Triple reassortment increases compatibility among viral ribonucleoprotein genes of contemporary avian and human influenza A viruses

Compatibility among the influenza A virus (IAV) ribonucleoprotein (RNP) genes affects viral replication efficiency and can limit the emergence of novel reassortants, including those with potential pandemic risks. In this study, we determined the polymerase activities of 2,451 RNP reassortants among three seasonal and eight enzootic IAVs by using a minigenome assay. Results showed that the 2009 H1N1 RNP are more compatible with the tested enzootic RNP than seasonal H3N2 RNP and that triple reassortment increased such compatibility. The RNP reassortants among 2009 H1N1, canine H3N8, and avian H4N6 IAVs had the highest polymerase activities. Residues in the RNA binding motifs and the contact regions among RNP proteins affected polymerase activities. Our data indicates that compatibility among seasonal and enzootic RNPs are selective, and enzoosis of multiple strains in the animal-human interface can facilitate emergence of an RNP with increased replication efficiency in mammals, including humans.     

An amino-terminal domain of the Sendai virus nucleocapsid protein is required for template function in viral RNA synthesis.

The nucleocapsid protein (NP) of Sendai virus encapsidates the genome RNA, forming a helical nucleocapsid which is the template for RNA synthesis by the viral RNA polymerase. The NP protein is thought to have both structural and functional roles, since it is an essential component of the NP0-P (P, phosphoprotein), NP-NP, nucleocapsid-polymerase, and RNA-NP complexes required during viral RNA replication. To identify domains in the NP protein, mutants were constructed by using clustered charge-to-alanine mutagenesis in a highly charged region from amino acids 107 to 129. Each of the mutants supported RNA encapsidation in vitro. The product nucleocapsids formed with three mutants, NP114, NP121, and NP126, however, did not serve as templates for further amplification in vivo, while NP107, NP108, and NP111 were nearly like wild-type NP in vivo. This template defect in the NP mutants from amino acids 114 to 129 was not due to a lack of NP0-P, NP-NP, or nucleocapsid-polymerase complex formation, since these interactions were normal in these mutants. We propose that amino acids 114 to 129 of the NP protein are required for the nucleocapsid to function as a template in viral genome replication.     

Isolation and Properties of an RNA Polymerase from Influenza Virus-Infected Cells

Structures with RNA polymerase activity were isolated from influenza virus-infected cells, and consisted of ribonucleoprotein (RNP) complexes, similar in morphology to the viral internal component or nucleocapsid. The isolation procedure involved fractionation of infected cells in a discontinuous sucrose gradient, in which enzyme activity was concentrated in a fraction of intermediate density which contains both smooth and rough cytoplasmic membranes. The RNPs with polymerase activity were further purified in a velocity gradient, after which the peak fractions showed a 35-fold purification of the polymerase activity when compared with cytoplasmic extracts. The NP polypeptide, which is the subunit of the virion RNP, was the only virus-specific polypeptide detected in these RNP structures.     

Borna disease virus matrix protein is an integral component of the viral ribonucleoprotein complex that does not interfere with polymerase activity

We have recently shown that the matrix protein M of Borna disease virus (BDV) copurifies with the affinity-purified nucleoprotein (N) from BDV-infected cells, suggesting that M is an integral component of the viral ribonucleoprotein complex (RNP). However, further studies were hampered by the lack of appropriate tools. Here we generated an M-specific rabbit polyclonal antiserum to investigate the intracellular distribution of M as well as its colocalization with other viral proteins in BDV-infected cells. Immunofluorescence analysis revealed that M is located both in the cytoplasm and in nuclear punctate structures typical for BDV infection. Colocalization studies indicated an association of M with nucleocapsid proteins in these nuclear punctate structures. In situ hybridization analysis revealed that M also colocalizes with the viral genome, implying that M associates directly with viral RNPs. Biochemical studies demonstrated that M binds specifically to the phosphoprotein P but not to N. Binding of M to P involves the N terminus of P and is independent of the ability of P to oligomerize. Surprisingly, despite P-M complex formation, BDV polymerase activity was not inhibited but rather slightly elevated by M, as revealed in a minireplicon assay. Thus, unlike M proteins of other negative-strand RNA viruses, BDV-M seems to be an integral component of the RNPs without interfering with the viral polymerase activity. We propose that this unique feature of BDV-M is a prerequisite for the establishment of BDV persistence.