Isolation and characterization of the positive-sense replicative intermediate of a negative-strand RNA virus

110

Retinoic acid-inducible gene I (RIG-I) is an important innate immune sensor that recognizes viral RNA in the cytoplasm. Its nonself recognition largely depends on the unique RNA structures imposed by viral RNA. The panhandle structure residing in the influenza A virus (IAV) genome, whose primary function is to serve as the viral promoter for transcription and replication, has been proposed to be a RIG-I agonist. However, this has never been proved experimentally. Here, we employed multiple approaches to determine if the IAV panhandle structure is directly involved in RIG-I activation and type I interferon (IFN) induction. First, in porcine alveolar macrophages, we demonstrated that the viral genomic coding region is dispensable for RIG-I-dependent IFN induction. Second, using in vitro-synthesized hairpin RNA, we showed that the IAV panhandle structure could directly bind to RIG-I and stimulate IFN production. Furthermore, we investigated the contributions of the wobble base pairs, mismatch, and unpaired nucleotides within the wild-type panhandle structure to RIG-I activation. Elimination of these destabilizing elements within the panhandle structure promoted RIG-I activation and IFN induction. Given the function of the panhandle structure as the viral promoter, we further monitored the promoter activity of these panhandle variants and found that viral replication was moderately affected, whereas viral transcription was impaired dramatically. In all, our results indicate that the IAV panhandle promoter region adopts a nucleotide composition that is optimal for balanced viral RNA synthesis and suboptimal for RIG-I activation. IMPORTANCEThe IAV genomic panhandle structure has been proposed to be an RIG-I agonist due to its partial complementarity; however, this has not been experimentally confirmed. Here, we provide direct evidence that the IAV panhandle structure is competent in, and sufficient for, RIG-I activation and IFN induction. By constructing panhandle variants with increased complementarity, we demonstrated that the wild-type panhandle structure could be modified to enhance RIG-I activation and IFN induction. These panhandle variants posed moderate influence on viral replication but dramatic impairment of viral transcription. These results indicate that the IAV panhandle promoter region adopts a nucleotide composition to achieve optimal balance of viral RNA synthesis and suboptimal RIG-I activation. Our results highlight the multifunctional role of the IAV panhandle promoter region in the virus life cycle and offer novel insights into the development of antiviral agents aiming to boost RIG-I signaling or virus attenuation by manipulating this conserved region.

Section: Resultssupporting

confidence: 54%

Section: Resultsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

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Influenza A Virus Panhandle Structure Is Directly Involved in RIG-I Activation and Interferon Induction

Liu¹,

Park²,

Pyo

et al. 2015

110

“…The viral RNA is wound around the surface of the nucleocapsid protein (NP) subunits and associates with the RNA polymerase complex, comprised of the PA, PB1, and PB2 proteins. NP binds single-stranded RNA without sequence specificity (16,17), but the virus packages only RNPs associated with viral RNA (18). The integrity of the viral RNP (vRNP) is maintained at least in part by NP-NP interactions, and RNP depleted of RNA maintains the characteristic morphology of vRNP (19).…”

mentioning

confidence: 99%

Lateral Organization of Influenza Virus Proteins in the Budozone Region of the Plasma Membrane

Leser

Lamb

2017

Influenza virus assembles and buds at the plasma membrane of virusinfected cells. The viral proteins assemble at the same site on the plasma membrane for budding to occur. This involves a complex web of interactions among viral proteins. Some proteins, like hemagglutinin (HA), NA, and M2, are integral membrane proteins. M1 is peripherally membrane associated, whereas NP associates with viral RNA to form an RNP complex that associates with the cytoplasmic face of the plasma membrane. Furthermore, HA and NP have been shown to be concentrated in cholesterol-rich membrane raft domains, whereas M2, although containing a cholesterol binding motif, is not raft associated. Here we identify viral proteins in planar sheets of plasma membrane using immunogold staining. The distribution of these proteins was examined individually and pairwise by using the Ripley K function, a type of nearest-neighbor analysis. Individually, HA, NA, M1, M2, and NP were shown to self-associate in or on the plasma membrane. HA and M2 are strongly coclustered in the plasma membrane; however, in the case of NA and M2, clustering depends upon the expression system used. Despite both proteins being raft resident, HA and NA occupy distinct but adjacent membrane domains. M2 and M1 strongly cocluster, but the association of M1 with HA or NA is dependent upon the means of expression. The presence of HA and NP at the site of budding depends upon the coexpression of other viral proteins. Similarly, M2 and NP occupy separate compartments, but an association can be bridged by the coexpression of M1.IMPORTANCE The complement of influenza virus proteins necessary for the budding of progeny virions needs to accumulate at budozones. This is complicated by HA and NA residing in lipid raft-like domains, whereas M2, although an integral membrane protein, is not raft associated. Other necessary protein components such as M1 and NP are peripherally associated with the membrane. Our data define spatial relationships between viral proteins in the plasma membrane. Some proteins, such as HA and M2, inherently cocluster within the membrane, although M2 is found mostly at the periphery of regions of HA, consistent with the proposed role of M2 in scission at the end of budding. The association between some pairs of influenza virus proteins, such as M2 and NP, appears to be brokered by additional influenza virus proteins, in this case M1. HA and NA, while raft associated, reside in distinct domains, reflecting their distributions in the viral membrane.

“…Recombinant influenza A/NT/60/68 virus RdRP with a protein A tag on the PB2 subunit was expressed using the MultiBac system in Sf9 insect cells (56) as previously described (55) [Roche], and 100 g/ml RNase A). The lysate was clarified by centrifugation at 35,000 ϫ g for 45 min at 4°C, and the supernatant was incubated with washed IgG Sepharose (approximately 2 ml bead slurry per liter of original culture; GE Healthcare) for 3 h at 4°C.…”

mentioning

confidence: 99%

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

York

Hutchinson

Fodor

2014

Self Cite

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...