Abstract
The poliovirus-specific polypeptide 3AB (B = VPg) was expressed in Escherichia coli and purified to near homogeneity. Corresponding to its known association with membranes in poliovirus-infected HeLa cells, 3AB expressed in E. coli was also membrane-associated, and it could be solubilized only in detergent-containing buffers. In soluble form, 3AB was resistant to digestion with the virus-specific proteinases 3Cpro and 3CDpro. However, it was cleaved by these enzymes to 3A and VPg when bound to the bacterial membranes, an observation suggesting that 3AB may deliver the genome-linked protein VPg to the membrane-associated poliovirus replication complex. The specific activity of 3CDpro in processing 3AB was significantly higher than that of 3Cpro. Soluble 3AB was found to stimulate nearly 100-fold poly (A)-dependent, primer-dependent poly(U) synthesis, catalyzed by purified poliovirus RNA polymerase 3Dpol. We propose that 3AB has a dual function in poliovirus genome replication: as a precursor for VPg, and as a co-factor for 3Dpol.
Highlights
Soluble 3AB was found to generated by cleavage of 3AB, a 12-kDa protein known to be stimulate nearly100-fold poly(A)-dependent, primer- tightly bound to membranes, presumably via astretch of dependentpoly(U) synthesis, catalyzedbypurified hydrophobic amino acids preceding the VPg sequence [11]
Viral RNAs synthesized in vitro in these replication complexes are VPglinked, and initiation of synthesis ceases if mild detergent is added [4, 5]
We have discovered that purified 3AB, in the absence of uridylylation, has the property to stimulate replication-specificpolypeptides, serves as template for minus poly(U) synthesis catalyzed by purified 3Dw', an observation strand synthesis
Summary
Service grants AI-15122and CA-28146.The costs of publication of this article were defrayed in part by the payment of page charges. This articlemust be hereby marked "advertisement" in Buffers-All pH values were adjusted at 24 "C. Tel.: 516-632- The abbreviations used are: PMSF, phenylmethylsulfonyl fluo-. In all the buffers except buffer D, with no pepstatin A, buffers DS Background values obtained from reactions in which 3Dp1 waasbsent and S-10, with no PMSF and pepstatin A, and buffers S-100 and S- were subtracted from the data.
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