The replication activity of influenza virus polymerase is linked to the capacity of the PA subunit to induce proteolysis

The PA subunit of the influenza virus polymerase complex is a phosphorylated protein that induces a proteolytic process that decreases its own accumulation levels and those of coexpressed proteins. The amino-terminal third of the protein is responsible for the induction of proteolysis. We mutated five potential casein kinase II phosphorylation sites located in the amino-terminal third of the protein. Mutations affecting position 157 almost completely abrogated proteolysis induction, whereas a mutation at position 162 produced a moderate decrease and mutations at positions 151, 200, and 224 did not affect proteolysis induction. Reconstitution of the influenza virus polymerase in vivo with viral model RNA containing the chloramphenicol acetyltransferase (CAT) gene indicated that the CAT activity obtained correlated with the capacity of each PA mutant to induce proteolysis. RNA protection assays of the products obtained with viral polymerase, reconstituted in vivo with model RNAs, indicated that mutations at position 157 led to a selective loss of the ability to synthesize cRNA from the viral RNA template but not to transcribe viral RNA, while a mutation affecting position 162 showed an intermediate phenotype. Collectively, these data provide a link between PA-mediated induction of proteolysis and the replication activity of the polymerase.     

Identification of a phosphorylation site within the P protein important for mRNA transcription and growth of parainfluenza virus 5

The viral RNA-dependent RNA polymerase (vRdRp) of paramyxovirus consists of the large (L) protein and the phosphoprotein (P). P is heavily phosphorylated, and it is thought that the phosphorylation of P plays a role in regulating viral RNA synthesis. However, no phosphorylation site within the P protein in paramyxovirus has been identified as playing a positive role in viral RNA synthesis in virus infection. Using mass spectrometry analysis, the threonine residue at position 286 of P of parainfluenza virus 5 (PIV5) was found phosphorylated. Mutation of T286 to alanine (T286A), aspartic acid (T286D), or glutamic acid (T286E) reduced minigenome activity. Recombinant virus containing a mutation at the T286 position (rPIV5-P-T286A) grew slower than wild-type virus; viral mRNA synthesis and protein expression of rPIV5-P-T286A were delayed. Biochemical studies showed that the binding of NP or L protein with the P mutants or tetramer formation by the mutant P proteins was unaltered from that for wild-type P. While we failed to rescue rPIV5-P-T286E virus, several revertant viruses were obtained. All non-wild-type revertants had mutations at T286 and showed defects in both minigenome activity and viral growth. This is the first time that a phosphorylation site within the P protein in paramyxovirus has been found to play a positive role in viral mRNA synthesis and virus growth.     

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.     

The C-terminal LCAR of host ANP32 proteins interacts with the influenza A virus nucleoprotein to promote the replication of the viral RNA genome

The segmented negative-sense RNA genome of influenza A virus is assembled into ribonucleoprotein complexes (RNP) with viral RNA-dependent RNA polymerase and nucleoprotein (NP). It is in the context of these RNPs that the polymerase transcribes and replicates viral RNA (vRNA). Host acidic nuclear phosphoprotein 32 (ANP32) family proteins play an essential role in vRNA replication by mediating the dimerization of the viral polymerase via their N-terminal leucine-rich repeat (LRR) domain. However, whether the C-terminal low-complexity acidic region (LCAR) plays a role in RNA synthesis remains unknown. Here, we report that the LCAR is required for viral genome replication during infection. Specifically, we show that the LCAR directly interacts with NP and this interaction is mutually exclusive with RNA. Furthermore, we show that the replication of a short vRNA-like template that can be replicated in the absence of NP is less sensitive to LCAR truncations compared with the replication of full-length vRNA segments which is NP-dependent. We propose a model in which the LCAR interacts with NP to promote NP recruitment to nascent RNA during influenza virus replication, ensuring the co-replicative assembly of RNA into RNPs.     

Monomeric Nucleoprotein of Influenza A Virus

Isolated influenza A virus nucleoprotein exists in an equilibrium between monomers and trimers. Samples containing only monomers or only trimers can be stabilized by respectively low and high salt. The trimers bind RNA with high affinity but remain trimmers, whereas the monomers polymerise onto RNA forming nucleoprotein-RNA complexes. When wild type (wt) nucleoprotein is crystallized, it forms trimers, whether one starts with monomers or trimers. We therefore crystallized the obligate monomeric R416A mutant nucleoprotein and observed how the domain exchange loop that leads over to a neighbouring protomer in the trimer structure interacts with equivalent sites on the mutant monomer surface, avoiding polymerisation. The C-terminus of the monomer is bound to the side of the RNA binding surface, lowering its positive charge. Biophysical characterization of the mutant and wild type monomeric proteins gives the same results, suggesting that the exchange domain is folded in the same way for the wild type protein. In a search for how monomeric wt nucleoprotein may be stabilized in the infected cell we determined the phosphorylation sites on nucleoprotein isolated from virus particles. We found that serine 165 was phosphorylated and conserved in all influenza A and B viruses. The S165D mutant that mimics phosphorylation is monomeric and displays a lowered affinity for RNA compared with wt monomeric NP. This suggests that phosphorylation may regulate the polymerisation state and RNA binding of nucleoprotein in the infected cell. The monomer structure could be used for finding new anti influenza drugs because compounds that stabilize the monomer may slow down viral infection.     

Ubiquitination and deubiquitination of NP protein regulates influenza A virus RNA replication

Influenza A virus RNA replication requires an intricate regulatory network involving viral and cellular proteins. In this study, we examined the roles of cellular ubiquitinating/deubiquitinating enzymes (DUBs). We observed that downregulation of a cellular deubiquitinating enzyme USP11 resulted in enhanced virus production, suggesting that USP11 could inhibit influenza virus replication. Conversely, overexpression of USP11 specifically inhibited viral genomic RNA replication, and this inhibition required the deubiquitinase activity. Furthermore, we showed that USP11 interacted with PB2, PA, and NP of viral RNA replication complex, and that NP is a monoubiquitinated protein and can be deubiquitinated by USP11 in vivo. Finally, we identified K184 as the ubiquitination site on NP and this residue is crucial for virus RNA replication. We propose that ubiquitination/deubiquitination of NP can be manipulated for antiviral therapeutic purposes.     

Discovery of novel antiviral agents directed against the influenza A virus nucleoprotein using photo-cross-linked chemical arrays

The nucleoprotein (NP) of the influenza virus is expressed in the early stage of infection and plays important roles in numerous steps of viral replication. NP is relatively well conserved compared with viral surface spike proteins. This study experimentally demonstrates that NP is a novel target for the development of new antiviral drugs against the influenza virus. First, artificial analogs of mycalamide A in a chemical array bound specifically with high affinity to NP. Second, the compounds inhibited multiplication of the influenza virus. Furthermore, surface plasmon resonance imaging experiments demonstrated that the binding activity of each compound to NP correlated with its antiviral activity. Finally, it was shown that these compounds bound NP within the N-terminal 110-amino acid region but their binding abilities were dramatically reduced when the N-terminal 13-amino acid tail was deleted, suggesting that the compounds might bind to this region, which mediates the nuclear transport of NP and its binding to viral RNA. These data suggest that compound binding to the N-terminal 13-amino acid tail region may inhibit viral replication by inhibiting the functions of NP. Collectively, these results strongly suggest that chemical arrays are convenient tools for the screening of viral product inhibitors.     

Mutational analysis identifies functional domains in the influenza A virus PB2 polymerase subunit.

A collection of influenza virus PB2 mutant genes was prepared, including N-terminal deletions, C-terminal deletions, and single-amino-acid insertions. These mutant genes, driven by a T7 promoter, were expressed by transfection into COS-1 cells infected with a vaccinia virus encoding T7 RNA polymerase. Mutant proteins accumulated to levels similar to that of wild-type PB2. Immunofluorescence analyses showed that the C-terminal region of the protein is essential for nuclear transport and that internal sequences affect nuclear localization, confirming previous results (J. Mukaijawa and D. P. Nayak, J. Virol. 65:245-253, 1991). The biological activity of these mutants was tested by determining their capacity to (i) reconstitute RNA polymerase activity in vivo by cotransfection with proteins NP, PB1, and PA and a virion-like RNA encoding the cat gene into vaccinia virus T7-infected COS-1 cells and (ii) complete with the wild-type PB2 activity. In addition, when tested at different temperatures in vivo, two mutant PB2 proteins showed a temperature-sensitive phenotype. The lack of interference shown by some N-terminal deletion mutants and the complete interference obtained with a C-terminal deletion mutant encoding only 124 amino acids indicated that this protein domain is responsible for interaction with another component of the polymerase, probably PB1. To further characterize the mutants, their ability to induce in vitro synthesis of viral cRNA or mRNA was tested by using ApG or beta-globin mRNA as a primer. One of the mutants, 1299, containing an isoleucine insertion at position 299, was able to induce cRNA and mRNA synthesis in ApG-primed reactions but required a higher beta-globin mRNA concentration than wild-type PB2 for detection of in vitro synthesis. This result suggested that mutant I299 has diminished cap-binding activity.     

Phosphorylation of rubella virus capsid regulates its RNA binding activity and virus replication

Rubella virus is an enveloped positive-strand RNA virus of the family TOGAVIRIDAE: Virions are composed of three structural proteins: a capsid and two membrane-spanning glycoproteins, E2 and E1. During virus assembly, the capsid interacts with genomic RNA to form nucleocapsids. In the present study, we have investigated the role of capsid phosphorylation in virus replication. We have identified a single serine residue within the RNA binding region that is required for normal phosphorylation of this protein. The importance of capsid phosphorylation in virus replication was demonstrated by the fact that recombinant viruses encoding hypophosphorylated capsids replicated at much lower titers and were less cytopathic than wild-type virus. Nonphosphorylated mutant capsid proteins exhibited higher affinities for viral RNA than wild-type phosphorylated capsids. Capsid protein isolated from wild-type strain virions bound viral RNA more efficiently than cell-associated capsid. However, the RNA-binding activity of cell-associated capsids increased dramatically after treatment with phosphatase, suggesting that the capsid is dephosphorylated during virus assembly. In vitro assays indicate that the capsid may be a substrate for protein phosphatase 1A. As capsid is heavily phosphorylated under conditions where virus assembly does not occur, we propose that phosphorylation serves to negatively regulate binding of viral genomic RNA. This may delay the initiation of nucleocapsid assembly until sufficient amounts of virus glycoproteins accumulate at the budding site and/or prevent nonspecific binding to cellular RNA when levels of genomic RNA are low. It follows that at a late stage in replication, the capsid may undergo dephosphorylation before nucleocapsid assembly occurs.     

Identification of the threonine phosphorylation sites on the polyomavirus major capsid protein VP1: relationship to the activity of middle T antigen.

Phosphorylation of the polyomavirus major capsid protein VP1 was examined after in vivo 32P labeling of virus-infected cells. Two phosphorylated peptide fragments of VP1 were identified by protease digestion, high-performance liquid chromatography purification, mass spectrometry, and N-terminal sequencing. The peptides from residues 58 to 78 and residues 153 to 173 were phosphorylated on threonine. Site-directed mutations were introduced at these threonine sites, and mutant viruses were reconstructed. A threonine-to-glycine change at residue 63 (mutant G63) and a threonine-to-alanine change at residue 156 (mutant A156) resulted in viruses defective in phosphorylation of the respective peptides after in vivo labeling. Growth of the mutant G63 virus was similar to that of the wild-type virus, but the mutant A156 was inefficient in assembly of 240S viral particles. Polyomavirus nontransforming host range (hr-t) mutants are defective in VP1 threonine phosphorylation when grown in nonpermissive cells (R. L. Garcea, K. Ballmer-Hofer, and T. L. Benjamin, J. Virol. 54:311-316, 1985). Proteolytic mapping of VP1 peptides after in vivo labeling from hr-t mutant virus infections demonstrated that both residues T-63 and T-156 were affected. These results suggest that the block in virion assembly in hr-t mutant viruses is associated with a defect in phosphorylation of threonine 156.     

Hyperphosphorylation of mutant influenza virus matrix protein, M1, causes its retention in the nucleus.

The matrix (M1) protein of influenza virus is a major structural component, involved in regulation of viral ribonucleoprotein transport into and out of the nucleus. Early in infection, M1 is distributed in the nucleus, whereas later, it is localized predominantly in the cytoplasm. Using immunofluorescence microscopy and the influenza virus mutant ts51, we found that at the nonpermissive temperature M1 was retained in the nucleus, even at late times after infection. In contrast, the viral nucleoprotein (NP), after a temporary retention in the nucleus, was distributed in the cytoplasm. Therefore, mutant M1 supported the release of the viral ribonucleoproteins from the nucleus, but not the formation of infectious virions. The point mutation in the ts51 M1 gene was predicted to encode an additional phosphorylation site. We observed a substantial increase in the incorporation of 32Pi into M1 at the nonpermissive temperature. The critical role of this phosphorylation site was demonstrated by using H89, a protein kinase inhibitor; it inhibited the expression of the mutant phenotype, as judged by M1 distribution in the cell. Immunofluorescence analysis of ts51-infected cells after treatment with H89 showed a wild-type phenotype. In summary, the data indicated that the ts51 M1 protein was hyperphosphorylated at the nonpermissive temperature and that this phosphorylation was responsible for its aberrant nuclear retention.     

Mutational analysis of the promoter required for influenza virus virion RNA synthesis.

An in vitro RNA synthesis system was established in which the influenza virus virion (minus-sense) RNA was made from the synthetic plus-sense RNA (cRNA) template by the purified viral polymerase complex. The cRNA promoter was studied by mutational analysis using the in vitro system, and on the basis of these experiments, the first 11 nucleotides of the 3' noncoding sequence were found to contain the minimum promoter required for virion RNA synthesis. The addition of extra nucleotides at the 3' end decreased the promoter activity of the templates, indicating that the viral polymerase does not recognize an internal promoter efficiently. The wild-type and mutated RNA templates were also tested in vivo by using the ribonucleoprotein transfection system. In contrast to the in vitro system, it was found that the majority of mutations at the 3'-terminal sequence significantly decreased or abolished chloramphenicol acetyltransferase (CAT) expression. These results suggest that the cRNA promoter overlaps other essential cis elements required for chloramphenicol acetyltransferase expression in vivo.     

Phosphorylation status of the parvovirus minute virus of mice particle: mapping and biological relevance of the major phosphorylation sites

The core of the VP-1 and VP-2 proteins forming the T=1 icosahedral capsid of the prototype strain of the parvovirus minute virus of mice (MVMp) share amino acids sequence and a common three-dimensional structure; however, the roles of these polypeptides in the virus infection cycle differ. To gain insights into this paradox, the nature, distribution, and biological significance of MVMp particle phosphorylation was investigated. The VP-1 and VP-2 proteins isolated from purified empty capsids and from virions containing DNA harbored phosphoserine and phosphothreonine amino acids, which in two-dimensional tryptic analysis resulted in complex patterns reproducibly composed by more than 15 unevenly phosphorylated peptides. Whereas secondary protease digestions and comigration of most weak peptides in the fingerprints revealed common phosphorylation sites in the VP-1 and VP-2 subunits assembled in capsids, the major tryptic phosphopeptides were remarkably characteristic of either polypeptide. The VP-2-specific peptide named B, containing the bulk of the (32)P label of the MVMp particle in the form of phosphoserine, was mapped to the structurally unordered N-terminal domain of this polypeptide. Mutations in any or all four serine residues present in peptide B showed that the VP-2 N-terminal domain is phosphorylated at multiple sites, even though none of them was essential for capsid assembly or virus formation. Chromatographic analysis of purified wild-type (wt) and mutant peptide B digested with a panel of specific proteases allowed us to identify the VP-2 residues Ser-2, Ser-6, and Ser-10 as the main phosphate acceptors for MVMp capsid during the natural viral infection. Phosphorylation at VP-2 N-terminal serines was not necessary for the externalization of this domain outside of the capsid shell in particles containing DNA. However, the plaque-forming capacity and plaque size of VP-2 N-terminal phosphorylation mutants were severely reduced, with the evolutionarily conserved Ser-2 determining most of the phenotypic effect. In addition, the phosphorylated amino acids were not required for infection initiation or for nuclear translocation of the expressed structural proteins, and thus a role at a late stage of MVMp life cycle is proposed. This study illustrates the complexity of posttranslational modification of icosahedral viral capsids and underscores phosphorylation as a versatile mechanism to modulate the biological functions of their protein subunits.     

Site-directed mutagenesis of the avian retrovirus nucleocapsid protein, pp 12, at Serine 40, the primary site of phosphorylation in vivo

The major nucleocapsid protein of avian retroviruses, pp 12, binds to single-stranded viral RNA with high affinity. Phosphorylation at Ser-40 is necessary for this binding. In order to examine the role of phosphorylation of serine 40 in the biological function of pp 12, we have introduced a series of amino acid substitutions at this position in the Rous sarcoma virus (Pr-C) protein. Substitution of threonine, alanine, or three other amino acids for Ser-40 had very little or no detectable effect on viral replication, nor did the control substitution of glycine for Ser-43, a nonphosphorylated residue. In vivo and in vitro, the Ala-40 and probably the Thr-40 substituted p 12 proteins are phosphorylated at alternative sites which are phosphorylated to a minor extent in vivo in the wild type protein. A study of the RNA binding properties of Ala-40 substituted p 12 has indicated that the protein has been stabilized in a high affinity RNA binding state which is independent of phosphorylation. The viability of the Ala-40 mutant virus indicates that this high binding affinity may be required for biological activity.