Isolation and Characterization of Canine Distemper Virus Nucleocapsid Variants

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Abstract
Summary Nucleocapsid (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 (CsCl) 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% 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 ± 0.0040 g/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 CsCl 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.