Assembly of recombinant Newcastle disease virus nucleocapsid protein into nucleocapsid-like structures is inhibited by the phosphoprotein.

Abstract

A recombinant baculovirus expressing the nucleocapsid gene (NP) of Newcastle disease virus (NDV), a member of the genus Rubulavirus, has been generated and shown to express the native protein to high levels in insect cells. In contrast to the NP protein of the rubulavirus human parainfluenza virus 2, the NDV protein has been demonstrated by electron microscopy and caesium chloride gradient analysis to be capable of self-assembly in vivo to form nucleocapsid-like structures in the absence of other NDV proteins. These structures, which contained RNA that was resistant to micrococcal nuclease digestion, were also observed when the protein was expressed in E. coli, a phenomenon which was not inhibited by the presence of a 40 amino acid fusion region at the amino terminus of the protein. Further, the formation of these structures was inhibited by the co-expression of the phosphoprotein (P). Therefore, we conclude that the P protein acts as a chaperone, preventing uncontrolled encapsidation of non-viral RNA by NP protein.Newcastle disease virus (NDV) is the aetiological agent for the devastating disease of poultry more commonly known as fowl pest. A member of the family Paramyxoviridae, NDV has recently been reclassified into the genus Rubulavirus (Murphy et al., 1995). Other members of this genus include mumps virus and simian virus 5 (SV5) in addition to the human parainfluenza viruses 2, 4a and 4b (hPIV2, 4a and 4b). The genome of NDV, which comprises a single strand of negative-sense RNA, encodes directly six structural genes in the order 3h-NP-P-M-

Requirements for Budding of Paramyxovirus Simian Virus 5 Virus-Like Particles

“…6A). These nucleocapsid-like structures varied in length from those of the single "washer" form to strands longer than 100 nm, observations consistent with previous studies on nonspecific assembly of paramyxovirus NP proteins with cellular RNA (7,42). SV5 M protein, in contrast to NP protein, would not be expected to form this type of dense structure, and M protein did not sediment through a similar CsCl gradient when expressed alone (Fig.…”

“…Of course, to obtain infectious virions from virus-infected cells, packaging of encapsidated viral genomes occurs and packaging of cellular RNAs is largely avoided. It is thought that in virusinfected cells, uncontrolled encapsidation of cellular RNA must be inhibited, and that the paramyxovirus P protein may play a role in this inhibition (2,6,7). This could result in a situation where there is no requirement to discriminate between encapsidated cellular and viral RNAs at budding sites, because virtually all encapsidated products in the infected cell would contain viral RNA.…”

“…Paramyxovirus NP proteins have been found previously to assemble into nucleocapsid-like structures when synthesized in either mammalian cells or in insect cells as a result of uncontrolled encapsidation of cellular RNA (2,5,7,8,28,42). We observed similar assembly of SV5 NP protein into nucleocapsid-like structures both when NP protein was expressed alone and when NP protein was coexpressed with other SV5 proteins.…”

“…The structures observed from VLP preparations measured 15 to 20 nm in diameter, similar to the SV5 RNPs, but were much shorter and more variable in length than authentic RNPs which encapsidate the 15,246-nucleotide RNA genome of SV5. It has been found for several other paramyxoviruses that the NP protein expressed by itself in cells encapsidates nonspecifically cellular RNA and forms structures similar in morphology to viral RNPs (2,5,7,8,28,42).…”

Complete nucleotide sequence of Newcastle disease virus: evidence for the existence of a new genus within the subfamily Paramyxovirinae.

“…However, some MAbs did react with SV5, SV41 and mumps virus, which are all members of the Rubulavirus genus. Finally, like SeV (Paramyxovirus genus) and unlike hPIV2 (Rubulavirus genus), the NP protein of NDV can bind to RNA in vivo to form nucleocapsid-like structures in the absence of the phosphoprotein (Errington & Emmerson, 1997).…”