The Viral Polymerase Complex Mediates the Interaction of Viral Ribonucleoprotein Complexes with Recycling Endosomes during Sendai Virus Assembly.

Paramyxoviruses are negative-sense single-stranded RNA viruses that comprise many important human and animal pathogens, including human parainfluenza viruses. These viruses bud from the plasma membrane of infected cells after the viral ribonucleoprotein complex (vRNP) is transported from the cytoplasm to the cell membrane via Rab11a-marked recycling endosomes. The viral proteins that are critical for mediating this important initial step in viral assembly are unknown. Here, we used the model paramyxovirus, murine parainfluenza virus 1, or Sendai virus (SeV), to investigate the roles of viral proteins in Rab11a-driven virion assembly. We previously reported that infection with SeV containing high levels of copy-back defective viral genomes (DVGs) (DVG-high SeV) generates heterogenous populations of cells. Cells enriched in full-length (FL) virus produce viral particles containing standard or defective viral genomes, while cells enriched in DVGs do not, despite high levels of defective viral genome replication. Here, we took advantage of this heterogenous cell phenotype to identify proteins that mediate interaction of vRNPs with Rab11a. We examined the roles of matrix protein and nucleoprotein and determined that their presence is not sufficient to drive interaction of vRNPs with recycling endosomes. Using a combination of mass spectrometry and comparative analyses of protein abundance and localization in DVG-high and FL-virus-high (FL-high) cells, we identified viral polymerase complex component protein L and, specifically, its cofactor C as interactors with Rab11a. We found that accumulation of L and C proteins within the cell is the defining feature that differentiates cells that proceed to viral egress from cells containing viruses that remain in replication phases.IMPORTANCE Paramyxoviruses are members of a family of viruses that include a number of pathogens imposing significant burdens on human health. In particular, human parainfluenza viruses are an important cause of pneumonia and bronchiolitis in children for which there are no vaccines or directly acting antivirals. These cytoplasmic replicating viruses bud from the plasma membrane and co-opt cellular endosomal recycling pathways to traffic viral ribonucleoprotein complexes from the cytoplasm to the membrane of infected cells. The viral proteins required for viral engagement with the recycling endosome pathway are still not known. Here, we used the model paramyxovirus Sendai virus, or murine parainfluenza virus 1, to investigate the role of viral proteins in this initial step of viral assembly. We found that the viral polymerase components large protein L and accessory protein C are necessary for engagement with recycling endosomes. These findings are important in identifying viral proteins as potential targets for development of antivirals.

Evidence that Receptor Destruction by the Sendai Virus Hemagglutinin-Neuraminidase Protein Is Responsible for Homologous Interference.

UNLABELLED: Receptor destruction has been considered one of the mechanisms of homologous Sendai virus (SeV) interference. However, direct evidence of receptor destruction upon virus infection and its relevance to interference is missing. To investigate a precise mechanism of homologous interference, we established SeV persistently infected cells. The persistently infected cells inhibited superinfection by homologous SeV but supported replication of human parainfluenza virus 2 (hPIV2) and influenza A virus (IAV). We confirmed that SeV particles could not attach to or penetrate the infected cells and that the hemagglutinin-neuraminidase (HN) protein of SeV was involved in the interference. Lectin blot assays showed that the α2,3-linked sialic acids were specifically reduced in the SeV-infected cells, but the level of α2,6-linked sialic acids had not changed. As infection with IAV removed both α2,3- and α2,6-linked sialic acids, especially α2,3-linked sialic acids, IAV-infected cells inhibited superinfection of SeV. These results provide concrete evidence that destruction of the specific SeV receptor, α2,3-linked sialic acids, is relevant to homologous interference by SeV. IMPORTANCE: Viral interference is a classically observed phenomenon, but the precise mechanism is not clear. Using SeV interference, we provide concrete evidence that reduction of the α2,3-linked sialic acid receptor by the HN of SeV is closely related with viral interference. Since SeV infection resulted in decrease of only α2,3-linked sialic acids, IAV, which also utilized α2,6-linked sialic acids to initiate infection, superinfected the SeV-infected cells. In contrast, SeV could not superinfect the IAV-infected cells because both α2,3- and α2,6-linked sialic acids were removed. These results indicate that receptor destruction critically contributes to viral interference.

Histochemical fluorescent staining of Sendai virus-infected cells with a novel sialidase substrate.

Sialidases, enzymes that remove terminal sialic acid residues, are pivotal in various biological processes such as malignancy and infection with pathogens. For histochemical staining of sialidase activity, we have developed a new synthetic sialidase substrate, sialic acid-conjugated fluorescent benzothiazolylphenol derivative (BTP3-Neu5Ac), for rapid, sensitive, and specific fluorescent staining of sialidase activity. Here, we showed the usefulness of BTP3-Neu5Ac for histochemical fluorescent staining of cells infected with Sendai virus (SV), which possesses sialidase activity. BTP3-Neu5Ac also visualised SV-infected regions of lung sections from SV-infected mice. We succeeded in histochemical fluorescent staining of SV both in vitro and in vivo. SV has been utilised in many virological and biotechnological studies such as developments of an oncolytic virus, a gene therapy vector, and a vaccine candidate. BTP3-Neu5Ac should contribute to rapid progress of such studies and researches on viral sialidase.

Intrinsic dynamics of the partly unstructured PX domain from the Sendai virus RNA polymerase cofactor P.

Despite their evident importance for function, dynamics of intrinsically unstructured proteins are poorly understood. Sendai virus phosphoprotein, cofactor of the RNA polymerase, contains a partly unstructured protein domain. The phosphoprotein X domain (PX) is responsible for binding the polymerase to the nucleocapsid assembling the viral RNA. For RNA synthesis, the interplay of the dynamics of the unstructured and structured PX subdomains is thought to drive progression of the RNA polymerase along the nucleocapsid. Here we present a detailed study of the dynamics of PX using hydrogen/deuterium exchange and different NMR relaxation measurements. In the unstructured subdomain, large amplitude fast motions were found to be fine-tuned by the presence of residues with short side chains. In the structured subdomain, where fast motions of both backbone and side chains are fairly restricted, the first helix undergoes slow conformational exchange corresponding to a local unfolding event. The other two helices, which represent the nucleocapsid binding site, were found to be more stable and to reorient with respect to each other, as probed by slow conformational exchange identified for residues on the third helix. The study illustrates the intrinsically differential dynamics of this partly unstructured protein and proposes the relation between these dynamics and its function.

Two N-terminal regions of the Sendai virus L RNA polymerase protein participate in oligomerization.

The RNA dependent RNA polymerase of Sendai virus consists of a complex of the large (L) and phosphoprotein (P) subunits where L is thought to be responsible for all the catalytic activities necessary for viral RNA synthesis. We previously showed that the L protein forms an oligomer [Smallwood, S., Cevik, B., Moyer, S.A., 2002. Intragenic complementation and oligomerization of the L subunit of the Sendai virus RNA polymerase. Virology 304, 235-245] and mapped the L oligomerization domain between amino acids 1 and 174 of the protein [Cevik, B., Smallwood, S., Moyer, S.A., 2003. The oligomerization domain resides at the very N-terminus of the Sendai virus L RNA polymerase protein. Virology 313, 525-536]. An internal deletion encompassing amino acids 20 to 178 of the L protein lost polymerase activity but still formed an L-L oligomer. The first 25 amino acids of paramyxovirus L proteins are highly conserved and site-directed mutagenesis within this region eliminated the biological activity of the L protein but did not have any effect on P-L or L-L interactions. Moreover deletion of amino acids 2-18 in L abolished biological activity, but again the L-L binding was normal demonstrating that the oligomerization domain of L protein resides in two N-terminal regions of the protein. Therefore, sequences between both aa 2-19 and aa 20-178 can independently mediate Sendai L oligomerization, however, both are required for the activity of the protein.

Sendai virus RNA polymerase scanning for mRNA start sites at gene junctions.

Mini-genomes expressing two reporter genes and a variable gene junction were used to study Sendai virus RNA polymerase (RdRp) scanning for the mRNA start signal of the downstream gene (gs2). We found that RdRp could scan the template efficiently as long as the initiating uridylate of gs2 (3' UCCCnnUUUC) was preceded by the conserved intergenic region (3' GAA) and the last 3 uridylates of the upstream gene end signal (ge1; 3' AUUCUUUUU). The end of the leader sequence (3' CUAAAA, which precedes gs1) could also be used for gene2 expression, but this sequence was considerably less efficient. Increasing the distance between ge1 and gs2 (up to 200 nt) led to the progressive loss of gene2 expression, in which half of gene2 expression was lost for each 70 nucleotides of intervening sequence. Beyond 200 nt, gene2 expression was lost more slowly. Our results suggest that there may be two populations of RdRp that scan at gene junctions, which can be distinguished by the efficiency with which they can scan the genome template for gs.

High throughput screening assay for negative single stranded RNA virus polymerase inhibitors.

The Paramyxoviridae form a large family of viruses containing many human and veterinary pathogens for which a need for antiviral treatment is emphasized, particularly following the recent emergence of new viruses. The viral RNA-dependent RNA polymerase constitutes an obvious target for antiviral compounds. An in vitro assay was developed that allows high throughput screening of compounds potentially inhibiting the Sendai virus RNA-dependent RNA polymerase. Screening relies on the detection of the Photinus pyralis luciferase produced in a transcription/translation coupled assay using a mini-replicon virus. It contains an internal control for possible adverse effects of the tested compounds on translation or on luciferase activity. It is estimated that the mini-replicon template produced in one fertilized egg is sufficient to run 5000-10,000 reactions. This assay constitutes a simple, sensitive and easily automated method to perform high throughput screening of Paramyxoviridae RNA-dependent RNA polymerase inhibitors.

Sendai virus RNA-dependent RNA polymerase L protein catalyzes cap methylation of virus-specific mRNA.

The Sendai virus (SeV) RNA-dependent RNA polymerase complex, which consists of L and P proteins, participates in the synthesis of viral mRNAs that possess a methylated cap structure. To identify the SeV protein(s) involved in mRNA cap methylation, we developed an in vitro assay system to detect mRNA (guanine-7-)methyltransferase (G-7-MTase) activity. Viral ribonucleoprotein complexes and purified recombinant L protein but not P protein exhibited G-7-MTase activity. On the other hand, mRNA synthesis in a reconstituted transcription system using purified N-RNA (N protein-genomic RNA) complex as a template required both the L and P proteins. The enzymatic properties of SeV G-7-MTase were different from those of cellular G-7-MTase. In particular, unlike cellular G-7-MTase, the SeV enzyme preferentially methylated capped RNA containing the viral mRNA 5'-end sequences (GpppApGpG-). The C-terminal part (amino acid residues 1,756-2,228) of the L protein catalyzed cap methylation, whereas the N-terminal half (residues 1-1,120) containing putative RNA polymerase subdomains did not. This is to our knowledge the first direct biochemical evidence that supports the idea that mononegavirus L protein catalyzes cap methylation as well as RNA synthesis.

Intragenic complementation and oligomerization of the L subunit of the sendai virus RNA polymerase.

The RNA-dependent RNA polymerase of Sendai virus consists of two subunits, the L and P proteins, where L is thought to be responsible for all the catalytic activities necessary for viral RNA synthesis. Sequence alignment of the L proteins of a variety of negative-stranded RNA viruses revealed six regions of good conservation, designated domains I-VI, which are thought to correspond to functional domains of the protein. Analysis of a number of site-directed mutants within the six domains of L allowed us to conclude that the activities of the polymerase are not simply compartmentalized and that each domain contributes to multiple steps in viral RNA synthesis. Nevertheless these domains can function in trans since we demonstrate here that intragenic complementation between pairs of coexpressed inactive L mutants can restore viral RNA synthesis on an added template. Although intragenic complementation is typically very inefficient, complementation to restore leader RNA synthesis was surprisingly very efficient for some pairs and complementation of mRNA synthesis and genome replication was less, but still significant. Complementation occurred with L mutants in five of the six domains, the exception being a domain III mutant, and required the cotranslation of the two L mutants. C-terminal truncations deleting up to half of L were capable of restoring transcription of an inactive domain I L mutant at amino acid 379. Oligomerization of L in the polymerase complex was demonstrated directly by the co-immunoprecipitation of differentially epitope-tagged full-length and truncated L proteins. These data are consistent with L protein being an oligomer with multiple independent domains each of which exhibits several functions.

Different substitutions at conserved amino acids in domains II and III in the Sendai L RNA polymerase protein inactivate viral RNA synthesis.

The Sendai virus RNA polymerase is a complex of two virus-encoded proteins, the phosphoprotein (P) and the large (L) protein, where L is believed to possess all the enzymatic activities necessary for viral transcription and replication. The alignment of amino acid sequences of L proteins from negative-sense RNA viruses shows six regions, designated domains I-VI, of good conservation which have been proposed to be important for the various enzymatic activities of the polymerase. To directly address the role(s) of domains II and III, site-directed mutations were constructed by the substitution of multiple amino acids at 13 highly or mostly conserved residues. Analysis of in vitro viral transcription and replication showed that the majority of the mutations completely inactivated the L protein for all aspects of RNA synthesis, thus conservation correlated with the essential nature of the amino acid. At some positions different phenotypes, from inactivation to partial activities, were observed which depended on the nature of the amino acid that was substituted. Two mutants, K543R and K666V, could synthesize some leader RNA, but were defective in mRNA synthesis and replication. K666R and G737E had significantly reduced replication compared to transcription in vitro, but replicated genome RNA much more efficiently in vivo. K666A gave transcription, but no replication. Representative inactive L mutants, however, were still able to bind P protein and the polymerase complex was capable of binding nucleocapsids, so the defect appeared to be in the initiation of RNA synthesis.