Interactome Analysis of the Influenza A Virus Transcription/Replication Machinery Identifies Protein Phosphatase 6 as a Cellular Factor Required for Efficient Virus Replication

Abstract

The 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 ( Viruses are obligate intracellular parasites that have been compelled by evolutionary processes to encode a minimal number of proteins for the completion of an infectious life cycle, in order to infect naive hosts and to persist in nature. This is exemplified by RNA viruses, the genomes of which are generally smaller than the genomes of DNA viruses, and therefore they greatly rely on the exploitation and subversion of host cellular proteins, structures, and pathways to facilitate virus replication (1). Transcription and replication of the influenza A virus genome are performed by the virally encoded RNA-dependent RNA polymerase (RdRP), a major determinant of species tropism and pathogenicity and a key player in the adaptation of avian influenza A viruses to mammalian hosts. These characteristics of the viral RdRP are thought to be governed by numerous interactions with cellular factors (reviewed in references 2 and 3). It is therefore of great importance to understand the relationship between the viral transcription/replication machinery and host cellular factors in order to understand how the host cell can influence the function of the viral transcription/replication machinery and vice versa. Insight into the molecular biology of these relationships could lead to novel antiviral strategies and has the potential to identify host-specific interactions that would act as a barrier to pandemic emergence.The genome of influenza A virus, like that of other members of the Orthomyxoviridae, is segmented and consists of eight individual viral ribonucleoprotein (vRNP) complexes. Each vRNP contains single-stranded viral RNA (vRNA) that is en...

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