP subunits (PB1 and PA) and the viral nucleoprotein and neuraminidase, as well as 171 cellular proteins. Label-free quantitative mass spectrometry revealed that the most abundant of these host proteins were chaperones, cytoskeletal proteins, importins, proteins involved in ubiquitination, kinases and phosphatases, and mitochondrial and ribosomal proteins. Among the phosphatases, we identified three subunits of the cellular serine/threonine protein phosphatase 6 (PP6), including the catalytic subunit PPP6C and regulatory subunits PPP6R1 and PPP6R3. PP6 was found to interact directly with the PB1 and PB2 subunits of the viral RdRP, and small interfering RNA (siRNA)-mediated knockdown of the catalytic subunit of PP6 in infected cells resulted in the reduction of viral RNA accumulation and the attenuation of virus growth. These results suggest that PP6 interacts with and positively regulates the activity of the influenza virus RdRP.ImportanceInfluenza A viruses are serious clinical and veterinary pathogens, causing substantial health and economic impacts. In addition to annual seasonal epidemics, occasional global pandemics occur when viral strains adapt to humans from other species. To replicate efficiently and cause disease, influenza viruses must interact with a large number of host factors. The reliance of the viral RNA-dependent RNA polymerase (RdRP) on host factors makes it a major host range determinant. This study describes and quantifies host proteins that interact, directly or indirectly, with a subunit of the RdRP. It increases our understanding of the role of host proteins in viral replication and identifies a large number of potential barriers to pandemic emergence. Identifying host factors allows their importance for viral replication to be tested. Here, we demonstrate a role for the cellular phosphatase PP6 in promoting viral replication, contributing to our emerging knowledge of regulatory phosphorylation in influenza virus biology.

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Activation of adenoviral gene expression by protein IX is not required for efficient virus replication.

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