Modulation of the RNA Binding and Protein Processing Activities of Poliovirus Polypeptide 3CD by the Viral RNA Polymerase Domain

Abstract

To study the role of the RNA polymerase domain (3D) in the proteinase substrate recognition and RNA binding properties of poliovirus polypeptide 3CD, we generated recombinant 3C and 3CD polypeptides and purified them to near homogeneity. By using these purified proteins in in vitro cleavage assays with structural and non-structural viral polyprotein substrates, we found that 3CD processes the poliovirus structural polyprotein precursor (P1) 100 to 1000 times more efficiently than 3C processes P1. We also found that trans-cleavage of other 3CD molecules and sites within the non-structural P3 precursor is more efficiently mediated by 3CD than 3C. However, 3C and 3CD appear to be equally efficient in the processing of a non-structural polyprotein precursor, 2C3AB. Four mutated 3CD polyproteins with site-directed lesions in the 3D domain of the proteinase were analyzed for their ability to process viral polyprotein precursors and to form a ternary complex with RNA sequences encoded in the 5 terminus of the viral genome. Analysis of mutated 3CD polypeptides revealed that specific mutations within the 3D amino acid sequences of 3CD confer differential effects on 3CD activity. All four mutated 3CD proteins tested were able to process the P1 structural precursor with wild type or near wild type efficiency. However, three of the mutated enzymes demonstrated an impaired ability to process some sites within the P3 non-structural precursor, relative to wild type 3CD. One of the mutant 3CD polypeptides, 3CD-3DK127A, also displayed a defect in its ability to form a ternary ribonucleoprotein complex with poliovirus 5 RNA sequences.The positive strand RNA genome of poliovirus contains a single open reading frame that codes for a 247-kDa viral polyprotein. Proper expression of poliovirus gene products requires specific protein processing of the viral polyprotein by the enzymatic activities of virally encoded proteinases. These enzymes cleave the polyprotein co-and post-translationally into the structural and non-structural viral gene products (Fig. 1). The majority of cleavage events within the viral polyprotein occur at Q-G bonds and is mediated by a proteinase function associated with the viral-encoded 3C protein (1-6). The rate at which 3C pro1 -mediated proteolytic processing occurs at various sites within the viral polyprotein controls the temporal expression of viral gene products (7). This regulation of gene expression is essential for viral replication since polyprotein precursors have functions in replication that are distinct from those of the mature cleavage products (8, 9). An example of a molecule exhibiting these differential functions is the viral polyprotein 3CD, which is an active proteinase containing the entire amino acid sequences of the 3C proteinase and the RNA-dependent RNA polymerase, 3D. Polypeptide 3CD is a multi-functional protein required for both proteolytic processing of the capsid precursor protein and, although it has no detectable elongation activity in vitro, viral RNA replication. The role of 3...

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“…For example, poliovirus pro- teins 3C and 3D function as a protease and an RNA-dependent RNA polymerase, respectively. However, the nominal precursor protein, 3CD, does not display polymerase activity, binds site specifically to the 5Ј noncoding region of the viral RNA, and displays proteolytic substrate specificity distinct from that of the 3C protein (30,58,59). For Sindbis virus, the uncleaved precursor nsP1/nsP2/nsP3, in combination with the nsP4 polymerase, supports negative-strand but not positivestrand RNA synthesis, whereas the cleaved forms show increased specificity for positive-strand synthesis (46,71).…”

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“…A major role of 3CD proteinase for enteroviruses and human rhinoviruses is the proteolytic processing of viral capsid precursors as well as the precursors leading to the production of mature nonstructural proteins (22,23). In addition, 3C/3CD cleaves the cellular RNA binding protein poly(rC) binding protein 2 (PCBP2), which is thought to contribute to a switch from viral translation to RNA replication during poliovirus infection (24).…”

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“…The recombinant 3CD pro utilized in these studies does not support cis-or trans-cleavage into 3C and 3D. Furthermore, the aliquots of 3C pro and 3CD pro used correspond to equimolar amounts and possess equal protease activity at the P2-P3 junction of the poliovirus polyprotein (44).…”

“…Proteins were then transferred to nitrocellulose and subjected to immunoblotting with either anti-eIF-4G or anti-DNA-PK CS antibodies, followed by the appropriate secondary antibody. For assessment of protease activity against the 2C3AB substrate, [ 35 S]methioninelabeled 2C3AB protein was generated by coupled in vitro transcription/translation of plasmid pTM1-2C3AB (kindly provided by B. Semler, University of California, Irvine) as previously described (44). One microliter of 2C3AB translation reaction mixture was then incubated with 100 g of HeLa cell S10 extract in the absence or presence of 100 ng of 2A pro or 3C pro or 300 ng of 3CD pro for 1 h at 37°C.…”