Charge heterogeneity in polypeptides of negative strand RNA viruses

Raghow, Rajendra; Portner, Allen; Hsu, C.H.; Clark, Sallie B.; Kingsbury, David W.

doi:10.1016/0042-6822(78)90305-7

Cited by 26 publications

(11 citation statements)

References 27 publications

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“…The relative positions of F P / R proteins were in good agreement with data obtained with other influenza strains (Raghow et al, 1978;Leavitt et al, 1979;Horisberger, 1980). However, there are major differences between our data and that of Privalsky & Penhoet (1978) who resolved N P but found M too basic to be focused.…”

Section: -2supporting

confidence: 80%

Phosphorylation of Influenza Virus Nucleoprotein in vivo

Petri

Dimmock

1981

Journal of General Virology

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Section: -2supporting

confidence: 80%

Phosphorylation of Influenza Virus Nucleoprotein in vivo

Petri

Dimmock

1981

Journal of General Virology

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“…However, the present finding of extensive, readily reversible, conformational changes in the nucleocapsid emphasizes the potential functional importance of such changes. These observations also have significant implications for other studies-for example, crosslinking experiments to examine the relationship of viral proteins to each other and the viral RNA (41,42).…”

Section: Discussionmentioning

confidence: 92%

Conformation of the helical nucleocapsids of paramyxoviruses and vesicular stomatitis virus: reversible coiling and uncoiling induced by changes in salt concentration.

Heggeness¹,

Scheid²,

Choppin³

1980

Proc. Natl. Acad. Sci. U.S.A.

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The conformations of the helical nucleocapsids of the paramyxoviruses Sendai virus and simian virus 5, and of a rhabdovirus, vesicular stomatitis virus, have been found to vary extensively with changes in salt concentration. In 10 mM sodium phosphate buffer at pH 7.2, the nucleocapsids are loosely coiled or almost completely extended; with increasing concentrations of NaCl they become more tightly coiled and less flexible. Under isotonic conditions (150 mM) the Sendai virus nucleocapsid is moderately tightly coiled but still curved and apparently flexible, whereas at 400 mM or higher it is very tightly coiled, with the appearance of a rigid rod. These saltdependent changes in conformation were also found with nucleocapsids composed of proteolytically cleaved protein subunits. Because of the effect of salt concentration, and the fact that it may change during the preparation of negatively stained samples for electron microscopy, it was necessary to fix the nucleocapsids before negative staining to preserve their original conformation. The striking changes in nucleocapsid conformation in response to the ionic milieu indicate the plasticity of its helical structure and suggest that changes in the microenvironment of the nucleocapsid could influence its conformation during viral RNA transcription and replication or during virus assembly by budding, processes in which changes in the coiling of the nucleocapsid or its flexibility could be important. The nucleocapsids of paramyxoviruses, such as Sendai virus, simian virus 5 (SV5), and Newcastle disease virus, are single, left-handed, helical structures which, when tightly coiled, have a diameter of 15-18 nm and a length of ;1 ,m (1-5). They are composed of single-stranded RNA and -2600 viral structural protein subunits (NP) with a molecular weight of t60,000 (6, 7). Two other proteins (L and P) are associated with the nucleocapsid and thought to be involved in the viral RNA transcriptase activity (8-12). The nucleocapsids of rhabdoviruses-e.g., vesicular stomatitis (VSV) and rabies viruses-also consist of a single-stranded RNA complexed with a single major structural protein (N) and two associated proteins (NS and L) (13-18). In the VSV virion, the nucleocapsid forms a coil t40 nm in diameter but, when separated from the viral membrane, it is often found in helices of considerably smaller diameter (13-17).The conformation of paramyxovirus nucleocapsids has shown wide variations as observed in the electron microscope. They have been seen to have tightly stacked turns and the overall appearance of relatively stiff rods ;1 ,um in length and also as loosely coiled helices extended to varying degrees (1-7, 19, 20). Proteolytic cleavage of the NP protein has been implicated as one of the causes of such variation in nucleocapsid conformation (6, 21); however, in the course of an electron microscopic study of Sendai virus nucleocapsids, we often observed a wide variation in the degree of coiling of duplicate samples that could not be attributed to this mechanism. When ...

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“…Minimally, this processing involves the removal of the initiating methionine followed by N ~-acetylation of the following amino acid. It should be noted that two-dimensional gels of the VSV nucleocapsid proteins show multiple spots, while the Sendai virus NP protein is phosphorylated (Roux & Kolakofsky, 1974;Lamb & Choppin, 1977;Raghow et al, 1978;Perrault et al, 1983). It remains unclear whether the amino-terminal modifications we have described are present in all these different forms of the N and NP proteins.…”

Section: Discussionmentioning

confidence: 96%

Preparation and Analysis of the Nucleocapsid Proteins of Vesicular Stomatitis Virus and Sendai Virus, and Analysis of the Sendai Virus Leader-NP Gene Region

Blumberg

Giorgi

Rose

et al. 1984

Journal of General Virology

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SUMMARYA procedure is presented for isolating the nucleocapsid proteins, N and NP from vesicular stomatitis virus and Sendai virus respectively, in soluble form. These proteins were suitable for the determination of their blocked amino-terminal peptide sequences by gas-liquid chromatography/mass spectrometry at the low nanomole level. The N protein prepared by this procedure was previously shown to retain some of its expected biological activity. The sequence of 626 nucleotides from the 3' end of the Sendal virus genome, which includes the first one-third of the NP gene, was determined. Using this information, primer extension studies on intracellular Sendai virus mRNAs allowed the determination of the structure of the leader-NP intervening sequence and the 5' end of the NP mRNA. Comparison of the amino termini of the nucleocapsid proteins with their respective mRNA sequences revealed that these proteins are similarly processed in vivo. INTRODUCTIONThe non-segmented minus-strand (-) RNA genome of the rhabdovirus vesicular stomatitis virus (VSV), and that of the paramyxovirus Sendal virus, are always found tightly complexed in helical nucleocapsid structures with their respective viral nucleocapsid proteins, N and NP. The nucleocapsid structure protects the genomes from nuclease attack, and in conjunction with two less tightly associated viral proteins which constitute the viral RNA polymerase (NS and L of VSV: Emerson & Yu, 1975; P and L of Sendal: Stone et al., 1972;Marx et al., 1974; Lamb & Mahy, 1975), displays all the enzyme activities required for synthesis of the viral mRNAs (Banerjee et al., 1977;Kingsbury, 1974).Because the nucleocapsids are of central importance in both virus structure and replication, it would be of interest to study the properties of the purified nucleocapsid proteins themselves. However, previous attempts to isolate the N protein from nucleocapsids by treatment with dimethyl urea (S. Emerson, personal communication) or with guanidinium salts (G. Wertz, personal communication) have resulted in preparations with intractable physical properties. Both proteins can be removed from their nucleocapsids by treatment with SDS or phenol, but these reagents are likely to denature the protein. The viral nucleocapsids are also stable to highsalt conditions. Indeed, banding in CsC1 gradients at a buoyant density of 1.33 g/ml (2-5 M-CsC1) is diagnostic for VSV and Sendai virus nucleocapsids, for under these conditions all other cellular RNA-protein complexes disaggregate (Leppert et al., 1979). Thus, although these proteins are major components of the mature virion (Wagner et al., 1969; Mountcastle et al., 197t), they have resisted attempts at purification. In this paper, we describe a simple procedure for the isolation of N and NP proteins from intracellular nucleocapsids, based on CsCI density gradient centrifugation in the presence of guanidinium chloride.

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Charge heterogeneity in polypeptides of negative strand RNA viruses

Cited by 26 publications

References 27 publications

Phosphorylation of Influenza Virus Nucleoprotein in vivo

Phosphorylation of Influenza Virus Nucleoprotein in vivo

Conformation of the helical nucleocapsids of paramyxoviruses and vesicular stomatitis virus: reversible coiling and uncoiling induced by changes in salt concentration.

Preparation and Analysis of the Nucleocapsid Proteins of Vesicular Stomatitis Virus and Sendai Virus, and Analysis of the Sendai Virus Leader-NP Gene Region

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