Analysis of Zika virus capsid-Aedes aegypti mosquito interactome reveals pro-viral host factors critical for establishing infection.

The escalating global prevalence of arboviral diseases emphasizes the need to improve our understanding of their biology. Research in this area has been hindered by the lack of molecular tools for studying virus-mosquito interactions. Here, we develop an Aedes aegypti cell line which stably expresses Zika virus (ZIKV) capsid proteins in order to study virus-vector protein-protein interactions through quantitative label-free proteomics. We identify 157 interactors and show that eight have potentially pro-viral activity during ZIKV infection in mosquito cells. Notably, silencing of transitional endoplasmic reticulum protein TER94 prevents ZIKV capsid degradation and significantly reduces viral replication. Similar results are observed if the TER94 ortholog (VCP) functioning is blocked with inhibitors in human cells. In addition, we show that an E3 ubiquitin-protein ligase, UBR5, mediates the interaction between TER94 and ZIKV capsid. Our study demonstrates a pro-viral function for TER94/VCP during ZIKV infection that is conserved between human and mosquito cells.

Single-particle measurements of filamentous influenza virions reveal damage induced by freezing.

Clinical isolates of influenza virus produce pleiomorphic virions, ranging from small spheres to elongated filaments. The filaments are seemingly adaptive in natural infections, but their basic functional properties are poorly understood and functional studies of filaments often report contradictory results. This may be due to artefactual damage from routine laboratory handling, an issue which has been noted several times without being explored in detail. To determine whether standard laboratory techniques could damage filaments, we used immunofluorescence microscopy to rapidly and reproducibly quantify and characterize the dimensions of filaments. Most of the techniques we tested had minimal impact on filaments, but freezing to -70 °C, a standard storage step before carrying out functional studies on influenza viruses, severely reduced their concentration, median length and the infectivity of the whole virion population. We noted that damage from freezing is likely to have affected most of the functional studies of filaments performed to date, and to address this we show that it can be mitigated by snap-freezing or incorporating the cryoprotectant DMSO. We recommend that functional studies of filaments characterize virion populations prior to analysis to ensure reproducibility, and that they use unfrozen samples if possible and cryoprotectants if not. These basic measures will support the robust functional characterizations of filaments that are required to understand their roles in natural influenza virus infections.

Understanding Influenza.

Influenza, a serious illness of humans and domesticated animals, has been studied intensively for many years. It therefore provides an example of how much we can learn from detailed studies of an infectious disease and of how even the most intensive scientific research leaves further questions to answer. This introduction is written for researchers who have become interested in one of these unanswered questions, but who may not have previously worked on influenza. To investigate these questions, researchers must not only have a firm grasp of relevant methods and protocols; they must also be familiar with the basic details of our current understanding of influenza. This article therefore briefly covers the burden of disease that has driven influenza research, summarizes how our thinking about influenza has evolved over time, and sets out key features of influenza viruses by discussing how we classify them and what we understand of their replication. It does not aim to be comprehensive, as any researcher will read deeply into the specific areas that have grasped their interest. Instead, it aims to provide a general summary of how we came to think about influenza in the way we do now, in the hope that the reader's own research will help us to understand it better.

Purification and Proteomics of Influenza Virions.

This chapter describes a basic workflow for analyzing the protein composition of influenza virions. In order to obtain suitable material, the chapter describes how to concentrate influenza virions from the growth media of infected cells and to purify them by ultracentrifugation through a density gradient. This approach is also suitable for purifying influenza virions from the allantoic fluid of embryonated chicken eggs. As a small quantity of microvesicles are co-purified with virions, optional steps are included to increase the stringency of purification by enriching material with viral receptor binding and cleaving activity. Material purified in this way can be used for a variety of downstream applications, including proteomics. As a detailed example of this, the chapter also describes a standard workflow for analyzing the protein composition of concentrated virions by liquid chromatography and tandem mass spectrometry.

Influenza Virus.

This infographic briefly summarises the natural history, replication cycle, and pathogenesis of influenza viruses, the cause of seasonal influenza and of influenza pandemics. Influenza viruses infect many vertebrates, with Influenza A, B and C viruses (IAV, IBV, and ICV) infecting humans. High mutation rates allow the evasion of immunity. IAV from different host species can 'reassort' their segmented genomes, producing pandemic strains that are antigenically novel but otherwise well adapted to humans. The 'Great Influenza' pandemic of 1918 remains the worst outbreak of infectious disease in history. There is concern that highly pathogenic avian influenza viruses of the H5 and H7 subtypes may evolve to cause similar pandemics. In humans, influenza viruses infect the respiratory epithelium. The haemagglutinin (HA) proteins of IAV and IBV, or the haemagglutinin-esterase-fusion (HEF) proteins of ICV, bind sialic acid, causing endocytosis. Unusually among RNA viruses, the viral genome replicates in the nucleus. New viruses assemble at the cell surface and are released by the receptor-cleaving neuraminidase (NA) proteins of IAV and IBV or the ICV HEF protein.

Filamentous influenza viruses.

Clinical isolates of influenza virus produce pleomorphic virus particles, including extremely long filamentous virions. In contrast, strains of influenza that have adapted to laboratory growth typically produce only spherical virions. As a result, the filamentous phenotype has been overlooked in most influenza virus research. Recent advances in imaging and improved animal models have highlighted the distinct structure and functional relevance of filamentous virions. In this review we summarize what is currently known about these strikingly elongated virus particles and discuss their possible roles in clinical infections.

MAIT cells are activated during human viral infections.

Mucosal-associated invariant T (MAIT) cells are abundant in humans and recognize bacterial ligands. Here, we demonstrate that MAIT cells are also activated during human viral infections in vivo. MAIT cells activation was observed during infection with dengue virus, hepatitis C virus and influenza virus. This activation-driving cytokine release and Granzyme B upregulation-is TCR-independent but dependent on IL-18 in synergy with IL-12, IL-15 and/or interferon-α/ß. IL-18 levels and MAIT cell activation correlate with disease severity in acute dengue infection. Furthermore, HCV treatment with interferon-α leads to specific MAIT cell activation in vivo in parallel with an enhanced therapeutic response. Moreover, TCR-independent activation of MAIT cells leads to a reduction of HCV replication in vitro mediated by IFN-γ. Together these data demonstrate MAIT cells are activated following viral infections, and suggest a potential role in both host defence and immunopathology.

Regulation of influenza A virus nucleoprotein oligomerization by phosphorylation.

In the influenza virus ribonucleoprotein complex, the oligomerization of the nucleoprotein is mediated by an interaction between the tail-loop of one molecule and the groove of the neighboring molecule. In this study, we show that phosphorylation of a serine residue (S165) within the groove of influenza A virus nucleoprotein inhibits oligomerization and, consequently, ribonucleoprotein activity and viral growth. We propose that nucleoprotein oligomerization in infected cells is regulated by reversible phosphorylation.

Interactome analysis of the influenza A virus transcription/replication machinery identifies protein phosphatase 6 as a cellular factor required for efficient virus replication.

UNLABELLED: 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 (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. IMPORTANCE: Influenza 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.

Conserved and host-specific features of influenza virion architecture.

Viruses use virions to spread between hosts, and virion composition is therefore the primary determinant of viral transmissibility and immunogenicity. However, the virions of many viruses are complex and pleomorphic, making them difficult to analyse in detail. Here we address this by identifying and quantifying virion proteins with mass spectrometry, producing a complete and quantified model of the hundreds of host-encoded and viral proteins that make up the pleomorphic virions of influenza viruses. We show that a conserved influenza virion architecture is maintained across diverse combinations of virus and host. This 'core' architecture, which includes substantial quantities of host proteins as well as the viral protein NS1, is elaborated with abundant host-dependent features. As a result, influenza virions produced by mammalian and avian hosts have distinct protein compositions. Finally, we note that influenza virions share an underlying protein composition with exosomes, suggesting that influenza virions form by subverting microvesicle production.

Transport of the influenza virus genome from nucleus to nucleus.

The segmented genome of an influenza virus is encapsidated into ribonucleoprotein complexes (RNPs). Unusually among RNA viruses, influenza viruses replicate in the nucleus of an infected cell, and their RNPs must therefore recruit host factors to ensure transport across a number of cellular compartments during the course of an infection. Recent studies have shed new light on many of these processes, including the regulation of nuclear export, genome packaging, mechanisms of virion assembly and viral entry and, in particular, the identification of Rab11 on recycling endosomes as a key mediator of RNP transport and genome assembly. This review uses these recent gains in understanding to describe in detail the journey of an influenza A virus RNP from its synthesis in the nucleus through to its entry into the nucleus of a new host cell.

Mapping the phosphoproteome of influenza A and B viruses by mass spectrometry.

Protein phosphorylation is a common post-translational modification in eukaryotic cells and has a wide range of functional effects. Here, we used mass spectrometry to search for phosphorylated residues in all the proteins of influenza A and B viruses--to the best of our knowledge, the first time such a comprehensive approach has been applied to a virus. We identified 36 novel phosphorylation sites, as well as confirming 3 previously-identified sites. N-terminal processing and ubiquitination of viral proteins was also detected. Phosphorylation was detected in the polymerase proteins (PB2, PB1 and PA), glycoproteins (HA and NA), nucleoprotein (NP), matrix protein (M1), ion channel (M2), non-structural protein (NS1) and nuclear export protein (NEP). Many of the phosphorylation sites detected were conserved between influenza virus genera, indicating the fundamental importance of phosphorylation for all influenza viruses. Their structural context indicates roles for phosphorylation in regulating viral entry and exit (HA and NA); nuclear localisation (PB2, M1, NP, NS1 and, through NP and NEP, of the viral RNA genome); and protein multimerisation (NS1 dimers, M2 tetramers and NP oligomers). Using reverse genetics we show that for NP of influenza A viruses phosphorylation sites in the N-terminal NLS are important for viral growth, whereas mutating sites in the C-terminus has little or no effect. Mutating phosphorylation sites in the oligomerisation domains of NP inhibits viral growth and in some cases transcription and replication of the viral RNA genome. However, constitutive phosphorylation of these sites is not optimal. Taken together, the conservation, structural context and functional significance of phosphorylation sites implies a key role for phosphorylation in influenza biology. By identifying phosphorylation sites throughout the proteomes of influenza A and B viruses we provide a framework for further study of phosphorylation events in the viral life cycle and suggest a range of potential antiviral targets.

Identification of a novel splice variant form of the influenza A virus M2 ion channel with an antigenically distinct ectodomain.

Segment 7 of influenza A virus produces up to four mRNAs. Unspliced transcripts encode M1, spliced mRNA2 encodes the M2 ion channel, while protein products from spliced mRNAs 3 and 4 have not previously been identified. The M2 protein plays important roles in virus entry and assembly, and is a target for antiviral drugs and vaccination. Surprisingly, M2 is not essential for virus replication in a laboratory setting, although its loss attenuates the virus. To better understand how IAV might replicate without M2, we studied the reversion mechanism of an M2-null virus. Serial passage of a virus lacking the mRNA2 splice donor site identified a single nucleotide pseudoreverting mutation, which restored growth in cell culture and virulence in mice by upregulating mRNA4 synthesis rather than by reinstating mRNA2 production. We show that mRNA4 encodes a novel M2-related protein (designated M42) with an antigenically distinct ectodomain that can functionally replace M2 despite showing clear differences in intracellular localisation, being largely retained in the Golgi compartment. We also show that the expression of two distinct ion channel proteins is not unique to laboratory-adapted viruses but, most notably, was also a feature of the 1983 North American outbreak of H5N2 highly pathogenic avian influenza virus. In identifying a 14th influenza A polypeptide, our data reinforce the unexpectedly high coding capacity of the viral genome and have implications for virus evolution, as well as for understanding the role of M2 in the virus life cycle.

Characterization of the interaction between the influenza A virus polymerase subunit PB1 and the host nuclear import factor Ran-binding protein 5.

The influenza A virus RNA polymerase is a heterotrimer that transcribes and replicates the viral genome in the cell nucleus. Newly synthesized RNA polymerase subunits must therefore be imported into the nucleus during an infection. While various models have been proposed for this process, the consensus is that the polymerase basic protein PB1 and polymerase acidic protein PA subunits form a dimer in the cytoplasm and are transported into the nucleus by the beta-importin Ran-binding protein 5 (RanBP5), with the PB2 subunit imported separately to complete the trimeric complex. In this study, we characterized the interaction of PB1 with RanBP5 further and assessed its importance for viral growth. In particular, we found that the N-terminal region of PB1 mediates its binding to RanBP5 and that basic residues in a nuclear localization signal are required for RanBP5 binding. Mutating these basic residues to alanines does not prevent PB1 forming a dimer with PA, but does reduce RanBP5 binding. RanBP5-binding mutations reduce, though do not entirely prevent, the nuclear accumulation of PB1. Furthermore, mutations affecting RanBP5 binding are incompatible with or severely attenuate viral growth, providing further support for a key role for RanBP5 in the influenza A virus life cycle.

Genome packaging in influenza A virus.

The negative-sense RNA genome of influenza A virus is composed of eight segments, which encode 12 proteins between them. At the final stage of viral assembly, these genomic virion (v)RNAs are incorporated into the virion as it buds from the apical plasma membrane of the cell. Genome segmentation confers evolutionary advantages on the virus, but also poses a problem during virion assembly as at least one copy of each of the eight segments is required to produce a fully infectious virus particle. Historically, arguments have been presented in favour of a specific packaging mechanism that ensures incorporation of a full genome complement, as well as for an alternative model in which segments are chosen at random but packaged in sufficient numbers to ensure that a reasonable proportion of virions are viable. The question has seen a resurgence of interest in recent years leading to a consensus that the vast majority of virions contain no more than eight segments and that a specific mechanism does indeed function to select one copy of each vRNA. This review summarizes work leading to this conclusion. In addition, we describe recent progress in identifying the specific packaging signals and discuss likely mechanisms by which these RNA elements might operate.

Characterisation of influenza A viruses with mutations in segment 5 packaging signals.

Influenza A virus vRNA segments contain specific packaging signals at their termini that overlap the coding regions. To further characterise segment 5 packaging signals, we introduced synonymous mutations into the terminal coding regions of the vRNA and characterised the replicative fitness of the resulting viruses. Most mutations tested were well-tolerated, but a virus with alterations to NP codons 464-466, near the 5'-end of the vRNA, produced small plaques and replicated to around one-tenth of the level of wild type virus. The mutant virus supported normal levels of NP and segment 5 vRNA synthesis but packaged reduced levels of both segment 5 and segment 3 into virus particles. This suggests an interaction between segments 3 and 5 during influenza A virus assembly.

Studies of an influenza A virus temperature-sensitive mutant identify a late role for NP in the formation of infectious virions.

The influenza A virus nucleoprotein (NP) is a single-stranded RNA-binding protein that encapsidates the virus genome and has essential functions in viral-RNA synthesis. Here, we report the characterization of a temperature-sensitive (ts) NP mutant (US3) originally generated in fowl plague virus (A/chicken/Rostock/34). Sequence analysis revealed a single mutation, M239L, in NP, consistent with earlier mapping studies assigning the ts lesion to segment 5. Introduction of this mutation into A/PR/8/34 virus by reverse genetics produced a ts phenotype, confirming the identity of the lesion. Despite an approximately 100-fold drop in the viral titer at the nonpermissive temperature, the mutant US3 polypeptide supported wild-type (WT) levels of genome transcription, replication, and protein synthesis, indicating a late-stage defect in function of the NP polypeptide. Nucleocytoplasmic trafficking of the US3 NP was also normal, and the virus actually assembled and released around sixfold more virus particles than the WT virus, with normal viral-RNA content. However, the particle/PFU ratio of these virions was 50-fold higher than that of WT virus, and many particles exhibited an abnormal morphology. Reverse-genetics studies in which A/PR/8/34 segment 7 was swapped with sequences from other strains of virus revealed a profound incompatibility between the M239L mutation and the A/Udorn/72 M1 gene, suggesting that the ts mutation affects M1-NP interactions. Thus, we have identified a late-acting defect in NP that, separate from its function in RNA synthesis, indicates a role for the polypeptide in virion assembly, most likely involving M1 as a partner.

Mutational analysis of cis-acting RNA signals in segment 7 of influenza A virus.

The genomic viral RNA (vRNA) segments of influenza A virus contain specific packaging signals at their termini that overlap the coding regions. To further characterize cis-acting signals in segment 7, we introduced synonymous mutations into the terminal coding regions. Mutation of codons that are normally highly conserved reduced virus growth in embryonated eggs and MDCK cells between 10- and 1,000-fold compared to that of the wild-type virus, whereas similar alterations to nonconserved codons had little effect. In all cases, the growth-impaired viruses showed defects in virion assembly and genome packaging. In eggs, nearly normal numbers of virus particles that in aggregate contained apparently equimolar quantities of the eight segments were formed, but with about fourfold less overall vRNA content than wild-type virions, suggesting that, on average, fewer than eight segments per particle were packaged. Concomitantly, the particle/PFU and segment/PFU ratios of the mutant viruses showed relative increases of up to 300-fold, with the behavior of the most defective viruses approaching that predicted for random segment packaging. Fluorescent staining of infected cells for the nucleoprotein and specific vRNAs confirmed that most mutant virus particles did not contain a full genome complement. The specific infectivity of the mutant viruses produced by MDCK cells was also reduced, but in this system, the mutations also dramatically reduced virion production. Overall, we conclude that segment 7 plays a key role in the influenza A virus genome packaging process, since mutation of as few as 4 nucleotides can dramatically inhibit infectious virus production through disruption of vRNA packaging.