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The study evaluated the effects of nucleoprotein viral and the infectious virus in SHK-1 cells. The results show a strong respiratory burst activation and the induction of p47phox, SOD, GLURED, and apoptotic genes. Additionally, the cells alter the profile of SUMOylated proteins by the effect of transfection and infection experiments. In silico analyses show a set of structural motifs in NP susceptible of post-translational modification by the SUMO protein. Interestingly, the inhibition of the NADPH oxidase complex blocked the production of reactive oxygen species and the high level of cellular ROS due to the nucleoprotein and the ISAv. At the same time, the blocking of the p38MAPK signaling pathway and the use of Aristotelia chilensis, decreased viral progeny production. These results suggest that the NP triggers a strong production of ROS and modifying the post-translational profile mediated by SUMO-2/3, a phenomenon that favors the production of new virions., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Cell division cycle 25 B (CDC25B) is a member of the CDC25 phosphatase family. It can dephosphorylate cyclin-dependent kinases and regulate the cell division cycle. Moreover, siRNA knockdown of CDC25B impairs influenza A virus (IAV) replication. Here, to further understand the regulatory mechanism of CDC25B for IAV replication, a CDC25B-knockout (KO) 293T cell line was constructed using CRISPR/Cas9. The present data indicated that the replication of IAV was decreased in CDC25B-KO cells. Additionally, CDC25B deficiency damaged viral polymerase activity, nucleoprotein (NP) self-oligomerization, and NP nuclear export. Most importantly, we found that the NP phosphorylation levels were significantly increased in CDC25B-KO cells. These findings indicate that CDC25B facilitates the dephosphorylation of NP, which is vital for regulating NP functions and the life cycle of IAV., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Maize mosaic virus (MMV), similar to other nucleorhabdoviruses, replicates in divergent hosts: plants and insects. To compare MMV protein localization and interactions, we visualized autofluorescent protein fusions in both cell types. Nucleoprotein (N) and glycoprotein (G) localized to the nucleus and cytoplasm, phosphoprotein (P) was only found in the nucleus, and 3 (movement) and matrix (M) were present in the cytoplasm. This localization pattern is consistent with the model of nucleorhabdoviral replication of N, P, L and viral RNA forming a complex in the nucleus and the subvirion associating with M and then G during budding into perinuclear space. The comparable localization patterns in both organisms indicates a similar replication cycle. Changes in localization when proteins were co-expressed suggested viral proteins interact thus altering organelle targeting. We documented a limited number of direct protein interactions indicating host factors play a role in the virus protein interactions during the infection cycle., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Zaire Ebolavirus (EBOV) causes a severe feverish disease with high case fatality rates. Transcription of EBOV is dependent on the activity of the nucleocapsid protein VP30 which represents an essential viral transcription factor. Activity of VP30 is regulated via phosphorylation at six N-terminal serine residues. Recent data demonstrated that dynamic phosphorylation and dephosphorylation of serine residue 29 is essential for transcriptional support activity of VP30. To analyze the spatio/temporal dynamics of VP30 phosphorylation, we generated a peptide antibody recognizing specifically VP30 phosphorylated at serine 29. Using this antibody we could demonstrate that (i) the majority of VP30 molecules in EBOV-infected cells is dephosphorylated at the crucial position serine 29, (ii) both, VP30 phosphorylation and dephosphorylation take place in viral inclusion bodies that are induced by the nucleoprotein NP and (iii) NP influences the phosphorylation state of VP30., (Copyright © 2017 Elsevier Inc. All rights reserved.)

An anti-influenza compound, DP2392-E10 based on inhibition of the nuclear export function of the viral nucleoprotein-nuclear export signal 3 (NP-NES3) domain was successfully identified by our previous high-throughput screening system. Here, we demonstrated that DP2392-E10 exerts its antiviral effect by inhibiting replication of a broad range of influenza A subtypes. In regard to the molecular mechanism, we revealed that DP2392-E10 inhibits nuclear export of both viral NP and nuclear export protein (NEP). More specifically, in vitro pull-down assays revealed that DP2392-E10 directly binds cellular CRM1, which mediates nuclear export of NP and NEP. In silico docking suggested that DP2392-E10 binds at a region close to the HEAT9 and HEAT10 domains of CRM1. Together, these results indicate that the CRM1-mediated nuclear export function of influenza virus represents a new potential target for antiviral drug development, and also provide a core structure for a novel class of inhibitors that target this function., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Several mechanisms underlying intertypic interference between co-infecting influenza types A and B viruses (IAV and IBV) have been proposed. We have recently described one in which IBV's nucleoprotein (BNP) sequestered IAV's nucleoprotein (ANP) and suppressed IAV polymerase and growth. However, its anti-IAV capacity and limitations have not been fully explored. Here, we showed that BNP's inhibitory effect was more potent toward a wide array of avian IAVs, whereas human IAVs revealed moderate resistance. BNP sensitivity was largely determined by ANP's residue 343 at the NP oligomerization interface. An avian IAV polymerase carrying an NP-V343L mutation switched from being highly BNP-sensitive to moderately BNP-resistant, and vice versa for a human IAV polymerase carrying a reverse mutation. To highlight its capacity, we demonstrated that the polymerases of highly-pathogenic H5N1 and the pandemic 2009 (H1N1) strains are strongly inhibited by BNP. Our work provides insights into lineage-specific sensitivity to BNP-mediated intertypic interference., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Influenza A viruses (IAVs) cause numerous pandemics and yearly epidemics resulting in ~500,000 annual deaths globally. IAV modulates cellular signaling pathways at every step of the infection cycle. Focal adhesion kinase (FAK) has been shown to play a critical role in endosomal trafficking of influenza A viruses, yet it is unclear how FAK kinase activity regulates IAV replication. Using mini-genomes derived from H1N1, H5N1 and H7N9 viruses, we dissected RNA replication by IAVs independent of viral entry or release. Our results show FAK activity promotes efficient IAV polymerase activity and inhibiting FAK activity with a chemical inhibitor or a kinase-dead mutant significantly reduces IAV polymerase activity. Using co-immunoprecipitations and proximity ligation assays, we observed interactions between FAK and the viral nucleoprotein, supporting a direct role of FAK in IAV replication. Altogether, the data indicates that FAK kinase activity is important in promoting IAV replication by regulating its polymerase activity., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Co-infection of influenza A and B viruses (IAV and IBV) results in marked decreases in IAV replication. Multiple mechanisms have been proposed for this phenomenon. Recently, we reported that IBV nucleoprotein (BNP) alone can suppress IAV replication and proposed an inhibition model in which BNP binds IAV nucleoprotein (ANP) and disrupts IAV polymerase complexes. Here, using mutagenesis and co-immunoprecipitation, we determined the protein motifs mediating the intertypic ANP-BNP complex and showed that it specifically interferes with ANP׳s interaction with the PB2 subunit of the IAV polymerase but not with the other subunit PB1. We further demonstrated that BNP only suppresses growth of IAVs but not other RNA viruses. However, different IAV strains display varied sensitivity toward the BNP׳s inhibitory effect. Together, our data provide mechanistic insights into intertypic nucleoprotein complex formation and highlight the role of BNP as a potential broad-spectrum anti-IAV agent., (Copyright © 2015 Elsevier Inc. All rights reserved.)

The nucleoprotein (N) of vesicular stomatitis virus (VSV) plays a central role in transcription and replication by encapsidating genome RNA to form a nucleocapsid as the template for the RNA synthesis. Using minigenome system we evaluated the roles of 21 amino acids of the N-terminal arm of N in forming functional N-RNA templates and found that three triple-amino-acid substitutions (TVK4-6A3, RII7-9A3, and VIV13-15A3) and one single-amino-acid substitution (R7A) resulted in RNA synthesis loss. But all the mutants maintain the ability to oligomerize N, interact with P, and encapsidate viral RNA for template formation. Further analysis showed that the nucleocapsid formed by these mutants failed to protect RNA from nuclease digestion. Then, we found that only recombinant viruses containing R7A could be recovered. Our results show that the several amino acids within the N-terminal arm of N contribute to the template function beyond its role in RNA encapsidation and viral growth., (Copyright © 2015 Elsevier Inc. All rights reserved.)

The highly conserved internal nucleoprotein (NP) is a promising antigen to develop a universal influenza A virus vaccine. In this study, mice were injected intramuscularly with Escherichia coli-derived NP protein alone or in combination with adjuvant alum (Al(OH)3), CpG or both. The results showed that the NP protein formulated with adjuvant was effective in inducing a protective immune response. Additionally, the adjuvant efficacy of Al(OH)3 was stronger than that of CpG. Optimal immune responses were observed in BALB/c mice immunized with a combination of NP protein plus Al(OH)3 and CpG. These mice also showed maximal resistance following challenge with influenza A virus PR8 strain. Most importantly, 10 µg NP formulated with Al(OH)3 and CpG induced higher protection than did 90 µg NP. These findings indicated that a combination of Al(OH)3 and CpG may be an efficient adjuvant in the NP formulation., (Copyright © 2014 Elsevier Inc. All rights reserved.)

The nucleoprotein (NP) of influenza A virus plays a critical role in the formation of viral ribonucleoprotein (vRNP) complex. However, it remains unclear which key residues in NP are associated with the assembly of vRNP and contribute to virus replication. Here, a highly conserved aspartic acid at residue 88 (D88) of NP was identified by molecular docking of NP with the Fv region of a broad-spectrum anti-NP mAb 19C10 and further demonstrated to be an important residue contributes to the RNP activity, virus growth in MDCK cells and replication in lungs of infected mice by comparing recombinant wild-type A/WSN/1933 virus to the mutant virus that contains an alanine instead of aspartic acid at NP residue 88. D88 was also predicted to interact with PB2 by molecular docking and further verified by immunoprecipitation. These findings provide new information for understanding the interaction between NP and other polymerase subunits in virus replication., (Copyright © 2014 Elsevier Inc. All rights reserved.)

The nucleoprotein of influenza B virus (BNP) shares several characteristics with its influenza A virus counterpart (ANP), including localization in the host's nucleus. However, while the nuclear localization signal(s) (NLS) of ANP are well characterized, little is known about those of BNP. In this study, we showed that the fusion protein bearing the BNP N-terminus fused with GFP (N70-GFP) is exclusively nuclear, and identified a highly conserved KRXR motif spanning residues 44-47 as a putative NLS. In addition, we demonstrated that residues 3-15 of BNP, though not an NLS, are also crucial for nuclear import. Results from mutational analyses of N70-GFP and the full-length BNP suggest that this region may be required for protection of the N-terminus from proteolytic cleavage. Altogether, we propose that the N-terminal region of BNP contains the NLS and cleavage-protection motif, which together drive its nuclear localization., (Copyright © 2013 Elsevier Inc. All rights reserved.)

The Ebola virus (EBOV) RNA-dependent RNA polymerase (RdRp) complex consists of the catalytic subunit of the polymerase, L, and its cofactor VP35. Using immunofluorescence analysis and coimmunoprecipitation assays, we mapped the VP35 binding site on L. A core binding domain spanning amino acids 280-370 of L was sufficient to mediate weak interaction with VP35, while the entire N-terminus up to amino acid 380 was required for strong VP35-L binding. Interestingly, the VP35 binding site overlaps with an N-terminal L homo-oligomerization domain in a non-competitive manner. N-terminal L deletion mutants containing the VP35 binding site were able to efficiently block EBOV replication and transcription in a minigenome system suggesting the VP35 binding site on L as a potential target for the development of antivirals., (Copyright © 2013 Elsevier Inc. All rights reserved.)

Given that co-infection of cells with equivalent titers of influenza A and B viruses (FluA and FluB) has been shown to result in suppression of FluA growth, it is possible that FluB-specific proteins might hinder FluA polymerase activity and replication. We addressed this possibility by individually determining the effect of each gene of FluB on the FluA polymerase assay and found that the nucleoprotein of FluB (NP(FluB)) inhibits polymerase activity of FluA in a dose-dependent manner. Mutational analyses of NP(FluB) suggest that functional NP(FluB) is necessary for this inhibition. Slower growth of FluA was also observed in MDCK cells stably expressing NP(FluB). Further analysis of NP(FluB) indicated that it does not affect nuclear import of NP(FluA). Taken together, these findings suggest a novel role of NP(FluB) in inhibiting replication of FluA, providing more insights into the mechanism of interference between FluA and FluB and the lack of reassortants between them., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Conventional assays of viral particle assembly and release are time consuming and laborious. We have developed an enzymatic virus-like particle (VLP) genesis assay that rapid and quantitative and is also versatile and applicable to diverse viruses including HIV-1 and Ebola virus. Using this assay, which has a dynamic range of several orders of magnitude, we show that the efficiency of VLP assembly and release, i.e., the fraction of the expressed protein that is assembled into extracellular particles, is dependent on the absolute level of expression of either HIV-1 Gag or Ebola virus VP40. We also demonstrate that the activity of the antiviral factor tetherin is dependent on the level of HIV-1 Gag expression and the numbers of VLPs generated, and appears to become saturated as these parameters are increased., (Copyright © 2012 Elsevier Inc. All rights reserved.)

To gain insight into the structural and functional properties of the vesicular stomatitis virus nucleocapsid-RNA complex (vN-RNA), we analyzed it by treatment with proteolytic enzymes. Chymotrypsin treatment to the vN-RNA results in complete digestion of the C-terminal 86 amino acids of the N protein. The residual chymotrypsin resistant vN-RNA complex (vDeltaN-RNA) carrying N-terminal 336 amino acids of the N protein (DeltaN) was inactive in transcription. The DeltaN protein retained its capability to protect the genomic RNA from nuclease digestion but failed to interact to the P protein. Interestingly, addition of excess amount of P protein rendered the vN-RNA complex resistant to the chymotrypsin digestion. Finally, our data revealed that the recombinant N-RNA complex purified from bacteria (bN-RNA) is resistant to chymotrypsin digestion, suggesting that the C-terminal unstructured domain (C-loop) remains inaccessible to protease digestion. Detailed comparative analyses of the vN-RNA and vDeltaN-RNA are discussed.

The Borna disease virus (BDV) nucleoprotein (N) monomer resembles the nucleoprotein structures from rabies virus (RABV) and vesicular stomatitis virus (VSV). We show that BDV N assembles into ring- and string-like structures in the presence of 5' genomic BDV RNA. RNA induced polymerization is partly RNA-specific since polymerization is inefficient in the presence of 3' genomic BDV RNA or E. coli RNA. Mutagenesis of basic residues located in the cleft made up by the N- and C-terminal domains of N abrogate RNA-induced polymerization indicating that BDV N binds RNA similarly as observed in case of RABV and VSV N-RNA complexes. Bound RNA is not protected and sensitive to degradation. N-RNA polymers form complexes with the phosphoprotein P as required for functional transcription or replication units. Our data indicate that BDV N utilizes similar structural principles for N-RNA and N-P-RNA complex formation as observed for related negative strand RNA viruses., (Copyright 2009 Elsevier Inc. All rights reserved.)

Cold-adapted (ca) B/Ann Arbor/1/66 is the master donor virus for influenza B (MDV-B) vaccine component of live attenuated influenza FluMist vaccine. The six internal protein gene segments of MDV-B confer the characteristic cold-adapted (ca), temperature-sensitive (ts) and attenuated (att) phenotypes to the reassortant vaccine strains that contain the HA and NA RNA segments from the circulating wild type strains. Previously, we have mapped the loci in the NP, PA and M genes that determine the ca, ts and att phenotypes of MDV-B. In this report, the ts mechanism of MDV-B was described by comparing replication of MDV-B with its wild type counterpart at permissive and restricted temperatures. We showed that the PA and NP proteins of MDV-B are defective in RNA polymerase function at the restricted temperature of 37 degrees C resulting in greatly reduced viral RNA and protein synthesis. In addition, the two M1 residues, Q159 and V183 that are unique to MDV-B, contribute to reduced virus replication at temperatures greater than 33 degrees C, possibly due to the reduced M1 membrane association and its reduced virion M1 incorporation. Thus, the previously identified MDV-B loci not only reduce viral polymerase function at the restricted temperature but also affect virus assembly and release.

Our previous results have shown that some residues of V protein-specific domain in human parainfluenza virus type 2 (hPIV2) are essential not only for STAT protein degradation but also for promoting virus growth. Here, we demonstrated that the virus growth of these recombinant hPIV2s (rPIV2) expressing mutated V proteins were improved in HeLa cell transiently expressing the wild-type V protein, but not in the cells constitutively expressing it. Consequently, we identified the region of the V protein that is essential for its oligomerization and for complex formation with NP protein. We also identified a host protein, AlP1/Alix, involved in apoptosis and efficient budding of several enveloped viruses as an interacting partner of the V and NP proteins. Depletion of AIP1/Alix by small interfering RNA suppressed virus growth. These data suggest that the conserved carboxyl terminus of the V protein plays an important role in virus growth.

An integrase dimer can process and integrate a single HIV-1 DNA end in vitro, whereas a tetramer is required to integrate two ends. LEDGF/p75 can potently stimulate integrase activity, but its effects on half- versus full-site integration have not been investigated. Stimulation of half-site but inhibition of full-site integration is revealed here. LEDGF/p75 seems to interfere with integrase oligomerization, but does not inhibit the catalytic activity of pre-assembled complexes. We therefore speculate that LEDGF/p75 function is restricted to a point in the viral lifecycle that occurs after the formation of the preintegration synaptic complex, for example, as a chromatin-associated tethering factor.

Plants viruses spread throughout their hosts using a number of pathways, the most common being movement cell to cell through plasmodesmata (PD), unique intercellular organelles of the plant kingdom, and between organs by means of the vascular system. Pioneering studies on plant viruses revealed that PD allow the cell-to-cell trafficking of virally encoded proteins, termed the movement proteins (MPs). This non-cell-autonomous protein (NCAP) pathway is similarly employed by the host to traffic macromolecules. Viral MPs bind RNA/DNA in a sequence nonspecific manner to form nucleoprotein complexes (NPC). Host proteins are then involved in the delivery of MPs and NPC to the PD orifice, and a role for the cytoskeleton has been implicated. Trafficking of NCAPs through the PD structure involves three steps in which the MP: (a) interacts with a putative PD docking complex, (b) induces dilation in the PD microchannels, and (c) binds to an internal translocation system for delivery into the neighboring cytoplasm. Viral genera that use this NCAP pathway have evolved a combination of a MP and ancillary proteins that work in concert to enable the formation of a stable NPC that can compete with endogenous NCAPs for the PD trafficking machinery. Incompatible MP-host protein interactions may underlie observed tissue tropisms and restricted infection domains. These pivotal discoveries are discussed in terms of the need to develop a more comprehensive understanding of the (a) three-dimensional structure of MPs, (b) PD supramolecular complex, and (c) host proteins involved in this cell-to-cell trafficking process.

Measles virus belongs to the Paramyxoviridae family within the Mononegavirales order. Its non-segmented, single stranded, negative sense RNA genome is encapsidated by the nucleoprotein (N) to form a helical nucleocapsid. This ribonucleoproteic complex is the substrate for both transcription and replication. The RNA-dependent RNA polymerase binds to the nucleocapsid template via its co-factor, the phosphoprotein (P). In this review, we summarize the main experimental data pointing out the abundance of structural disorder within measles virus N and P. We also describe studies indicating that structural disorder is a widespread property in the replicative complex of Paramyxoviridae and, more generally, of Mononegavirales. The functional implications of structural disorder are also discussed. Finally, we propose a model where the flexibility of the disordered N and P domains allows the formation of a tripartite complex (N degrees-P-L) during replication, followed by the delivery of N monomers to the newly synthesized genomic RNA chain.

We have identified a putative coiled-coil motif within the amino-terminal half of the ebolavirus VP35 protein. Cross-linking studies demonstrated the ability of VP35 to form trimers, consistent with the presence of a functional coiled-coil motif. VP35 mutants lacking the coiled-coil motif or possessing a mutation designed to disrupt coiled-coil function were defective in oligomerization, as deduced by co-immunoprecipitation studies. VP35 inhibits signaling that activates interferon regulatory factor 3 (IRF-3) and inhibits (IFN)-alpha/beta production. Experiments comparing the ability of VP35 mutants to block IFN responses demonstrated that the VP35 amino-terminus, which retains the putative coiled-coil motif, was unable to inhibit IFN responses, whereas the VP35 carboxy-terminus weakly inhibited the activation of IFN responses. IFN-antagonist function was restored when a heterologous trimerization motif was fused to the carboxy-terminal half of VP35, suggesting that an oligomerization function at the amino-terminus facilitates an "IFN-antagonist" function exerted by the carboxy-terminal half of VP35.

Several viruses in the Arenavirus genus of the family Arenaviridae cause severe, often fatal, hemorrhagic fever. One such virus, Lassa virus (LV), is a frequent cause of disease in Africa, and survivors often are left with substantial neurological impairment. The feasibility of protective immunization against LV infection, and the associated disease, has been demonstrated in animal models, using recombinant vaccinia viruses to deliver Lassa proteins. Circumstantial evidence implicates cellular immunity in this Lassa-induced protection, but this has not been confirmed. Here, we describe DNA vaccines that encode LV proteins. A single inoculation of a plasmid encoding full-length Lassa nucleoprotein (LNP) can induce CD8(+) T cell responses in mice and can protect against challenge with two arenaviruses, lymphocytic choriomeningitis virus (LCMV) and Pichinde virus (PV). A DNA minigene vaccine encoding a 9 amino acid sequence from LNP also induces CD8(+) T cells and protects against arenavirus challenge, thus confirming prior speculation that protective cellular immunity is induced by LV proteins.

During influenza virus infection, transcription and replication of the viral RNA take place in the cell nucleus. Directly after entry in the nucleus the viral ribonucleoproteins (RNPs, the viral subunits containing vRNA, nucleoprotein and the viral polymerase) are tightly associated with the nuclear matrix. Here, we have analysed the binding of RNPs, M1 and NS2/NEP proteins to purified nucleosomes, reconstituted histone octamers and purified single histones. RNPs and M1 both bind to the chromatin components but at two different sites, RNP to the histone tails and M1 to the globular domain of the histone octamer. NS2/NEP did not bind to nucleosomes at all. The possible consequences of these findings for nuclear release of newly made RNPs and for other processes during the infection cycle are discussed.

To catalyze RNA synthesis, the Sendai virus P-L RNA polymerase complex first binds the viral nucleocapsid (NC) template through an interaction of the P subunit with NP assembled with the genome RNA. For replication, the polymerase utilizes an NP(0)-P complex as the substrate for the encapsidation of newly synthesized RNA which involves both NP-RNA and NP-NP interactions. Previous studies showed that the C-terminal 124 amino acids of NP (aa 401-524) contain the P-NC binding site. To further delineate the amino acids important for this interaction, C-terminal truncations and site-directed mutations in NP were characterized for their replication activity and protein-protein interactions. This C-terminal region was found in fact to be necessary for several different protein interactions. The C-terminal 492-524 aa were nonessential for the complete activity of the protein. Deletion of amino acids 472-491, however, abolished replication activity due to a specific defect in the formation of the NP(0)-P complex. Binding of the P protein of the polymerase complex to NC required aa 462-471 of NP, while self-assembly of NP into NC required aa 440-461. Site-directed mutations from aa 435 to 491 showed, however, that the charged amino acids in this region were not essential for these defects.

The matrix protein VP40 from Ebola virus plays an important role in the assembly process of virus particles by interacting with cellular factors, cellular membranes, and the ribonuclearprotein particle complex. Here we show that the N-terminal domain of VP40 folds into a mixture of two different oligomeric states in vitro, namely hexameric and octameric ringlike structures, as detected by gel filtration chromatography, chemical cross-linking, and electron microscopy. Octamer formation depends largely on the interaction with nucleic acids, which in turn confers in vitro SDS resistance. Refolding experiments with a nucleic acid free N-terminal domain preparation reveal a mostly dimeric form of VP40, which is transformed into an SDS resistant octamer upon incubation with E. coli nucleic acids. In addition, we demonstrate that the N-terminal domain of Marburg virus VP40 also folds into ringlike structures, similar to Ebola virus VP40. Interestingly, Marburg virus VP40 rings reveal a high tendency to polymerize into rods composed of stacked rings. These results may suggest distinct roles for different oligomeric forms of VP40 in the filovirus life cycle.

The influenza virus copies its genomic RNA in the nuclei of host cells, but the viral particles are formed at the plasma membrane. Thus, the export of new genome from the nucleus into the cytoplasm is essential for viral production. Several viral proteins, such as nucleoprotein (NP) and RNA polymerases, synthesized in the cytoplasm, are imported into the nucleus, and form viral ribonucleoprotein (vRNP) with new genomic RNA. vRNP is then exported into the cytoplasm from the nucleus to produce new viral particles. M1, a viral matrix protein, is suggested to participate in the nuclear export of vRNP. It was found unexpectedly that the production of influenza virus was suppressed in MDCK cells at 41 degrees C, although viral proteins were synthesized and the cytopathic effect was observed in host cells. Indirect immunofluorescent staining with anti-NP or M1 monoclonal antibody showed that NP and M1 remained in the nuclei of infected cells at 41 degrees C, suggesting that a suppression of viral production was caused by inhibition of the nuclear export of these proteins. The cellular machinery for nuclear export depending on CRM1, which mediates the nuclear export of influenza viral RNP, functioned normally at 41 degrees C. Glycerol-density gradient centrifugation demonstrated that vRNP also formed normally at 41 degrees C. However, an examination of the interaction between vRNP and M1 by immunoprecipitation indicated that M1 did not associate with vRNP at 41 degrees C, suggesting that the association is essential for the nuclear export of vRNP. Furthermore, when infected cells incubated at 41 degrees C were cultured at 37 degrees C, the interaction between vRNP and M1 was no longer detected even at 37 degrees C. The results suggest that M1 synthesized at 41 degrees C is unable to interact with vRNP and the dissociation of M1 from vRNP is one of the reasons that the transfer of vRNP into the cytoplasm from the nucleus is prevented at 41 degrees C.

FluMist influenza A vaccine strains contain the PB1, PB2, PA, NP, M, and NS gene segments of ca A/AA/6/60, the master donor virus-A strain. These gene segments impart the characteristic cold-adapted (ca), attenuated (att), and temperature-sensitive (ts) phenotypes to the vaccine strains. A plasmid-based reverse genetics system was used to create a series of recombinant hybrids between the isogenic non-ts wt A/Ann Arbor/6/60 and MDV-A strains to characterize the genetic basis of the ts phenotype, a critical, genetically stable, biological trait that contributes to the attenuation and safety of FluMist vaccines. PB1, PB2, and NP derived from MDV-A each expressed determinants of temperature sensitivity and the combination of all three gene segments was synergistic, resulting in expression of the characteristic MDV-A ts phenotype. Site-directed mutagenesis analysis mapped the MDV-A ts phenotype to the following four major loci: PB1(1195) (K391E), PB1(1766) (E581G), PB2(821) (N265S), and NP(146) (D34G). In addition, PB1(2005) (A661T) also contributed to the ts phenotype. The identification of multiple genetic loci that control the MDV-A ts phenotype provides a molecular basis for the observed genetic stability of FluMist vaccines.

The nucleoprotein (N) of measles virus encapsidates viral genomic RNA to form a helical nucleocapsid. Its strong self-association is a major hurdle in determining its high-resolution structure using X-ray crystallography. We report the bacterial expression, purification, and characterization of a variant N that has lost its ability to form nucleocapsid-like structures after substitution of two residues by polar residues. Using immunoprecipitation, circular dichroism, and limited proteolysis studies, we show that this nucleoprotein retains a folding similar to wild-type N. Furthermore, the variant N binds the phosphoprotein, indicating that it retains biochemical relevance. We also present evidence indicating that the N-terminus of N lies at the surface of the nucleocapsid. Beyond the identification of one region of N involved in self-association, our results should facilitate structural studies of N using X-ray crystallography.

Morphogenesis of influenza virus is a poorly understood process that produces two types of enveloped virion: approximately 100-nm spheres and similar diameter filaments that reach 20 microm in length. Spherical particles assemble at plasma membrane lipid rafts in a process independent of microfilaments. The budding site of filamentous virions is hitherto uncharacterised but their formation involves the actin cytoskeleton. We confirm microfilament involvement in filamentous budding and show that after disruption of cortical actin by jasplakinolide, HA, NP, and M1 redistributed around beta-actin clusters to form novel annular membrane structures. HA in filamentous virions and jasplakinolide-induced annuli was detergent insoluble at 4 degrees C. Furthermore, in both cases HA partitioned into low buoyant density detergent-insoluble glycolipid domains, indicating that filamentous virions and annuli contain reorganised lipid rafts. We propose that the actin cytoskeleton is required to maintain the correct organisation of lipid rafts for incorporation into budding viral filaments.

The formation of influenza virus ribonucleoprotein (RNP) is a necessary step in viral assembly and maturation in infected cells, but the mechanism remains incompletely understood. Influenza virus proteins such as matrix (M1) and cellular proteins have been implicated in assembly and transport of RNP. To study the assembly of RNP and the translocation of RNP complexes in cells, RNPs were reconstituted from nucleoprotein (NP), M1, and viral RNA (vRNA) synthesized in vitro. The syntheses were accomplished using specific plasmids in a system coupling transcription and translation under the control of the T7 promoter. The density of the resulting RNP complexes was analyzed by glycerol gradient centrifugation and the morphology was examined by transmission electron microscopy. Protomers of NP self-assembled into circular oligomers regardless of the presence of vRNA or M1. However, helical structures similar in conformation and density to RNPs purified directly from influenza virus were formed only when M1 and vRNA were also present. In the absence of vRNA, no helical structures were formed from NP and M1. The plasmids also contained the CMV promoter, which permitted expression of M1, NP, and vRNA in Madin-Darby canine kidney (MDCK). M1 and NP were both present in the cytoplasm of MDCK also expressing vRNA, but NP was retained in the nucleus of cells expressing M1 without vRNA. Our data demonstrate for the first time that vRNA and M1 together promote the self-assembly of influenza virus NP into the quaternary helical structure typical of the viral RNP. The results also indicate that the interaction of NP with vRNA and M1 in a system devoid of other viral proteins can lead to translocation of RNP from nucleus to cytoplasm.

The phosphoprotein (P) of bovine respiratory syncytial virus (BRSV) is a multifunctional protein that plays a central role in transcription and replication of the viral genomic RNA. To investigate the domains and specific residues involved in different activities of the P protein, we generated a total of 22 deletion and 17 point mutants of the P protein. These mutants were characterized using an intracellular BRSV-CAT minigenome replication system for the ability to (1) direct minigenome transcription, (2) direct minigenome replication, and (3) form complexes with nucleocapsid protein (N) and large polymerase protein (L). These studies revealed that all the regions of P protein except amino acids 41-80 are essential for minigenome transcription and replication. Interestingly, amino acids 41-60 appeared to contain sequences that negatively regulate transcription and replication. Analysis of the N- or C-terminal ends indicated that deletion of up to 3 amino acids from the N- or C-terminus completely ablated the replication, while leaving substantial residual transcription. Single amino acid substitutions within the N-terminal 4 or C-terminal 13 amino acids showed that substitution at position 2, 4, 234, 236, 238, 240, or 241 was highly inhibitory to both transcription and replication, whereas substitution at position 3 was highly inhibitory to replication while leaving substantial residual transcription. Substitution of serine residues at the C-terminus indicated that loss of phosphorylation sites did not appear to have any effect on transcription and replication. Coimmunoprecipitation of P-N and P-L complexes with P-specific antiserum revealed that substitution mutations at the N- or C-terminus did not affect binding to N and L proteins, except that substitution mutation at C-terminus position 234, 236, 238, 240, or 241 affected binding to N protein by 10-fold., (Copyright 2001 Academic Press.)

We have analysed the expression and cellular localisation of the matrix protein VP40 from Ebola virus. Full-length VP40 and an N-terminal truncated construct missing the first 31 residues [VP40(31-326)] both locate to the plasma membrane of 293T cells when expressed transiently, while a C-terminal truncation of residues 213 to 326 [VP40(31-212)] shows only expression in the cytoplasm, when analysed by indirect immunofluorescence and plasma membrane preparations. In addition, we find that full-length VP40 [VP40(1-326)] and VP40(31-326) are both released into the cell culture supernatant and float up in sucrose gradients. The efficiency of their release, however, is dependent on the presence of the N-terminal 31 residues. VP40 that is released into the supernatant is resistant to trypsin digestion, a finding that is consistent with the formation of viruslike particles detected by electron microscopy. Together, these results provide strong evidence that Ebola virus VP40 is sufficient for virus assembly and budding from the plasma membrane., (Copyright 2001 Academic Press.)

Infection of mouse L.CD46 fibroblasts with measles virus resulted in a poor virus yield, although no defects in the steps of virus binding, entry or fusion, were detected. Two days post-infection, the level of expression of the viral F protein was found to be similar on the surface of infected L.CD46 and HeLa cells using a virus multiplicity enabling an equal number of cells to be infected. After immunofluorescence labelling and confocal microscopy, L.CD46 cells also displayed a significant increase in the co-localisation of the N protein with the cell surface H and F proteins. Immunogold labelling and transmission electron microscopy demonstrated the accumulation of numerous nucleocapsids near the plasma membrane of L. CD46 cells with little virus budding, in contrast to infected HeLa cells which displayed fewer cortical nucleocapsids and more enveloped viral particles. Purified virus particles from infected L. CD46 contained a reduced amount of H, F and M protein. Altogether, these data indicate that, in L.CD46 cells, the late stage of measles virus assembly is defective. This cellular model will be helpful for the identification of cellular factors controlling measles virus maturation., (Copyright 1999 Academic Press.)

Previous studies have predicted the presence of a small open reading frame (ORFx1) located between ORF-1 and ORF-2 of the Borna disease viral (BDV) genome. The ORFx1 is expressed as a p10 protein that is localized in the nucleus and cytoplasm of BDV-infected cells. In this study, we cloned the nucleotide sequence of ORFx1 into expression vectors and showed that it is expressed as p10. An anti-p10 serum gave nuclear and cytoplasmic staining of cells persistently infected with BDV. Immunoprecipitation of p10 from BDV-infected cells coprecipitated the p40 nucleoprotein N and the 24-kDa viral phosphoprotein P. Transient transfection of noninfected cells showed that p10 and p40 can be coprecipitated and revealed that p10 localized in the cytoplasm was imported into the nucleus in the presence of the BDV p40 N. In vitro protein-protein interaction studies on solid phase showed the direct interaction of the p10 with the BDV N protein. The subcellular distribution of p10 and its interaction with p40 suggest that this protein may play a role in the nuclear replication and/or transcription of BDV., (Copyright 1999 Academic Press.)

The nucleoprotein (NP) of Marburg virus is phosphorylated at serine and threonine residues in a ratio of 85:15, regardless of whether the protein is isolated from virions or from eukaryotic expression systems. Phosphotyrosine is absent. Although many potential phosphorylation sites are located in the N-terminal half of NP, this part of the protein is not phosphorylated. Analyses of phosphorylation state and phosphoamino acid content of truncated NPs expressed in HeLa cells using the vaccinia virus T7 expression system led to the identification of seven phosphorylated regions (region I*, amino acids 404-432; II*, amino acids 446-472; III*, amino acids 484-511; IV*, amino acids 534-543; V*, amino acid 549; VI*, amino acids 599-604; and VII*, amino acid 619) with a minimum of seven phosphorylated amino acid residues located in the C-terminal half of NP. All phosphothreonine residues and consensus recognition sequences for protein kinase CKII are located in regions I*-V*. Regions VI* and VII* contain only phosphoserine with three of four serine residues in consensus recognition motifs for proline-directed protein kinases. Mutagenesis of proline-adjacent serine residues to alanine or aspartic acid did not influence the function of NP in a reconstituted transcription/replication system; thus it is concluded that serine phosphorylation in the most C-terminal part of NP is not a regulatory factor in viral RNA synthesis., (Copyright 1999 Academic Press.)

Infectious human respiratory syncytial virus (RSV) was produced from a cDNA clone that contains 15,222 nucleotides of RSV genome derived from the A2 strain of subgroup A. Recovery of infectious RSV from cDNA required cotransfection of only three expression plasmids encoding the nucleoprotein (N), the phosphoprotein (P), and the major polymerase protein (L). Inclusion of the M2-1 plasmid was not required in the transfection reaction and if included did not significantly increase the rescue efficiency. However, a single nucleotide substitution in the RSV leader region (C to G at position 4 in the antigenomic sense), greatly increased the amount of infectious virus recovered from cDNA. A recombinant RSVA2 virus that expresses an additional structural G protein derived from a subgroup B RSV was also obtained. Both A2 and B strain G glycoproteins were expressed in cells infected with the chimeric RSV. A chimeric RSV that expresses a heterologous subgroup antigen in a live attenuated vaccine candidate may be important for prevention of diseases associated with both RSV subgroup A and subgroup B infection., (Copyright 1998 Academic Press.)

We have previously shown that the nucleoprotein (NP) of Thogoto virus (THOV), a tick-borne member of the Orthomyxoviridae family, accumulates in the cell nucleus. Here we demonstrate that THOV NP contains a motif (KRxxxxxxxxxKTKK) at amino acid positions 179-193 that represents a classical bipartite nuclear localization signal (NLS). This sequence motif (named cNLS) was able to translocate a cytoplasmic 80-kDa reporter protein into the nucleus. Targeted mutations substituting lysines for alanines in the downstream cluster of the bipartite motif abolished the capacity of cNLS to mediate nuclear import. In contrast, identical mutations had no effect on nuclear localization when introduced into THOV NP, indicating that additional transport signals are present in NP. Amino-acid sequence comparisons revealed that THOV NP lacks the N-terminal nonconvential NLS (named here nNLS), which has been implicated in nuclear import of influenza A virus NP. Accordingly, THOV NP failed to interact in coprecipitation assays with the cellular NPI-1/3 transport factors of the karyopherin alpha family. A highly conserved motif identified in THOV NP was the so-called nuclear accumulation sequence (NAS). Mutating NAS alone, or in combination with cNLS, had no gross effect on the intracellular distribution of the protein, indicating that a functional NAS is not required for nuclear accumulation of THOV NP in mammalian cells. We also studied nuclear transport of influenza A/PR/8/34 virus NP. Interestingly, we found a cNLS motif at amino acid positions 198-216 in addition to the previously described nonconventional nNLS. To further assess the functional role of cNLS, nNLS, and NAS, we analyzed single, double, and triple mutants of influenza A virus NP. When nNLS was destroyed, the protein stayed in the cytoplasm as expected. When NAS was disrupted in addition to nNLS, the double mutant accumulated in the nucleus, suggesting that cNLS was active. Indeed, when cNLS was also inactivated, the triple mutant protein localized again predominantly to the cytoplasm. These findings suggest that NP of orthomyxoviruses have two independent NLSs, namely cNLS and nNLS. They further suggest that NAS and NLSs may assume opposing roles in nucleocytoplasmic transport of NP., (Copyright 1998 Academic Press.)

The small subunit of bacteriophages SPP1 and SF6 terminase, G1P, share 71% identity clustered in three conserved segments (I, II, and III). Within segment I the helix-turn-helix DNA-binding domain was mapped, whereas segment III was found to be nonessential. For terminase activity, chimeric G1Ps, obtained by domain swapping between gene 1 of SPP1 and the SF6 origin (Chi1 to Chi4), were purified. The chimeric proteins behave in all respects similarly to the G1P of SPP1 or SF6. The major determinant for G1P:G1P interactions was found to lie within segment II. We showed that a G1P derivative (G1P*) lacking the 62 N-terminal residues (segment I), and Chi1 lacking the 45 C-terminal residues (segment III) interact with G1P. The N-terminal domain of G1P is necessary for terminase subunit assembly, because the large subunit of the terminase (G2P) interacts only with G1P and Chi1, but fails to do so with G1P*. These results suggest that segment III and the extended C-terminal part of SPP1 G1P do not play a major role in DNA recognition and that G1P recognizes an extended nucleotide sequence and DNA structure.

Reovirus nonstructural protein sigma NS exhibits a ssRNA-binding activity thought to be involved in assembling the reovirus mRNAs for genome replication and virion morphogenesis. To extend analysis of this activity, recombinant sigma NS (r sigma NS) was expressed in insect cells using a recombinant baculovirus. In infected-cell extracts, r sigma NS was found in large complexes (> or = 30 S) that were disassembled into smaller, 13-19 S complexes upon treatment with RNase A. R sigma NS also bound to poly(A)-Sepharose beads both before and after purification. Treatment with high salt during purification caused r sigma NS to sediment in even smaller, 7-9 S complexes, consistent with more complete loss of RNA. To localize the RNA-binding site, limited proteolysis was used to fragment the r sigma NS protein. Upon mild treatment with thermolysin, 11 amino acids were removed from the amino terminus of r sigma NS, and the resulting protein no longer bound to poly(A). In addition, when r sigma NS in cell extracts was treated with thermolysin to generate the amino-terminally truncated from, it sedimented at 7-9 S, also consistent with the loss of RNA-binding capacity. To confirm these findings, a deletion mutant lacking amino acids 2-11 was constructed and expressed in insect cells from a recombinant baculovirus. The mutant protein in cell extracts showed greatly reduced poly(A)-binding activity and sedimented as 7-9 S complexes. These data suggest that the first 11 amino acids of sigma NS, which are predicted to form an amphipathic alpha-helix, are important for both ssRNA binding and formation of complexes larger than 7-9 S.

Measles virus nucleoprotein encoded from the vaccinia virus genome assembles into nucleocapsids similar in many respects to those observed during a natural measles virus infection. The influence of the measles virus phosphoprotein on nucleocapsid assembly has been studied using a vaccinia virus recombinant encoding both the nucleoprotein and the phosphoprotein. Infection of cells with the virus recombinant resulted in the formation of cytoplasmic inclusions in which the nucleoprotein and the phosphoprotein colocalized. Electron microscopic examination suggested that these inclusions contained characteristic nucleocapsid filaments. The buoyant density of nucleocapsids assembled in the presence of the phosphoprotein was found to be slightly higher than that of nucleocapsids assembled in its absence. Furthermore, the phosphoprotein partially inhibited the formation of nucleocapsids, a process which was extremely efficient when the nucleoprotein was expressed alone. Analysis of the nucleic acid content of nucleocapsids showed that they packaged heterologous RNA into a micrococcal nuclease-resistant form. These experiments demonstrate that the measles virus phosphoprotein regulates the efficiency with which the nucleoprotein assembles into nucleocapsids and the structural conformation they acquire.

It has previously been shown that the purified influenza virus nucleoprotein (NP) forms the oligomers in vitro in NP preparations obtained from virions (Wiley et al., 1977, Virology, 79, 446-448; Ruigrok and Baudin, 1995, J. Gen. Virol., 76, 1009-1014) and infected cells (Becht and Weiss, 1991, Behring Inst Mitt., Justus-Liebig Universitat, Giessen, 89, 1-11). We have shown in this report that boiling-sensitive NP oligomers (di- and trimers) are formed in vivo in the course of intracellular influenza virus replication. They are detected by PAGE about 10 min after monomeric 56-kDa NP molecules are synthesized. NP oligomers are formed by different strains of influenza virus in different cell lines. Some influenza virus strains are characterized by complete conversion of NP monomers into oligomers and others by only partial conversion. In the Triton X-114 phase partitioning system NP oligomers show more hydrophobicity than NP monomers. NP oligomers are detected in the sedimentable and soluble fractions of both cell lysate and extracellular medium. The possibility is discussed that oligomeric NP is a native and functionally significant form of influenza virus NP.

Integration of reverse transcribed retroviral cDNA is not restricted to particular host DNA sequences. However, the frequency of integration into a particular phosphodiester bond is influenced by the local sequence. Here we examine the target-sequence preferences of purified HIV integrase and viral nucleoprotein complexes (preintegration complexes) isolated from freshly infected cells. We find that the three-base sequence including the integration site is not the major factor determining the frequency of integration, since identical triplets embedded in different sequences are used with very different efficiencies. However, there is a statistically significant bias against integration upstream of a pyrimidine nucleotide. The target-sequence preferences of purified integrase and preintegration complexes are very different. Strong integration sites on opposite DNA strands occur in pairs separated by five residues when preintegration complexes are used but not with purified integrase. These studies highlight the difference between the two sources of HIV integration activity and may provide the basis for a simple assay for the correct assembly of viral nucleoprotein complexes.

To illuminate the molecular basis for host range restriction of parainfluenza virus replication, we have examined the types of virus macromolecules produced during abortive infection of nonpermissive MDBK cells with human parainfluenza virus type 1 (hPIV1). While these cells do not support production of hPIV1 virus, they can be infected by hPIV1 as evidenced by accumulation of intracellular viral NP and HN proteins. HPIV1 is also able to drive transcription of a synthetic analog of Sendai virus (SV) genome RNA transfected into virus-infected MDBK cells. In contrast to transcription, hPIV1 genome replication does not occur in MDBK cells. Intracellular full-length genome RNA was detected only in trace amounts 2 days after infection, and was undetectable 4 days after infection. Full-length antigenome (+) sense RNA was not detectable. Nucleocapsid complexes failed to accumulate in the cytoplasm of nonpermissive cells, and no detectable nucleocapsids were released into the medium as virus particles. The data indicate that defective vRNA synthesis and/or nucleocapsid formation is responsible for the inability of hPIV1 to grow in MDBK cells. Our data also show that hPIV1 is capable of providing all helper functions for packaging SV synthetic genome analogs into infectious particles, but these SV-specific RNAs encapsidated with hPIV1 proteins are in turn not replicated by SV proteins. These results suggest that functional protein-protein interactions between parainfluenza virus strains have more stringent requirements than do protein-RNA interactions.

The nucleocapsid protein (N, 525 amino acids) of measles virus plays a central role in the replication of the viral genomic RNA. Its functions require interactions with itself and with other viral components. The N protein encapsidates genomic RNA, a function reflected in its ability to self-assemble into nucleocapsid-like particles in the absence of other viral proteins. The substrate for the packaging of nascent RNA during RNA replication is a complex between the N and phosphoprotein (P). The domains on the N protein that promote binding to P protein and self-assembly have been identified utilizing a series of N protein deletions. Two noncontiguous regions, amino acids 4-188 and 304-373 of N protein, are required for the formation of the soluble N-P complex, while deletion of amino acids 189-239 did not affect N-P binding. Amino acids 240-303 appear to be necessary for the stability of the protein. The N-terminal 398 amino acids are all required for the formation of organized nucleocapsid-like particles, since deletion of the central region from amino acids 189-373 completely abolished N-N interaction, and deletion of amino acids 4-188 and 374-492 caused the formation of unstructured aggregates.

We used the yeast interactive trap system to identify a cellular protein which interacts with the nucleoprotein of influenza A viruses. This protein, nucleoprotein interactor 1 (NPI-1) is the human homolog of the yeast protein SRP1. SRP1 was previously identified as a suppressor of temperature-sensitive RNA polymerase I mutations (R. Yano, M. Oakes, M. Yamaghishi, J. Dodd, and M. Nomura, Mol. Cell. Biol. 12, 5640-5651, 1992). A full-length cDNA clone of NPI-1 was generated from HeLa cell poly A + RNA. The viral nucleoprotein, which had been partially purified from influenza A/PR/8/34 virus-infected embryonated eggs, could be coprecipitated from solution by glutathione agarose beads complexed with a bacterially expressed glutathione-S-transferase-NPI-1 fusion protein, confirming the results of the yeast genetic system. Antisera raised against NPI-1 identified a 60-kDa polypeptide from total cellular extracts of both HeLa and MDBK cells. The viral nucleoprotein was coimmunoprecipitated from influenza A/WSN/33 virus-infected MDBK cells by anti-NPI-1 sera, demonstrating an interaction of these two proteins in infected cells. Similarly, NPI-1 was coimmunoprecipitated from MDBK cells by anti-NP sera. These experiments suggest that NPI-1 plays a role during influenza virus replication.