No abstract

The paramyxovirus nucleocapsid proteins (NPs) are relatively well conserved, except for the C-terminal 20%o (or ca. 100 amino acids), referred to as the tail. We have examined whether this hypervariable tail is required for genome synthesis, both in vitro, where synthesis is predominantly from the input templates, and in vivo, where multiple rounds of amplification occur. In these viruses, genome synthesis and assembly of the nascent chain are coupled. We find that the tail is required in vivo but not in vitro. Closer examination of the in vivo system showed that the tailless NP could encapsidate the genome chain but that amplification did not occur. We interpret these results as indicating that the tail is not required for RNA assembly but is required for the template to function in RNA synthesis. Relatively small deletions within the conserved N-terminal 80%o of the protein, on the other hand, rendered the protein nonfunctional in either system. The possible functions of the tail in RNA synthesis are discussed.

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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

Based on the observation that the growth of solid tumors is dependent on the formation of new blood vessels, therapeutic strategies aimed at inhibiting angiogenesis have been proposed. A number of proteins with angiostatic activity have been described, but their development as therapeutic agents has been hampered by difficulties in their production and their poor pharmacokinetics. These limitations may be resolved using a gene therapy approach whereby the genes are delivered and expressed in vivo. Here we compared adenoviral delivery of endostatin, proliferin-related protein (PRP), and interferon-inducible protein 10 (IP10) genes. Recombinant adenoviruses carrying the three angiostatic genes express biologically active gene products as determined in vitro in endothelial cell proliferation and migration assays, and in vivo by inhibition of neoangiogenesis in rat chambers. Eradication of established tumors in vivo, in the murine B16F10 melanoma model in immunocompetent mice, was not achieved by intratumoral injection of the different vectors. However, the combination of intravenous plus intratumoral injections allowed rejection of tumors. Ad-PRP or Ad-IP10 were significantly more efficient than Ad-endostatin, leading to complete tumor rejection and prolonged survival in a high proportion of treated animals. These data support the use of in vivo gene delivery approaches to produce high-circulating and local levels of antiangiogenic agents for the therapy of local and metastatic human tumors.