The poliovirus-encoded, membrane-associated polypeptide 2C is required for viral replication. We have expressed 2C, its precursor 2BC, and a number of 2C deletion mutants in eukaryotic (HeLa) cells and examined their localization using indirect immunofluorescence. Results presented here demonstrate that proteins 2C and 2BC are capable of localizing to the endoplasmic reticulum area in transfected cells in the absence of other poliovirus proteins. Additionally, 2C binds tightly to microsomal membranes in a direct in vitro membrane binding assay. Although the 2C protein lacks a defined membrane binding domain, we demonstrate that the N-terminal region encompassing amino acids 21-54 and containing a putative amphipathic helix plays an important role in membrane binding both in vivo and in vitro. In contrast, most of the C-terminus portion of the protein appears to be unnecessary for membrane association. The susceptibility of membrane-associated 2C to proteinase K digestion in vitro suggests that all or most of 2C is exposed to the cytoplasmic face of the membrane. Furthermore, unlike most proteins targeted to the endoplasmic reticulum, 2C does not appear to be glycosylated or cleaved by signal peptidases. The implication of the membrane binding domain of 2C in viral RNA replication is discussed.
The poliovirus-encoded, membrane-associated polypeptide 2C is believed to be required for initiation and elongation of RNA synthesis. We have expressed and purified recombinant, histidine-tagged 2C and examined its ability to bind to the first 100 nucleotides of the poliovirus 5 untranslated region of the positive strand and its complementary 3-terminal negative-strand RNA sequences. Results presented here demonstrate that the 2C polypeptide specifically binds to the 3-terminal sequences of poliovirus negative-strand RNA. Since this region is believed to form a stable cloverleaf structure, a number of mutations were constructed to examine which nucleotides and/or structures within the cloverleaf are essential for 2C binding. Binding of 2C to the 3-terminal cloverleaf of the negative-strand RNA is greatly affected when the conserved sequence, UGUUUU, in stem a of the cloverleaf is altered. Mutational studies suggest that interaction of 2C with the 3-terminal cloverleaf of negative-strand RNA is facilitated when the sequence UGUUUU is present in the context of a double-stranded structure. The implication of 2C binding to negative-strand RNA in viral replication is discussed.
Poliovirus-encoded nonstructural polypeptide 2C is a multifunctional protein that plays an important role in viral RNA replication. 2C interacts with both intracellular membranes and virus-specific RNAs and has ATPase and GTPase activities. Extensive computer analysis of the 2C sequence revealed that in addition to the known ATPase-, GTPase-, membrane-, and RNA-binding domains it also contains several "serpin" (serine protease inhibitor) motifs. We provide experimental evidence suggesting that 2C is indeed capable of regulating virus-encoded proteases. The purified 2C protein inhibits Poliovirus is the prototype member of the picornavirus family with a plus-sense RNA genome of 7,440 nucleotides, which is covalently linked to a small viral protein (VP g ) at its 5Ј end and polyadenylated at its 3Ј end (34, 56). The positive-strand mRNA, which lacks VP g , codes for a single large polyprotein, which is processed into mature proteins by viral proteases 2A pro , 3C pro and 3CD pro (reviewed in references 39 and 71). Biochemical and genetic evidence suggests that most of the poliovirus nonstructural proteins are involved in viral RNA replication in the form of precursors, mature polypeptides, or both (71). The viral RNA polymerase precursors 3CD pro , the VP g precursor 3AB and a cellular polypeptide, poly(rC)-binding protein, have been shown to interact with the 5Ј-cloverleaf structure of viral RNA, leading to the formation of a functional complex important for viral RNA replication (3,4,31,47,72). The precursor proteins 3AB and 3CD pro also interact with the 3Ј-untranslated region of viral RNA in the absence of other proteins (31). The initiation of poliovirus RNA synthesis appears to be primed by a protein-nucleotidyl covalent complex (VP g -pU or VP g -pUpU) (10, 50, 52).Although it was initially thought to be a member of the cysteine protease family, mutational analyses, amino acid sequence comparison, and three-dimensional modeling have suggested that 3C pro adopts a fold similar to that found in serine proteases such as chymotrypsin (12,28,30,32,33,37,45). The other viral protease, 2Apro , also possesses a chymotrypsin-like fold that is related to smaller serine proteases such as ␣-lytic proteinase (12). Although the major function of these proteases is to process viral precursor polypeptides, both proteases are also involved in the shutoff of host cell metabolism. Although 2A pro is involved in the shutoff of host cell translation (13, 29, 41), 3C pro has been shown to cleave and inactivate a number of host cell transcription factors leading to the inhibition of cellular transcription (19,61,70,75).The 2C protein of poliovirus is 329 amino acids long (37.5 kDa) and is highly conserved among picornaviruses (5). Genetic analyses have implicated the 2C polypeptide in a number of functions during viral replication such as uncoating (40), host cell membrane rearrangement (18), RNA replication (reviewed in reference 71), and encapsidation (69). The exact role of 2C in these processes, however, is not known. Many ...
HHV-8 vIL-6 clearly has the potential to upregulate HIV-1 expression in monocytic cells, and therefore may play a role in AIDS pathogenesis in individuals infected with both viruses.
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