The hypervariable C-terminal tail of the Sendai paramyxovirus nucleocapsid protein is required for template function but not for RNA encapsidation

112

Sendai virus nucleocapsid protein NP synthesized in the absence of other viral components assembled into nucleocapsidlike particles. They were identical in density and morphology to authentic nucleocapsids but were smaller in size. The reduction in size was probably due to the fact that they contained RNA only 0.5 to 2 kb in length. Nucleocapsid assembly requires NP-NP and NP-RNA interactions. To identify domains on NP protein involved in nucleocapsid formation, 29 NP protein mutants were tested for the ability to assemble. Any deletion between amino acid residues 1 and 399 abolished formation of nucleocapsidlike particles, but mutants within this region exhibited two different phenotypes. Deletions between positions 83 and 384 completely abolished all interactions. Deletions between residues 1 and 82 and between residues 385 and 399, at the N-and C-terminal ends of the region from 1 to 399, resulted in unstructured aggregates of NP protein, indicating only a partial loss of function. Deletions within the C-terminal 124 amino acids were the only ones that did not affect assembly. The results suggest that NP protein can be divided into at least two separate domains which function independently of each other. Domain I (residues 1 to 399) seems to contain all of the structural information necessary for assembly, while domain II (residues 400 to 524) is not involved in nucleocapsid formation. Nucleocapsids of Sendai virus, a model paramyxovirus, are large, helical, highly flexible ribonucleoprotein particles, approximately 15 nm in diameter and 1 ,um in length. They contain the 15,384-nucleotide-long negative-stranded RNA genome and three different viral proteins. The major structural component of the nucleocapsid is the nucleocapsid protein NP (58 kDa), of which about 2,600 molecules tightly encapsidate the RNA. Encapsidation renders the RNA inaccessible to RNases and is required for it to serve as a template for the viral RNA-dependent RNA polymerase complex. The two proteins necessary for polymerase functions are the large protein L (251 kDa), which is considered the core of the polymerase complex, and the phosphoprotein P (65 kDa). Both of these proteins are present in only minor amounts on nucleocapsids (4). NP also binds M protein (7). Binding of M protein to nucleocapsids seems to modify polymerase activities (12, 13) and may also be required to mediate interactions between nucleocapsids and the viral envelope proteins during virus budding. The paramyxovirus RNA polymerase is able to work in two different modes, and NP protein may be regarded as a regulatory factor that switches the polymerase from the transcriptive to the replicative mode. During transcription, intergenic start/stop signals on the viral genome are recognized, and thus a 54-nucleotide-long leader RNA and six capped, polyadenylated mRNAs are generated. Replication, as opposed to transcription, results in full-size antigenomic RNA, encapsidated by NP protein. Replication, but not

Section: Discussionsupporting

confidence: 90%

The conserved N-terminal region of Sendai virus nucleocapsid protein NP is required for nucleocapsid assembly

Buchholz

Spehner

Drillien

et al. 1993

112

“…Thus, the plasmid-supplied N, P, and L proteins appear to be fully sufficient to support RNA replication, a finding which has also been made with two rhabdoviruses, vesicular stomatitis virus and rabies virus (9,31,37), and the paramyxovirus Sendai virus (2,13). RSV is sufficiently dissimilar that this confirmation is useful.…”

Section: Discussionmentioning

confidence: 65%

“…Rescue means that the minigenome participates in one or more of the major features of the intracellular viral replicative cycle, including RNA replication, transcription, and production of transmissible particles. Studies to date have employed minigenomes such as a cDNAencoded copy of naturally occurring defective interfering RNA (2,13,31,40) and an engineered version of genome RNA containing a large internal deletion and the insertion of a marker gene, usually that for bacterial chloramphenicol acetyltransferase (CAT), under the control of putative transcription signals (6-9, 14, 15, 19, 24-26, 29, 30).…”

mentioning

confidence: 99%

RNA replication by respiratory syncytial virus (RSV) is directed by the N, P, and L proteins; transcription also occurs under these conditions but requires RSV superinfection for efficient synthesis of full-length mRNA

Grosfeld

Hill

Collins

1995

194

128

Previously, a cDNA was constructed so that transcription by T7 RNA polymerase yielded a ϳ1-kb negativesense analog of genomic RNA of human respiratory syncytial virus (RSV) containing the gene for chloramphenicol acetyltransferase (CAT) under the control of putative RSV transcription motifs and flanked by the RSV genomic termini. When transfected into RSV-infected cells, this minigenome was ''rescued,'' as evidenced by high levels of CAT expression and the production of transmissible particles which propagated and expressed high levels of CAT expression during serial passage (P. L. Collins, M. A. Mink, and D. S. Stec, Proc. Natl. Acad. Sci. USA, 88:9663-9667, 1991). Here, this cDNA, together with a second one designed to yield an exact-copy positive-sense RSV-CAT RNA antigenome, were each modified to contain a self-cleaving hammerhead ribozyme for the generation of a nearly exact 3 end. Each cDNA was transfected into cells infected with a vaccinia virus recombinant expressing T7 RNA polymerase, together with plasmids encoding the RSV N, P, and L proteins, each under the control of a T7 promoter. When the plasmid-supplied template was the mini-antigenome, the minigenome was produced. When the plasmid-supplied template was the minigenome, the products were mini-antigenome, subgenomic polyadenylated mRNA and progeny minigenome. Identification of progeny minigenome made from the plasmid-supplied minigenome template indicates that the full RSV RNA replication cycle occurred. RNA synthesis required all three RSV proteins, N, P, and L, and was ablated completely by the substitution of Asn for Asp at position 989 in the L protein. Thus, the N, P, and L proteins were sufficient for the synthesis of correct minigenome and antigenome, but this was not the case for subgenomic mRNA, indicating that the requirements for RNA replication and transcription are not identical. Complementation with N, P, and L alone yielded an mRNA pattern containing a large fraction of molecules of incomplete, heterogeneous size. In contrast, complementation with RSV (supplying all of the RSV gene products) yielded a single discrete mRNA band. Superinfection with RSV of cells staging N/P/L-based RNA synthesis yielded the single discrete mRNA species. Some additional factor supplied by RSV superinfection appeared to be involved in transcription, the most obvious possibility being one or more additional RSV gene products.

“…Failure to recover infectious virus from the construct with extensive deletion of 48 residues in NDV-⌬48 might be due to the loss of important functional domains in addition to structural constraints. Indeed, the carboxy-terminal region of the NP of Sendai paramyxovirus was shown to be required for template function (8). Whether the corresponding region of the NP of NDV possesses similar function remains to be elucidated.…”

Section: Discussionmentioning

confidence: 99%

“…Moreover, NP regulates transcription and replication of the viral genome by interacting with P alone, with P and L, or with itself (NP-NP interaction). For Sendai paramyxovirus, it was shown that a conserved N-terminal region of NP was involved in NP-RNA and NP-NP interaction (5), whereas the carboxyterminal domain was shown to be required for template function (8). Most of the NP is thus absolutely essential for virus replication due to multifold engagement of NP in the assembly and biologic activity of the RNP.…”

mentioning

confidence: 99%

Newcastle Disease Virus (NDV) Marker Vaccine: an Immunodominant Epitope on the Nucleoprotein Gene of NDV Can Be Deleted or Replaced by a Foreign Epitope

Mebatsion¹,

Koolen²,

Vaan³

et al. 2002

The nucleoprotein (NP) of Newcastle disease virus (NDV) functions primarily to encapsidate the virus genome for the purpose of RNA transcription, replication, and packaging. This conserved multifunctional protein is also efficient in inducing NDV-specific antibody in chickens. Here, we localized a conserved B-cell immunodominant epitope (IDE) spanning residues 447 to 455 and successfully generated a recombinant NDV lacking the IDE by reverse genetics. Despite deletion of NP residues 443 to 460 encompassing the NP-IDE, the mutant NDV propagated in embryonated specific-pathogen-free chicken eggs to a level comparable to that of the parent virus. In addition, a B-cell epitope of the S2 glycoprotein of murine hepatitis virus (MHV) was inserted in-frame to replace the NP-IDE. Recombinant viruses properly expressing the introduced MHV epitope were successfully generated, demonstrating that the NP-IDE not only is dispensable for virus replication but also can be replaced by foreign sequences. Chickens immunized with the hybrid recombinants produced specific antibodies against the S2 glycoprotein of MHV and completely lacked antibodies directed against the NP-IDE. These marked-NDV recombinants, in conjunction with a diagnostic test, enable serological differentiation of vaccinated animals from infected animals and may be useful tools in ND eradication programs. The identification of a mutation-permissive region on the NP gene allows a rational approach to the insertion of protective epitopes and may be relevant for the design of NDV-based cross-protective marker vaccines.The negative-strand RNA virus genome of Newcastle disease virus (NDV) contains six genes encoding six major structural proteins: nucleoprotein (NP), phosphoprotein (P), matrix protein (M), fusion protein (F), hemagglutinin-neuraminidase (HN), and RNA-dependent RNA polymerase (L). The RNA together with NP, P, and L proteins forms the ribonucleoprotein complex (RNP), which serves as a template for RNA synthesis (15). The NP together with the polymerase proteins, P and L, plays an eminent role in encapsidating the RNA. Moreover, NP regulates transcription and replication of the viral genome by interacting with P alone, with P and L, or with itself (NP-NP interaction). For Sendai paramyxovirus, it was shown that a conserved N-terminal region of NP was involved in NP-RNA and NP-NP interaction (5), whereas the carboxyterminal domain was shown to be required for template function (8). Most of the NP is thus absolutely essential for virus replication due to multifold engagement of NP in the assembly and biologic activity of the RNP. In addition, NPs of negativestrand RNA viruses are highly immunogenic in nature and have been used as antigens for diagnostic purposes, including