David W. Kingsbury

A temperature-sensitive mutant of Sendai virus with an altered hemagglutinin-neuraminidase polypeptide: Consequences for virus assembly and cytopathology

Abstract Sendai virus mutant ts 271 was previously shown to be an RNA positive mutant with a temperature-sensitive hemagglutinin. We now report that noninfectious virus particles are produced when this mutant is grown at the nonpermissive temperature. These virus particles appear to have only one defect: they are devoid of a single polypeptide, the 70,000 dalton HN glycopolypeptide responsible for hemagglutinin and neuraminidase activities. Consequently, the noninfectious particles lack these activities and they are incapable of attaching to cells. Moreover, cells producing the particles contain neither hemagglutinin nor neuraminidase activities, suggesting that the relevant glycopolypeptide does not assume a functional conformation when it is synthesized under nonpermissive conditions. We conclude that Sendai virus morphogenesis does not require HN as a structural element or any function supplied by HN. Other consequences of the mutation were marked lessenings of cytopathology and cell protein synthesis inhibition during infections at nonpermissive temperature, indicating that the native HN glycopolypeptide is an important factor in cell killing by Sendai virus.

Newcastle disease virus RNA: I. Isolation and preliminary characterization of RNA from virus particles

Exposure of Newcastle disease virus particles to 1% sodium dodecyl sulfate resulted in release of an RNA which sedimented more rapidly than chick embryo ribosomal RNA in sucrose density-gradients. Shaking virus with phenol after addition of detergent did not change the sedimentation velocity of the rapidly sedimenting viral RNA. This viral RNA was separated from contaminants of smaller size by gel filtration on 2% agarose columns. Viral RNA isolated in this manner had a sedimentation coefficient S 20,w of 49·2 s in 0·01 M -sodium acetate, 0·05 M -NaCl, 0·5% sodium dodecyl sulfate (pH 5·1). It had a bouyant density of 1·68 g/cc in cesium sulfate. Its base composition was: cytosine, 23·2%; adenine, 20·1%; guanine, 25·4%; uracil, 31·2%. The high molecular weight RNA isolated from Newcastle disease virus was not shown to be infective.

Newcastle disease virus RNA: II. Preferential synthesis of RNA complementary to parental viral RNA by chick embryo cells

Following infection by strain “C” of Newcastle disease virus, and during incubation in the presence of actinomycin D, chick embryo cells synthesized RNA which had a base composition complementary to the base composition of RNA from virus particles. Evidence for base-sequence complementarity between RNA from virus particles and RNA synthesized by infected cells was obtained by in vitro hybridization. Of the total radioisotopically labeled RNA synthesized by infected cells in the presence of actinomycin D, 80% or more became insensitive to pancreatic ribo-nuclease after annealing with RNA from virus particles. The ribonuclease-resistant RNA had properties of a double-stranded polynucleotide. RNA from chick embryo cells or from Escherichia coli cells did not render labeled RNA from Newcastle disease virus-infected cells resistant to ribonuclease, and RNA from virus particles did not confer ribonuclease resistance on RNA from uninfected chick embryo cells. From 18 to 40% of the labeled RNA obtained from infected cells became RNase-resistant when annealed in the absence of RNA from virus particles. Cells infected by any of four other strains of Newcastle disease virus synthesized RNA, which was complementary in base composition to RNA from strain “C” virus particles. Virus particle RNA from all of these strains hybridized with RNA from cells infected by the “C” strain.

Complete sequence of the sendai virus NP gene from a cloned insert

A DNA molecule representing all but the three terminal bases of the Sendai virus nucleoprotein (NP) gene, copied from viral mRNA, was inserted into pBR322. The NP insert comprised 1673 bases. The first AUG protein initiation codon, at position 65, began an open reading frame of 1551 bases, encoding a protein of 517 amino acids with an amino acid composition corresponding to previously published data. The NP gene sequence determined in the present work is similar to that described by Shioda et al. [ Nucl . Acids Res. 11, 7317 (1983)], but there are 14 amino acid differences that probably reflect differences in virus strains. The predicted secondary structure of the NP molecule and the locations within that structure of potential protease cleavage sites are in accord with structural domains previously defined by controlled protease digestion.

Assembly of influenza ribonucleoprotein in vitro using recombinant nucleoprotein

The influenza A virus nucleoprotein previously expressed in Escherichia coli after fusion to 32 heterologous amino acids has now been purified and tested for its ability to form complexes with RNA in vitro. By using a simple filter binding assay, we show that ribonucleoprotein (RNP) complexes form readily with single-stranded RNA of viral or nonviral origin but not with double-stranded RNA. The RNP complexes formed were similar to authentic influenza virus RNPs in appearance under the electron microscope, in buoyant density in gradients of cesium chloride, and in sensitivities to pancreatic ribonuclease, to chaotropic reagents, and to high salt. We conclude that nucleoprotein synthesized in E. coli has all the properties required for correct assembly into ribonucleoprotein.

Regulatory events in the synthesis of Sendai virus polypeptides and their assembly into virions.

Abstract The synthesis and assembly of Sendai virus polypeptides into virions were studied by pulse-chase and long-term labeling late in infection. The transfer of polypeptides from cells into virions was modeled as a stochastic process, yielding a distinctive first-order rate constant for the turnover of each major polypeptide. The data revealed unexpected kinetic properties of the lesser nucleocapsid polypeptide, P, implicated earlier in virus RNA transcription. The available pool of P for virus assembly was depleted four times faster than the available pool of NP, the nucleocapsid structure unit. Furthermore, P was synthesized about fourfold in excess of its steady-state representation in virions, and it was incorporated into virions without dilution by a large preexistent pool. It was concluded that newly synthesized P associates rapidly with intracellular nucleocapsids and then departs into an unavailable pool, consistent with a transient function of P in nucleocapsid assembly or in RNA synthesis. In contrast to the overproduction of P, production of M, the nonglycosylated polypeptide in the virus envelope, was deficient about two-fold, relative to NP. This suggests a control device to insure that at least half of the nucleocapsids produced can never exit as components of virions, but must remain within the cell to serve as RNA-synthesizing complexes. Although the three nonglycosylated virus polypeptides, P, NP, and M, entered virions without delay, the entry of the glycosylated polypeptides did not begin until about 15 to 30 min after their synthesis, presumably reflecting the time necessary for their glycosylation and transport to sites of virus assembly.

Antibodies against Sendai virus L protein: distribution of the protein in nucleocapsids revealed by immunoelectron microscopy

Antibodies against the L protein of Sendai virus were made by immunizing rabbits with a synthetic peptide representing a carboxyl-terminal region of the protein predicted from the base sequence of its gene. These antibodies were used to localize the L protein in viral nucleocapsids by electron microscopy. Immunogold labeling revealed that L protein molecules were distributed in clusters along nucleocapsids, suggesting that L molecules act cooperatively in viral RNA synthesis. Immunogold double-labeling showed that all L clusters were associated with clusters of P molecules. We believe that this morphological association reflects the functional cooperation of the L and P proteins in viral RNA synthesis.

Carboxyl-terminal region of sendai virus P protein is required for binding to viral nucleocapsids

The Sendai virus P protein is a component of the viral nucleocapsid, where it participates in RNA synthesis. To identify domains of the protein involved in nucleocapsid recognition, deleted P protein molecules were generated from a cDNA clone of its gene. In vitro transcription of the complete gene and translation of the transcript generated a protein with electrophoretic mobility and immunoreactivity indistinguishable from those of authentic P protein. The in vitro product bound specifically to nucleocapsids when mixed with extracts from infected cells. However, a product lacking only 30 carboxyl-terminal amino acid residues (5% of the molecule) did not bind. Residues within a 195 amino acid region, adjacent to and overlapping by one amino acid with the carboxyl-terminal 30 residues, were also required for binding. No other protein region was required. Therefore, the 224-residue region which includes the carboxyl terminus appears to contain the nucleocapsid attachment site, and the 30 terminal residues either form part of the site or are required to maintain an active conformation.

Topography of phosphate residues in Sendai virus proteins.

Abstract Phosphorylation is a common post-translational modification of Sendai virus proteins, occurring in all species except L. Phosphate residues in the envelope proteins FIN, F, and M were completely removed by the action of bacterial alkaline phosphatase on virions disrupted with nonionic detergent, whereas intact virions were entirely resistant to the enzyme. These results suggest that phosphate residues are located in domains of the native envelope proteins that lie within the virion. The phosphate residues in nucleocapsid proteins P and NP were also removed efficiently when intact nucleocapsids were treated with the phosphatase. Indeed, most of the phosphates in P and NP were located in the limited external domains that were cleaved from these protein species by controlled proteolysis. Phosphorylation of these domains may, therefore, regulate nucleocapsid functions and/or mediate electrostatic interactions with the nascent virus envelope during virion assembly.

Properties of incomplete Sendai virions and subgenomic viral RNAs

Abstract A strain of Sendai virus encapsidated 19 S and 25 S RNAs in addition to 50 S RNA genomes. The 19 S and 25 S RNAs hybridized with Sendai virus-specific RNAs from infected cells, but not with 50 S viral RNA, indicating that they contained nucleotide sequences similar to 50 S viral genomes. Sendai virions were separated by rate zonal centrifugation into groups sedimenting at about 400 S, 1000 S, and greater than 1000 S. Virions sedimenting at 1000 S or greater were infectious and contained 19 S, 25 S, and 50 S RNAs. The 400 S virions contained only 19 S and 25 S RNAs; they were not infectious, and they interfered with replication of infectious virions. Passage of Sendai virus at low multiplicity gave progeny lacking 400 S virions and 19 S and 25 S RNAs. The evidence indicates that noninfectious virions containing incomplete genomes occur in Sendai virus replication.