Abstract:
:Purification of the putative cowpea mosaic virus (CPMV) RNA replicase previously started with an RNA-dependent RNA polymerase activity which had been solubilized from a crude membrane fraction of CPMV-infected cowpea leaves by extraction with a Mg2+-deficient buffer. This led to the identification of a host-encoded, 130,000-dalton monomeric enzyme, the activity of which was highly enhanced upon infection. As the role of this enzyme in viral replication was questionable, we reverted to the template-associated RNA-dependent RNA polymerase in the crude membrane fraction in order to characterize in detail its in vitro products. We now demonstrate that the crude membrane fraction of CPMV-infected cowpea leaves harbors two functionally different, RNA-dependent RNA polymerase activities that are both associated with endogenous template RNA and can be separated from each other without affecting their distinct properties. One of the RNA polymerase activities was specific for CPMV-infected leaves and constituted a CPMV RNA replication complex; enzyme activity in vitro allowed for the completion of nascent chains initiated in vivo. Full-length viral RNAS (B- and M-RNA) were produced which were recovered mainly in double-stranded form. Solution- and Northern blot hybridization demonstrated that the in vitro-labeled RNA chains were viral RNAs of positive polarity. The other template-associated RNA-dependent RNA polymerase activity occurred in both uninfected and infected leaves and transcribed in vitro endogenous plant and viral RNAs only into small RNAs (4-5 S) of negative polarity. Northern blot analysis revealed the RNA products of plant origin to be transcribed from two major RNA templates of approximately 0.26 and 0.14 X 106 daltons, respectively. Washing of the crude membrane fraction in a Mg2+-deficient buffer did accomplish the complete release of the low-molecular-weight RNA-synthesizing activity but did not solubilize the CPMV RNA replication complex. We tentatively conclude that the RNA-dependent RNA polymerase which has previously been purified from the buffer-soluble fraction and has been identified as a host-encoded enzyme is not involved in viral RNA replication. Solubilization of the viral replication complex was achieved with Triton X-100. By taking advantage of the characteristic conformation of the replication complex, we applied Sepharose 2B chromatography as a highly efficient and simple means for purifying the detergent-solubilized complex. We anticipate that this purification step should also be applicable to replication complexes of other RNA viruses.
journal_name
Virologyjournal_title
Virologyauthors
Dorssers L,van der Meer J,Van Kammen A,Zabel Pdoi
10.1016/0042-6822(83)90071-5subject
Has Abstractpub_date
1983-02-01 00:00:00pages
155-74issue
1eissn
0042-6822issn
1096-0341pii
0042-6822(83)90071-5journal_volume
125pub_type
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