The major ribonucleoprotein-associated protein kinase of vesicular stomatitis virus is a host cell protein.

S A Harmon,L L Marnell,D F Summers

doi:10.1016/s0021-9258(17)43804-x

Abstract

Ribonucleoprotein particles (RNPs) of vesicular stomatitis virus (VSV) were fractionated by column chromatography through Fractogel TSK HW-55F and by centrifugation through KCl sucrose. Analyses of fractions for protein content and for protein kinase activity indicated that the major peak of kinase activity did not correspond exactly with any of the VSV-specific proteins. Neither anti-NS nor anti-M IgG preparations inhibited protein kinase activity, and IgG did not act as an exogenous phosphate acceptor. Reconstitution of an RNP-enzyme complex did not result in a restoration of protein kinase activity. In vitro translation of VSV-specific poly(A)-containing RNA did not result in any detectable production of kinase activity. Thus, the major RNP-associated kinase is a host cell protein which is tightly bound to the RNP particle.

Highlights

Ribonucleoprotein particles (RNPs) of vesicular sto- VSV virion and RNPparticle have only indirectly implicated matitis virus (VSV) were fractionated by column chro- this enzyme activity as a possible means of regulating the matography through Fractogel TSK HW-55F and by virus-specific RNA polymerase during replication
Clinton et al [6], using immunological techniques, tentatively identified one protein kinase associated with VSV as Isolation of RNPs from Virions-RNPs were isolated by mixing virions with an equal volume of lysis solution (3.8% Triton N 101, 0.8 M NaCI, 1.2 mM DTT) and subsequent centrifugation through a discontinuous glycerol gradient [12]
Fractionation of VSV RNPs by Fractogel Column Chromatography-Virion R N P s were incubated in the presence of 2

Summary

VSV Indiana would then be amenable to structural analyses

VSV’ has a virion-associated protein kinase activity which is CAMP independent and has been suggested to be a hostderiveil protein [6, 7, 24]. In addition to aliquots of column or gradient fractions, the reaction mixture (100 pl) contained 10 mM Tris, pH 8.0, 10 mM MgCl,, 5 mM DTT, 1 X M ATP, 6 pCi of [y3'P]ATP (New England Nuclear; specific activity, 8.7 Ci/mmol), and 20 pg ofcasein as an exogenousphosphate acceptor [8].After incubation of the protein kinase reaction mixtures at 37 "C for 45 min, aliquots were precipitated with 10% trichloroacetic acid containing 50 mM monosodium phosphate and 50 mM sodium pyrophosphate, precipitates were collected on GF/C Whatman filters, and radioactivity was determined in a Beckman liquid scintillation spectrometer. The fractions were dialyzed overnight against 10 mM Tris, pH 8.0,l mM DTT, and a sample was assayed for protein kinase activity using casein as the phosphate acceptor, as described above.

RESULTS

Protein kinase actioitiesof purified proteins

FRACTION NUMBER

DISCUSSION

Reconstitution of purifiedL andNSproteins with the