Isolation and Characterization of Canine Distemper Virus Nucleocapsid Variants

Abstract

SUMMARYNucleocapsid (NC) variants expressed by the Onderstepoort strain of canine distemper virus (CDV) were ultrastructurally and biochemically characterized. Three isolated variants were defined which corresponded to the three variants observed within the cytoplasm of infected cells. Dense NC (D-NC), isolated on discontinuous caesium chloride (CsCI) isopycnic gradients, had an average density of 1.2971 + 0.0042 g/ml. Ultrastructurally, D-NC were 1620.0 + 112.1 nm in length with a 20.1 _+ 1-3 nm outer and a 5.8 + 0.7 nm inner core diameter. The D-NC protein composition was 89-7 ~ of a 61K protein (N), 8-4 ~ of a 75K protein (P) and 1-9 ~o of a 160K to 200K protein (L). A single species of nucleic acid, 15 kb in length, was isolated from D-NC. Light NC (L-NC), similarly isolated, had an average density of 1-2894+0-0040g/ml. L-NC differed ultrastructurally from D-NC in that poor resolution of NC subunits, a larger outer diameter (32.0 + 2.8 nm), and a greater inner core diameter (10.4 + 0.6 nm) were observed. The average L-NC strand length was 1574.4 + 115.8 nm. The protein composition was the same as D-NC with the exception of an additional 70K protein, representing 4.0 to 7.7 ~ of the total L-NC protein mass. A 15 kb nucleic acid was also identified in L-NC, although heightened sensitivity of encapsidated L-NC nucleic acid to non-specific nuclease degradation was observed. The ratio of D-NC to L-NC isolated from individual virus preparations varied and was independent of viral infectivity. A third NC variant, defective-NC (Df-NC), was also identified. This had the lowest density on CsC1 gradients (1.2460 + 0.0046 g/ml). The Df-NC structures were truncated to a uniform length of 87.0 + 5.8 nm. Diameter measurements were between those of D-NC and L-NC, being 24.4 + 1-4 nm (outer) and 6.9 + 0.4 nm (inner core). Like L-NC, the 70K protein was present but in greater amounts, representing as much as 43-7~ of the total Df-NC protein mass. RNase A-sensitive nucleic acid was isolated from Df-NC which ranged in size from 1.16 to 0-67 kb with a majority of the material being 0.86 kb in length. For both L-NC and Df-NC, canine CDV convalescent serum reacted with viral N and P proteins in Western blot analyses but not with the 70K protein, suggesting a host cellular origin for the latter.

Interaction of canine distemper virus nucleocapsid variants with 70K heat-shock proteins

“…Reports in the literature attribute greater biological activity to L-NC (granular NC) than to D-NC (Robbins et al, 1980). In addition, there is a predominance of L-NC and Df-NC ultrastructural in situ equivalents within the cytoplasm of lytically infected cells (Oglesbee et al, 1989).…”

“…when a different technique for N C processing is used which excludes free hsp70 from the final CsC1 gradient (Oglesbee et al, 1989). In addition, hsp70 from un-…”

“…Recently, we described the isolation and biochemical characterization of nucleocapsid (NC) variants of Onderstepoort canine distemper virus (Ond-CDV), designated light-NC (L-NC) and dense-NC (D-NC), which contain an encapsidated full-length RNA genome, and defective-NC (Df-NC), containing encapsidated RNAs of heterogeneous length (Oglesbee et al, 1989). L-NC and Df-NC incorporated a 70K protein of host cell origin into the basic capsid structure of N, P and L proteins.…”

“…Cytoplasmic NC was purified from the crude cytoplasmic extracts of Vero (V) cells infected lyticaUy with Ond-CDV by caesium chloride isopycnic density centrifugation (Oglesbee et al, 1989). Gradients of uninfected cellular material were similarly processed.…”

“…SDS-PAGE analysis of this material demonstrated a heterogeneous population of proteins, with prominent representation of a 70K band. Samples were examined by Western blot analysis (Oglesbee et al, 1989) using polyclonal canine anti-CDV antibody, mouse monoclonal antibody (N27) with specific reactivity to primate 70K hsps (72K and 73K hsps as well as the major 70K hsp degradation products 0000-9240 © 1990 SGM are detected by this reagent; Riabowol et al, 1988), and mouse monoclonal antibody (C92) with specific reactivity to primate 72K hsp (Welch & Suhan, 1986). Analysis of NC variant total protein, using canine CDV convalescent serum as a source of primary antibody, highlighted viral N and P proteins but not the 70K species present in L-NC and Df-NC variants ( Fig.…”