Time-Resolved Visualisation of Nearly-Native Influenza A Virus Progeny Ribonucleoproteins and Their Individual Components in Live Infected Cells

Influenza viruses are a global health concern because of the permanent threat of novel emerging strains potentially capable of causing pandemics. Viral ribonucleoproteins (vRNPs) containing genomic RNA segments, nucleoprotein oligomers, and the viral polymerase, play a central role in the viral replication cycle. Our knowledge about critical events such as vRNP assembly and interactions with other viral and cellular proteins is poor and could be substantially improved by time lapse imaging of the infected cells. However, such studies are limited by the difficulty to achieve live-cell compatible labeling of active vRNPs. Previously we designed the first unimpaired recombinant influenza WSN-PB2-GFP11 virus allowing fluorescent labeling of the PB2 subunit of the viral polymerase (Avilov et al., J.Virol. 2012). Here, we simultaneously labeled the viral PB2 protein using the above-mentioned strategy, and virus-encoded progeny RNPs through spontaneous incorporation of transiently expressed NP-mCherry fusion proteins during RNP assembly in live infected cells. This dual labeling enabled us to visualize progeny vRNPs throughout the infection cycle and to characterize independently the mobility, oligomerization status and interactions of vRNP components in the nuclei of live infected cells.     

Affinity purification of influenza virus ribonucleoprotein complexes from the chromatin of infected cells

Like all negative-strand RNA viruses, the genome of influenza viruses is packaged in the form of viral ribonucleoprotein complexes (vRNP), in which the single-stranded genome is encapsidated by the nucleoprotein (NP), and associated with the trimeric polymerase complex consisting of the PA, PB1, and PB2 subunits. However, in contrast to most RNA viruses, influenza viruses perform viral RNA synthesis in the nuclei of infected cells. Interestingly, viral mRNA synthesis uses cellular pre-mRNAs as primers, and it has been proposed that this process takes place on chromatin. Interactions between the viral polymerase and the host RNA polymerase II, as well as between NP and host nucleosomes have also been characterized. Recently, the generation of recombinant influenza viruses encoding a One-Strep-Tag genetically fused to the C-terminus of the PB2 subunit of the viral polymerase (rWSN-PB2-Strep) has been described. These recombinant viruses allow the purification of PB2-containing complexes, including vRNPs, from infected cells. To obtain purified vRNPs, cell cultures are infected, and vRNPs are affinity purified from lysates derived from these cells. However, the lysis procedures used to date have been based on one-step detergent lysis, which, despite the presence of a general nuclease, often extract chromatin-bound material only inefficiently. Our preliminary work suggested that a large portion of nuclear vRNPs were not extracted during traditional cell lysis, and therefore could not be affinity purified. To increase this extraction efficiency, and to separate chromatin-bound from non-chromatin-bound nuclear vRNPs, we adapted a step-wise subcellular extraction protocol to influenza virus-infected cells. Briefly, this procedure first separates the nuclei from the cell and then extracts soluble nuclear proteins (here termed the "nucleoplasmic" fraction). The remaining insoluble nuclear material is then digested with Benzonase, an unspecific DNA/RNA nuclease, followed by two salt extraction steps: first using 150 mM NaCl (termed "ch150"), then 500 mM NaCl ("ch500") (Fig. 1). These salt extraction steps were chosen based on our observation that 500 mM NaCl was sufficient to solubilize over 85% of nuclear vRNPs yet still allow binding of tagged vRNPs to the affinity matrix. After subcellular fractionation of infected cells, it is possible to affinity purify PB2-tagged vRNPs from each individual fraction and analyze their protein and RNA components using Western Blot and primer extension, respectively. Recently, we utilized this method to discover that vRNP export complexes form during late points after infection on the chromatin fraction extracted with 500 mM NaCl (ch500).     

RAB11A is essential for transport of the influenza virus genome to the plasma membrane

Influenza A virus assembly is a complex process that requires the intersection of pathways involved in transporting viral glycoproteins, the matrix protein, and viral genomes, incorporated in the viral ribonucleoprotein (vRNP) complex, to plasma membrane sites of virion formation. Among these virion components, the mechanism of vRNP delivery is the most incompletely understood. Here, we reveal a functional relationship between the cellular Rab11 GTPase isoform, RAB11A, and vRNPs and show that RAB11A is indispensable for proper vRNP transport to the plasma membrane. Using an immunofluorescence-based assay with a monoclonal antibody that recognizes nucleoprotein in the form of vRNP, we demonstrate association between RAB11A and vRNPs at all stages of vRNP cytoplasmic transport. Abrogation of RAB11A expression through small interfering RNA (siRNA) treatment or disruption of RAB11A function by overexpression of dominant negative or constitutively active proteins caused aberrant vRNP intracellular accumulation, retention in the perinuclear region, and lack of accumulation at the plasma membrane. Complex formation between RAB11A and vRNPs was further established biochemically. Our results uncover a critical host factor with an essential contribution to influenza virus genome delivery and reveal a potential role for RAB11A in the transport of ribonucleoprotein cargo.     

Apical transport of influenza A virus ribonucleoprotein requires Rab11-positive recycling endosome

Influenza A virus RNA genome exists as eight-segmented ribonucleoprotein complexes containing viral RNA polymerase and nucleoprotein (vRNPs). Packaging of vRNPs and virus budding take place at the apical plasma membrane (APM). However, little is known about the molecular mechanisms of apical transport of newly synthesized vRNP. Transfection of fluorescent-labeled antibody and subsequent live cell imaging revealed that punctate vRNP signals moved along microtubules rapidly but intermittently in both directions, suggestive of vesicle trafficking. Using a series of Rab family protein, we demonstrated that progeny vRNP localized to recycling endosome (RE) in an active/GTP-bound Rab11-dependent manner. The vRNP interacted with Rab11 through viral RNA polymerase. The localization of vRNP to RE and subsequent accumulation to the APM were impaired by overexpression of Rab binding domains (RBD) of Rab11 family interacting proteins (Rab11-FIPs). Similarly, no APM accumulation was observed by overexpression of class II Rab11-FIP mutants lacking RBD. These results suggest that the progeny vRNP makes use of Rab11-dependent RE machinery for APM trafficking.     

Host factor Rab11a is critical for efficient assembly of influenza A virus genomic segments

It is well documented that influenza A viruses selectively package 8 distinct viral ribonucleoprotein complexes (vRNPs) into each virion; however, the role of host factors in genome assembly is not completely understood. To evaluate the significance of cellular factors in genome assembly, we generated a reporter virus carrying a tetracysteine tag in the NP gene (NP-Tc virus) and assessed the dynamics of vRNP localization with cellular components by fluorescence microscopy. At early time points, vRNP complexes were preferentially exported to the MTOC; subsequently, vRNPs associated on vesicles positive for cellular factor Rab11a and formed distinct vRNP bundles that trafficked to the plasma membrane on microtubule networks. In Rab11a deficient cells, however, vRNP bundles were smaller in the cytoplasm with less co-localization between different vRNP segments. Furthermore, Rab11a deficiency increased the production of non-infectious particles with higher RNA copy number to PFU ratios, indicative of defects in specific genome assembly. These results indicate that Rab11a+ vesicles serve as hubs for the congregation of vRNP complexes and enable specific genome assembly through vRNP:vRNP interactions, revealing the importance of Rab11a as a critical host factor for influenza A virus genome assembly.     

Purification and Visualization of Influenza A Viral Ribonucleoprotein Complexes

The influenza A viral genome consists of eight negative-sense, single stranded RNA molecules, individually packed with multiple copies of the influenza A nucleoprotein (NP) into viral ribonulceoprotein particles (vRNPs). The influenza vRNPs are enclosed within the viral envelope. During cell entry, however, these vRNP complexes are released into the cytoplasm, where they gain access to the host nuclear transport machinery. In order to study the nuclear import of influenza vRNPs and the replication of the influenza genome, it is useful to work with isolated vRNPs so that other components of the virus do not interfere with these processes. Here, we describe a procedure to purify these vRNPs from the influenza A virus. The procedure starts with the disruption of the influenza A virion with detergents in order to release the vRNP complexes from the enveloped virion. The vRNPs are then separated from the other components of the influenza A virion on a 33-70% discontinuous glycerol gradient by velocity sedimentation. The fractions obtained from the glycerol gradient are then analyzed on via SDS-PAGE after staining with Coomassie blue. The peak fractions containing NP are then pooled together and concentrated by centrifugation. After concentration, the integrity of the vRNPs is verified by visualization of the vRNPs by transmission electron microscopy after negative staining. The glycerol gradient purification is a modification of that from Kemler et al. (1994)¹, and the negative staining has been performed by Wu et al. (2007).²Open in a separate windowClick here to view.^{(60M, flv)}     

Avian Influenza A Virus Polymerase Association with Nucleoprotein, but Not Polymerase Assembly, Is Impaired in Human Cells during the Course of Infection

Strong determinants of the host range of influenza A viruses have been identified on the polymerase complex formed by the PB1, PB2, and PA subunits and on the nucleoprotein (NP). In the present study, molecular mechanisms that may involve these four core proteins and contribute to the restriction of avian influenza virus multiplication in human cells have been investigated. The efficiencies with which the polymerase complexes of a human and an avian influenza virus isolate assemble and interact with the viral NP and cellular RNA polymerase II proteins were compared in mammalian and in avian infected cells. To this end, recombinant influenza viruses expressing either human or avian-derived core proteins with a PB2 protein fused to the One-Strep purification tag at the N or C terminus were generated. Copurification experiments performed on infected cell extracts indicate that the avian-derived polymerase is assembled and interacts physically with the cellular RNA polymerase II at least as efficiently as does the human-derived polymerase in human as well as in avian cells. Restricted growth of the avian isolate in human cells correlates with low levels of the core proteins in infected cell extracts and with poor association of the NP with the polymerase compared to what is observed for the human isolate. The NP-polymerase association is restored by a Glu-to-Lys substitution at residue 627 of PB2. Overall, our data point to viral and cellular factors regulating the NP-polymerase interaction as key determinants of influenza A virus host range. Recombinant viruses expressing a tagged polymerase should prove useful for further studies of the molecular interactions between viral polymerase and host factors during the infection cycle.     

Influenza virus ribonucleoprotein complexes gain preferential access to cellular export machinery through chromatin targeting

In contrast to most RNA viruses, influenza viruses replicate their genome in the nucleus of infected cells. As a result, newly-synthesized vRNA genomes, in the form of viral ribonucleoprotein complexes (vRNPs), must be exported to the cytoplasm for productive infection. To characterize the composition of vRNP export complexes and their interplay with the nucleus of infected cells, we affinity-purified tagged vRNPs from biochemically fractionated infected nuclei. After treatment of infected cells with leptomycin B, a potent inhibitor of Crm1-mediated export, we isolated vRNP export complexes which, unexpectedly, were tethered to the host-cell chromatin with very high affinity. At late time points of infection, the cellular export receptor Crm1 also accumulated at the same regions of the chromatin as vRNPs, which led to a decrease in the export of other nuclear Crm1 substrates from the nucleus. Interestingly, chromatin targeting of vRNP export complexes brought them into association with Rcc1, the Ran guanine exchange factor responsible for generating RanGTP and driving Crm1-dependent nuclear export. Thus, influenza viruses gain preferential access to newly-generated host cell export machinery by targeting vRNP export complexes at the sites of Ran regeneration.     

Role of the influenza virus M1 protein in nuclear export of viral ribonucleoproteins

The protein kinase inhibitor H7 blocks influenza virus replication, inhibits production of the matrix protein (M1), and leads to a retention of the viral ribonucleoproteins (vRNPs) in the nucleus at late times of infection (K. Martin and A. Helenius, Cell 67:117-130, 1991). We show here that production of assembled vRNPs occurs normally in H7-treated cells, and we have used H7 as a biochemical tool to trap vRNPs in the nucleus. When H7 was removed from the cells, vRNP export was specifically induced in a CHO cell line stably expressing recombinant M1. Similarly, fusion of cells expressing recombinant M1 from a Semliki Forest virus vector allowed nuclear export of vRNPs. However, export was not rescued when H7 was present in the cells, implying an additional role for phosphorylation in this process. The viral NS2 protein was undetectable in these systems. We conclude that influenza virus M1 is required to induce vRNP nuclear export but that cellular phosphorylation is an additional factor.     

HMGB1 Protein Binds to Influenza Virus Nucleoprotein and Promotes Viral Replication

Influenza virus has evolved replication strategies that hijack host cell pathways. To uncover interactions between viral macromolecules and host proteins, we applied a phage display strategy. A library of human cDNA expression products displayed on filamentous phages was submitted to affinity selection for influenza viral ribonucleoproteins (vRNPs). High-mobility-group box (HMGB) proteins were found to bind to the nucleoprotein (NP) component of vRNPs. HMGB1 and HMGB2 bind directly to the purified NP in the absence of viral RNA, and the HMG box A domain is sufficient to bind the NP. We show that HMGB1 associates with the viral NP in the nuclei of infected cells, promotes viral growth, and enhances the activity of the viral polymerase. The presence of a functional HMGB1 DNA-binding site is required to enhance influenza virus replication. Glycyrrhizin, which reduces HMGB1 binding to DNA, inhibits influenza virus polymerase activity. Our data show that the HMGB1 protein can play a significant role in intranuclear replication of influenza viruses, thus extending previous findings on the bornavirus and on a number of DNA viruses.     

MxA GTPase blocks reporter gene expression of reconstituted Thogoto virus ribonucleoprotein complexes

Human MxA protein accumulates in the cytoplasm of interferon-treated cells and inhibits the multiplication of several RNA viruses, including Thogoto virus (THOV), a tick-borne orthomyxovirus that transcribes and replicates its genome in the cell nucleus. The antiviral mechanism of MxA was investigated by using two alternative minireplicon systems in which recombinant viral ribonucleoprotein complexes (vRNPs) of THOV were reconstituted from cloned cDNAs. A chloramphenicol acetyltransferase reporter minigenome RNA was expressed either by T7 RNA polymerase in the cytoplasm of transfected cells or, alternatively, by RNA polymerase I in the nucleus. The inhibitory effect of MxA was studied in both cellular compartments by coexpressing wild-type MxA or TMxA, an artificial nuclear form of MxA. Our results indicate that both MxA proteins recognize the assembled vRNP rather than the newly synthesized unassembled components. The present findings are consistent with previous data which indicated that cytoplasmic MxA prevents transport of vRNPs into the nucleus, whereas nuclear MxA directly inhibits the viral polymerase activity in the nucleus.     

甲型流感病毒蛋白和遗传物质在宿主细胞质内顺向转运过程及其机制

流感病毒的蛋白和基因组在宿主细胞内能否正确地转运到相关部位,直接影响到病毒颗粒的形态发生。流感病毒跨膜蛋白(HA、NA和M2)主要通过宿主细胞的运输膜泡实现转运,而宿主细胞的蛋白转运机器参与了这一过程。新合成的流感病毒核糖核蛋白复合物(vRNPs)出核后,通过与活化的Rab11相结合,聚集于邻近微管组织中心(MTOC)的胞内体。然后以运输小膜泡的形式,沿着MTOC的微管网络向细胞膜方向转运。跨膜蛋白和基因组在细胞质内的转运受一些宿主因子的调控,如ARHGAP21和小G蛋白Cdc42能够调节NA蛋白向细胞膜转运,Rab11协助vRNPs从MTOC向细胞膜转运。文中主要讨论新合成的流感病毒跨膜蛋白和遗传物质在宿主细胞质内的顺向转运(Anterograde transport)过程与调控。