Nonsegmented Negative-strand RNA Research Articles

Functional analysis of the L Protein of Nipah Virus Using Minigenome System

Nipah virus (NiV) was identified in Malaysia in late September 1998 as the etiological agent of an outbreak of acute encephalitis with the high mortality rate in human. NiV polymerase gene (L) encodes RNA-dependent RNA polymerase (RdRp) which is an enzyme required for viral replication. The aim of this study is to investigate the role of the N terminal of polymerase protein in replication of NiV. Based on the extensive deduced amino acid sequence analyses of a number of L proteins of non-segmented negative-strand (NNS) RNA viruses, a cluster of high-homology sequence segments have been identified within the body of the L proteins. The functional characterization of the NiV L polymerase was addressed in this research by generating a series of progressive N- deletions in the cloned gene and testing them in a minigenome assay. Five mutations that delete increasing amounts of the amino terminus of the L protein (20 amino acids) were generated at the amino-terminal. The ability of new recombinants plasmids in viral replication were tested by using minigenome system which is based on an intracellular and plasmid-based replication assay. This system contains an inserted chloramphenicol acetyltransferase (CAT) gene, for readout of the replication of the mini genome directed by wild type or mutant L protein. The results demonstrated that the first 100 amino acids of the NH2-terminal domain spanning a highly conserved motif are directly involved in transcription of the genome RNA. The possible functional significance of the NH2-terminal domain of paramyxovirus L protein is discussed.

In vitro capping and transcription of rhabdoviruses

The RNA-dependent RNA polymerase L protein of vesicular stomatitis virus (VSV), a prototypic nonsegmented negative strand (NNS) RNA virus classified into the Rhabdoviridae family, has been used to investigate the fundamental molecular mechanisms of NNS RNA viral mRNA synthesis and processing. In vitro studies on mRNA cap formation with the VSV L protein eventually led to the discovery of the unconventional mRNA capping pathway catalyzed by the guanosine 5′-triphosphatase and RNA:GDP polyribonucleotidyltransferase (PRNTase) activities. The PRNTase activity is a novel enzymatic activity, which transfers 5′-monophosphorylated (p-) RNA from 5′-triphosphorylated (ppp-) RNA to GDP to form 5′-capped RNA (GpppRNA) in a viral mRNA-start sequence-dependent manner. This unconventional capping (pRNA transfer) reaction with PRNTase can be experimentally distinguished from the conventional capping (GMP transfer) reaction with eukaryotic GTP:RNA guanylyltransferase (GTase) on the basis of the following differences in their substrate specificity for the cap formation: PRNTase uses GDP and pppRNA, but not ppRNA, whereas GTase employs GTP, but not GDP, and ppRNA. The pRNA transfer reaction with PRNTase proceeds through a covalent enzyme-pRNA intermediate with a phosphoamide bond. Hence, to prove the PRNTase activity, it is necessary to demonstrate the following consecutive steps separately: (1) the enzyme forms a covalent enzyme-pRNA intermediate, and (2) the intermediate transfers pRNA to GDP. This article describes the methods for in vitro transcription and capping with the recombinant VSV L protein, which permit detailed characterization of its enzymatic reactions and mapping of active sites of its enzymatic domains. It is expected that these systems are adaptable to rhabdoviruses and, by extension, other NNS RNA viruses belonging to different families.

Down-regulation of viral replication by lentiviral-mediated expression of short-hairpin RNAs against vesicular stomatitis virus ribonuclear complex genes

Vesicular stomatitis virus (VSV) causes great economic impact to livestock industry and is a prototype for studying non-segmented negative-stranded RNA (NSNR) viruses. In this study, we evaluated the antiviral potential of unique short-hairpin RNA (shRNA) targeting genes that form the ribonuclear protein (RNP) complex of VSV serotype Indiana (VSIV). We used lentiviral vectors to construct cell lines that stably expressed one of seven shRNAs targeting the RNP genes of VSIV, namely nucleocapsid (N), phosphoprotein (P), or polymerase (L). We reported two N-shRNA sequences targeting the 5′ or 3′ end of N that significantly reduced N, P, and L viral transcripts (p<0.001), reduced viral protein expression, and reduced the viral particles shed in Vero cells (p<0.01). When we analyzed the sequence diversity in the target region of this N-shRNA from two field isolates, we detected a single base substitution outside the seed region. We also reported five other shRNA sequences targeting components of the viral RNA that significantly reduce N, P, and L viral transcripts (p<0.001) but failed to efficiently impair viral replication. The differences in the efficiency of the shRNAs tested were not due to mismatches within the target region in the genome of VSIV. Although partial silencing of viral transcripts by single shRNAs impaired but did not block VSIV replication, the combination of the shRNAs identified here into a multiple shRNA vector may result in inhibition of viral replication. These data contribute to ongoing development of RNAi-based technologies to combat viral diseases.

Interaction of chandipura virus N and P proteins: identification of two mutually exclusive domains of N involved in interaction with P.

The nucleocapsid protein (N) and the phosphoprotein (P) of nonsegmented negative-strand (NNS) RNA viruses interact with each other to accomplish two crucial events necessary for the viral replication cycle. First, the P protein binds to the aggregation prone nascent N molecules maintaining them in a soluble monomeric (N0) form (N0-P complex). It is this form that is competent for specific encapsidation of the viral genome. Second, the P protein binds to oligomeric N in the nucleoprotein complex (N-RNA-P complex), and thereby facilitates the recruitment of the viral polymerase (L) onto its template. All previous attempts to study these complexes relied on co-expression of the two proteins in diverse systems. In this study, we have characterised these different modes of N-P interaction in detail and for the first time have been able to reconstitute these complexes individually in vitro in the chandipura virus (CHPV), a human pathogenic NNS RNA virus. Using a battery of truncated mutants of the N protein, we have been able to identify two mutually exclusive domains of N involved in differential interaction with the P protein. An unique N-terminal binding site, comprising of amino acids (aa) 1–180 form the N0-P interacting region, whereas, C-terminal residues spanning aa 320–390 is instrumental in N-RNA-P interactions. Significantly, the ex-vivo data also supports these observations. Based on these results, we suggest that the P protein acts as N-specific chaperone and thereby partially masking the N-N self-association region, which leads to the specific recognition of viral genome RNA by N0.

Independent Structural Domains in Paramyxovirus Polymerase Protein

All enzymatic activities required for genomic replication and transcription of nonsegmented negative strand RNA viruses (or Mononegavirales) are believed to be concentrated in the viral polymerase (L) protein. However, our insight into the organization of these different enzymatic activities into a bioactive tertiary structure remains rudimentary. Fragments of Mononegavirales polymerases analyzed to date cannot restore bioactivity through trans-complementation, unlike the related L proteins of segmented NSVs. We investigated the domain organization of phylogenetically diverse Paramyxovirus L proteins derived from measles virus (MeV), Nipah virus (NiV), and respiratory syncytial virus (RSV). Through a comprehensive in silico and experimental analysis of domain intersections, we defined MeV L position 615 as an interdomain candidate in addition to the previously reported residue 1708. Only position 1708 of MeV and the homologous positions in NiV and RSV L also tolerated the insertion of epitope tags. Splitting of MeV L at residue 1708 created fragments that were unable to physically interact and trans-complement, but strikingly, these activities were reconstituted by the addition of dimerization tags to the fragments. Equivalently split fragments of NiV, RSV, and MeV L oligomerized with comparable efficiency in all homo- and heterotypic combinations, but only the homotypic pairs were able to trans-complement. These results demonstrate that synthesis as a single polypeptide is not required for the Mononegavirales polymerases to adopt a proper tertiary conformation. Paramyxovirus polymerases are composed of at least two truly independent folding domains that lack a traditional interface but require molecular compatibility for bioactivity. The functional probing of the L domain architecture through trans-complementation is anticipated to be applicable to all Mononegavirales polymerases.

Detecting protein–protein interactions in vesicular stomatitis virus using a cytoplasmic yeast two hybrid system

Protein–protein interactions play an important role in many virus-encoded functions and in virus–host interactions. While a “classical” yeast two-hybrid system (Y2H) is one of the most common techniques to detect such interactions, it has a number of limitations, including a requirement for the proteins of interest to be relocated to the nucleus. Modified Y2H, such as the Sos recruitment system (SRS), which detect interactions occurring in the cytoplasm rather than the nucleus, allow proteins from viruses replicating in the cytoplasm to be tested in a more natural context. In this study, a SRS was used to detect interactions involving proteins from vesicular stomatitis virus (VSV), a prototypic non-segmented negative strand RNA (NNS) virus. All five full-length VSV proteins, as well as several truncated proteins, were screened against each other. Using the SRS, most interactions demonstrated previously involving VSV phosphoprotein, nucleocapsid (N) and large polymerase proteins were confirmed independently, while difficulties were encountered using the membrane associated matrix and glycoproteins. A human cDNA library was also screened against VSV N protein and one cellular protein, SFRS18, was identified which interacted with N in this context. The system presented can be redesigned easily for studies in other less tractable NNS viruses.

Molecular architecture of the vesicular stomatitis virus RNA polymerase

Nonsegmented negative-strand (NNS) RNA viruses initiate infection by delivering into the host cell a highly specialized RNA synthesis machine comprising the genomic RNA completely encapsidated by the viral nucleocapsid protein and associated with the viral polymerase. The catalytic core of this protein-RNA complex is a 250-kDa multifunctional large (L) polymerase protein that contains enzymatic activities for nucleotide polymerization as well as for each step of mRNA cap formation. Working with vesicular stomatitis virus (VSV), a prototype of NNS RNA viruses, we used negative stain electron microscopy (EM) to obtain a molecular view of L, alone and in complex with the viral phosphoprotein (P) cofactor. EM analysis, combined with proteolytic digestion and deletion mapping, revealed the organization of L into a ring domain containing the RNA polymerase and an appendage of three globular domains containing the cap-forming activities. The capping enzyme maps to a globular domain, which is juxtaposed to the ring, and the cap methyltransferase maps to a more distal and flexibly connected globule. Upon P binding, L undergoes a significant rearrangement that may reflect an optimal positioning of its functional domains for transcription. The structural map of L provides new insights into the interrelationship of its various domains, and their rearrangement on P binding that is likely important for RNA synthesis. Because the arrangement of conserved regions involved in catalysis is homologous, the structural insights obtained for VSV L likely extend to all NNS RNA viruses.

Protein Expression Redirects Vesicular Stomatitis Virus RNA Synthesis to Cytoplasmic Inclusions

Positive-strand and double-strand RNA viruses typically compartmentalize their replication machinery in infected cells. This is thought to shield viral RNA from detection by innate immune sensors and favor RNA synthesis. The picture for the non-segmented negative-strand (NNS) RNA viruses, however, is less clear. Working with vesicular stomatitis virus (VSV), a prototype of the NNS RNA viruses, we examined the location of the viral replication machinery and RNA synthesis in cells. By short-term labeling of viral RNA with 5′-bromouridine 5′-triphosphate (BrUTP), we demonstrate that primary mRNA synthesis occurs throughout the host cell cytoplasm. Protein synthesis results in the formation of inclusions that contain the viral RNA synthesis machinery and become the predominant sites of mRNA synthesis in the cell. Disruption of the microtubule network by treatment of cells with nocodazole leads to the accumulation of viral mRNA in discrete structures that decorate the surface of the inclusions. By pulse-chase analysis of the mRNA, we find that viral transcripts synthesized at the inclusions are transported away from the inclusions in a microtubule-dependent manner. Metabolic labeling of viral proteins revealed that inhibiting this transport step diminished the rate of translation. Collectively those data suggest that microtubule-dependent transport of viral mRNAs from inclusions facilitates their translation. Our experiments also show that during a VSV infection, protein synthesis is required to redirect viral RNA synthesis to intracytoplasmic inclusions. As viral RNA synthesis is initially unrestricted, we speculate that its subsequent confinement to inclusions might reflect a cellular response to infection.

Viruses know more than one way to don a cap

Capping of mRNA is an early posttranscriptional event, unique to eukaryotes, that strongly influences subsequent processing, nuclear export, stability, and translation of mRNA. Accordingly, viruses of eukaryotes, whether they reside in the nucleus or cytoplasm, must solve the capping problem for efficient replication. In a recent issue of PNAS, Ogino et al. (1) demonstrate a distinct mechanism used by vesicular stomatitis virus (VSV), and likely other nonsegmented negative strand RNA viruses (Mononegavirales), to cap the 5′ end of mRNA. The finding is of interest from biochemical and evolutionary perspectives and exemplifies the diverse ways that viruses adapt to their hosts.

Histidine-mediated RNA transfer to GDP for unique mRNA capping by vesicular stomatitis virus RNA polymerase

The RNA-dependent RNA polymerase L protein of vesicular stomatitis virus, a prototype of nonsegmented negative-strand (NNS) RNA viruses, forms a covalent complex with a 5'-phosphorylated viral mRNA-start sequence (L-pRNA), a putative intermediate in the unconventional mRNA capping reaction catalyzed by the RNA:GDP polyribonucleotidyltransferase (PRNTase) activity. Here, we directly demonstrate that the purified L-pRNA complex transfers pRNA to GDP to produce the capped RNA (Gpp-pRNA), indicating that the complex is a bona fide intermediate in the RNA transfer reaction. To locate the active site of the PRNTase domain in the L protein, the covalent RNA attachment site was mapped. We found that the 5'-monophosphate end of the RNA is linked to the histidine residue at position 1,227 (H1227) of the L protein through a phosphoamide bond. Interestingly, H1227 is part of the histidine-arginine (HR) motif, which is conserved within the L proteins of the NNS RNA viruses including rabies, measles, Ebola, and Borna disease viruses. Mutagenesis analyses revealed that the HR motif is required for the PRNTase activity at the step of the enzyme-pRNA intermediate formation. Thus, our findings suggest that an ancient NNS RNA viral polymerase has acquired the PRNTase domain independently of the eukaryotic mRNA capping enzyme during evolution and PRNTase becomes a rational target for designing antiviral agents.

De novo generation of a non-segmented negative strand RNA virus with a bicistronic gene

Reverse genetics has facilitated the use of non-segmented negative strand RNA viruses (NNSV) as vectors. Currently, heterologous gene expression necessitates insertion of extra-numeral transcription units (ENTUs), which may alter the NNSV polar transcription gradient and attenuate growth relative to wild-type (Wt). We hypothesized that rescuing recombinant Sendai Virus (rSeV) with a bicistronic gene might circumvent this attenuation but still allow heterologous open reading frame (ORF) expression. Therefore, we used a 9-nucleotide sequence previously described with internal ribosome entry site (IRES) activity, which, when constructed as several repeats, synergistically increased the level of expression of the second cistron [Chappell, S.A., Edelman, G.M., Mauro, V.P., 2000. A 9-nt segment of a cellular mRNA can function as an internal ribosome entry site (IRES) and when present in linked multiple copies greatly enhances IRES activity. Proc. Natl. Acad. Sci. U.S.A. 97, 1536–1541] . We inserted the Renilla luciferase ( rLuc) ORF, preceded by 1, 3 or 7 IRES copies, downstream of the SeV N ORF in an infectious clone. Corresponding rSeVs were successfully rescued. Interestingly, bicistronic rSeVs grew as fast as or faster than Wt rSeV. Furthermore, SeV gene transcription downstream of the N/rLuc gene was either equivalent to, or slightly enhanced, compared to Wt rSeV. Importantly, all rSeV/rLuc viruses efficiently expressed rLuc. IRES repetition increased rLuc expression at a multiplicity of infection of 0.1, although without evidence of synergistic enhancement. In conclusion, our approach provides a novel way of insertion and expression of foreign genes in NNSVs.

What Are the Risks—Hypothetical and Observed—of Recombination Involving Live Vaccines and Vaccine Vectors Based on Nonsegmented Negative-Strain RNA Viruses?

Newcastle disease virus (NDV) is a nonsegmented negative-strand RNA virus (NNSV) that is being developed as a potential vaccine vector for use in poultry (7, 12, 17) and humans (5, 6). The primary proposed human use would be to express protective antigens of highly pathogenic agents for outbreak control. We noted (1) that one of the advantages of NDV is that “gene exchange seems to be rare for nonsegmented negative strand RNA viruses, with few reported instances. This differs to the frequent gene reassortment observed for segmented viruses, such as influenza virus and rotavirus, and the high frequency of recombination observed for certain viruses, such as coronavirus and poliovirus.” In a recent letter to the editor (9), Han et al. criticized our studies, suggesting that we (i) dismissed the possibility of recombination, (ii) failed to recognize the potentially adverse consequences of recombination, and (iii) did not experimentally address the potential for the instability of the inserted foreign gene.

Cytosolic 5′-Triphosphate Ended Viral Leader Transcript of Measles Virus as Activator of the RIG I-Mediated Interferon Response

BackgroundDouble stranded RNA (dsRNA) is widely accepted as an RNA motif recognized as a danger signal by the cellular sentries. However, the biology of non-segmented negative strand RNA viruses, or Mononegavirales, is hardly compatible with the production of such dsRNA.Methodology and Principal FindingsDuring measles virus infection, the IFN-β gene transcription was found to be paralleled by the virus transcription, but not by the virus replication. Since the expression of every individual viral mRNA failed to activate the IFN-β gene, we postulated the involvement of the leader RNA, which is a small not capped and not polyadenylated RNA firstly transcribed by Mononegavirales. The measles virus leader RNA, synthesized both in vitro and in vivo, was efficient in inducing the IFN-β expression, provided that it was delivered into the cytosol as a 5′-trisphosphate ended RNA. The use of a human cell line expressing a debilitated RIG-I molecule, together with overexpression studies of wild type RIG-I, showed that the IFN-β induction by virus infection or by leader RNA required RIG-I to be functional. RIG-I binds to leader RNA independently from being 5-trisphosphate ended; while a point mutant, Q299A, predicted to establish contacts with the RNA, fails to bind to leader RNA. Since the 5′-triphosphate is required for optimal RIG-I activation but not for leader RNA binding, our data support that RIG-I is activated upon recognition of the 5′-triphosphate RNA end.Conclusions/SignificanceRIG-I is proposed to recognize Mononegavirales transcription, which occurs in the cytosol, while scanning cytosolic RNAs, and to trigger an IFN response when encountering a free 5′-triphosphate RNA resulting from a mislocated transcription activity, which is therefore considered as the hallmark of a foreign invader.

Recombinant parainfluenza virus 5 (PIV5) expressing the influenza A virus hemagglutinin provides immunity in mice to influenza A virus challenge

Parainfluenza virus type 5 (PIV5), formerly known as simian virus 5 (SV5), is a non-segmented negative strand RNA virus that offers several advantages as a vaccine vector. PIV5 infects many cell types causing little cytopathic effect, it replicates in the cytoplasm of infected cells, and does not have a DNA phase in its life cycle thus avoiding the possibility of introducing foreign genes into the host DNA genome. Importantly, PIV5 can infect humans but it is not associated with any known human illness. PIV5 grows well in tissue culture cells, including Vero cells, which have been approved for vaccine production, and the virus can be obtained easily from the media. To test the feasibility of using PIV5 as a live vaccine vector, the hemagglutinin (HA) gene from influenza A virus strain A/Udorn/72 (H3N2) was inserted into the PIV5 genome as an extra gene between the hemagglutinin-neuraminidase (HN) gene and the large (L) polymerase gene. Recombinant PIV5 containing the HA gene of Udorn (rPIV5-H3) was recovered and it replicated similarly to wild type PIV5, both in vitro and in vivo. The HA protein expressed by rPIV5-H3-infected cells was incorporated into the virions and addition of the HA gene did not increase virus virulence in mice. The efficacy of rPIV5-H3 as a live vaccine was examined in 6-week-old BALB/c mice. The results show that a single dose inoculation provides broad and considerable immunity against influenza A virus infection.

Unconventional Mechanism of mRNA Capping by the RNA-Dependent RNA Polymerase of Vesicular Stomatitis Virus

All known eukaryotic and some viral mRNA capping enzymes (CEs) transfer a GMP moiety of GTP to the 5'-diphosphate end of the acceptor RNA via a covalent enzyme-GMP intermediate to generate the cap structure. In striking contrast, the putative CE of vesicular stomatitis virus (VSV), a prototype of nonsegmented negative-strand (NNS) RNA viruses including rabies, measles, and Ebola, incorporates the GDP moiety of GTP into the cap structure of transcribing mRNAs. Here, we report that the RNA-dependent RNA polymerase L protein of VSV catalyzes the capping reaction by an RNA:GDP polyribonucleotidyltransferase activity, in which a 5'-monophosphorylated viral mRNA-start sequence is transferred to GDP generated from GTP via a covalent enzyme-RNA intermediate. Thus, the L proteins of VSV and, by extension, other NNS RNA viruses represent a new class of viral CEs, which have evolved independently from known eukaryotic CEs.

451. Matrix Metalloproteinase-Targeted Oncolytic Sendai Virus Vector “Armed” with a Sucide Gene “Yeast Cytosine Deaminase”; Remarkable Combinational Effects

Malignant tumor cells often express matrix metalloproteinases (MMPs) at a high level to enable their dissemination and metastasis. Sendai virus (SeV), a non-segmented negative strand RNA virus, spreads in the target tissues in vivo via cleavage activation of the viral fusion (F) glycoprotein by a tissue-specific, trypsin-like protease. By deleting the viral matrix (M) protein, we had generated a genetically modified SeV that does not bud to mature virons, but is highly fusogenic and spreads extensively from cell to cell in a trypsin-dependent manner. Moreover, we had changed the tryptic cleavage site of the F glycoprotein of this vector to a site susceptible to MMPs. The resulting recombinant SeV vector was no longer activated by trypsin but spread efficiently in cultured cells supplemented with MMP2 or MMP9, and in human tumor cell lines expressing these MMPs. This vector spread extensively in tumor cells xenotransplanted to nude mice without disseminating to the surrounding normal cells, leading to the strong inhibition of the tumor growth in the mice. We call this vector as the |[ldquo]|MMP-targeted oncolytic Sendai virus vector; SeV/F(MMP-sub)|[Delta]|M|[rdquo]|.

107. Sustained Stable Gene Expression Induced by a Cytoplasmic RNA-Virus Vector Based on Mutant Sendai Virus Strain cl.151

Top of pageAbstract Sendai virus (SeV)|[mdash]|mouse para-influenza virus type 1|[mdash]|is a prototype virus of the Paramyxoviridae with a non-segmented negative strand RNA genome. SeV can infect a wide variety of mammalian cells in vitro and in vivo, and directs high-level gene expression from cytoplasmic viral genome RNA. As it is not pathogenic to humans, it promises to be a unique potential viral vector for human gene therapy while avoiding genotoxicity. However, current SeV vectors induce gene expression only transiently, partly because they are based on the SeV Z strain with lytic replication. To overcome this transient nature of gene expression, we have developed a vector based on the mutant SeV strain, which is capable of persistent infection. The temperature-sensitive mutant cl.151 strain, originally isolated from an SeV carrier culture (Yoshida et al., Virology 92, 139|[ndash]|154, 1979), has a defect in the late step of viral assembly (Kondo, et al., J. Biol. Chem., 268, 21924|[ndash]|21930, 1993). This mutant form readily establishes persistent infection in various cultured cells at the nonpermissive temperature (38 |[deg]|C), and expresses the viral antigen stably. We cloned the cDNA covering the entire RNA genome of this strain (15,384 nt). It has 50 nucleotide substitutions and 35 amino acid substitutions compared with the genome of the parental Nagoya strain. Using this cDNA as a starting material, we prepared recombinant SeVs carrying marker genes (for green fluorescent protein, GFP, and luciferase, Luc) by standard reverse genetics. These marker genes, flanked by transcription initiation and termination signals, were inserted immediately upstream of the NP (nucleocapsid protein) gene. The resulting cl.151-based recombinant SeV forms (r151-Luc and r151-GFP) showed temperature-dependent replication and infected various cultured cells persistently. Thus, this cloned cDNA contains all the information required for these characteristics of the cl.151 strain. These recombinant SeVs expressed the marker genes strongly as the Z strain-based vectors, and showed sustained gene expression stably for more than one month. These results strongly suggest the potential utility of this cl.151-based SeV vector as a valuable and non-toxic tool for human gene therapy. Characteristics of marker gene expression induced by cl.151-based SeV vectors are now under examination in animal tissue cells in vivo.

Overlap of Interaction Domains Indicates a Central Role of the P Protein in Assembly and Regulation of the Borna Disease Virus Polymerase Complex

The active polymerase complex of Borna disease virus is composed of the viral proteins N, P, and L. The viral X (negative regulatory factor) protein acts as a regulator of polymerase activity. Interactions of P with N and X were previously studied, but interactions with L were poorly defined. Using a mammalian two-hybrid system, we observed that L specifically interacts with P but not with N, X, or itself. Mapping of the L-binding domain in the P molecule revealed that it overlaps with two adjacent domains required for multimerization and interaction with N. Competition experiments showed that the interaction between L and P was inefficient when N was present, indicating that L may preferentially interact with free P in infected cells. Interestingly, a multimerization-defective P mutant maintained the ability to interact with L, N, and X but failed to support reporter gene expression from an artificial Borna disease virus minigenome. Furthermore, dominant negative effects on minigenome activity were only observed when P mutants with an intact multimerization domain were used, suggesting that P multimers, rather than monomers, exhibit biological activity. P mutants lacking functional interaction domains for L or N still formed complexes with these viral proteins when wild-type P was available as a bridging molecule, indicating that P multimers have the potential to act as scaffolds on which the RNA polymerase complex is assembled.

Structure and Function of the C-terminal Domain of the Polymerase Cofactor of Rabies Virus

The phosphoprotein (P) of rabies virus binds the viral polymerase to the nucleoprotein (N)-RNA template for transcription and replication. By limited protease digestion we defined a monomeric C-terminal domain of P that can bind to N-RNA. The atomic structure of this domain was determined and previously described mutations that interfere with binding of P to N-RNA could now be interpreted. There appears to be two features involved in this activity situated at opposite surfaces of the molecule: a positively charged patch and a hydrophobic pocket with an exposed tryptophan side-chain. Other previously published work suggests a conformational change in P when it binds to N-RNA, which may imply the repositioning of two helices that would expose a hydrophobic groove for interaction with N. This domain of rabies virus P is structurally unrelated to the N-RNA binding domains of the phosphoproteins of Sendai and measles virus that are members of the same order of viruses, the non-segmented negative strand RNA viruses. The implications of this finding for the evolution of this virus group are discussed.

722. SeV RNA Replicon Vector: An Efficient Cytoplasmic Expression System Derived from Sendai Virus Self-Replicating Ribonucleoprotein Complexes

Sendai virus (SeV) is an enveloped virus with a nonsegmented negative strand RNA belonging to Paramyxoviridae family. We have previously shown that recombinant SeV as well as its modified non-transmissible variants can efficiently transfer and express foreign genes for a broad range of mammalian cells and tissues in vitro and in vivo and are suitable for use in gene therapy. SeV has two envelope glycoproteins HN and F, which mediate the virus attachment to and penetration into target cells, respectively. These envelope glycoproteins are not only important factors for viral tropism but also major determinants of immunogenicity in infected animals. Here we report the development of a novel SeV RNA replicon vector system that takes advantage of SeV self-replicating ribonucleoprotein complexes (RNP). In the vector system, RNP itself is non-infectious and can be infectious by the addition of lipofection reagents. In this system, nuclear entry of SeV RNA genome which forms an RNP complex with SeV NP, P and L proteins is not required and high expression of transgene is expected in transduced cells by self-replication of RNP in the cytoplasm. In addition, no effect of transduction of transgene in the presence of neutral antibody against SeV is expected. As the source of RNP, non-infectious virus-like particles (VLP) were recovered by infecting F-defective SeV vector into LLC-MK2 cells and harvesting VLP from the supernatants of the infected cells. To qualitatively and quantitatively compare this new vector system with conventional SeV vector or plasmid DNA, we introduced β-galactosidase, luciferase or enhanced green fluorescent protein (EGFP) reporter gene into the F-defective SeV genome, and recovered VLP from cloned cDNAs. The SeV RNA replicon vector mixed with lipofection reagents efficiently transferred foreign genes to several mammalian cell lines in vitro, and dose-dependently expressed luciferase or EGFP genes in tranduced cells. The expression level of SeV RNA replicon vector was comparable to that of SeV F-defective vector and remarkably higher than plasmid DNA-liposome complexes. Transduction of a foreign gene by SeV RNA replicon vector was not inhibited by the addition of an anti-SeV-HN antibody, although transduction by conventional SeV vector was completely inhibited. Therefore, the SeV RNA replicon vector system is useful for cytoplasmic expression of foreign genes in vitro and may be of help to its application to human gene therapy such as repeated administration and targeting of the vector.