Poliovirus protein 2C contains a predicted N-terminal amphipathic helix that mediates association of the protein with the membranes of the viral RNA replication complex. A chimeric virus that contains sequences encoding the 18-residue core from the orthologous amphipathic helix from human rhinovirus type 14 (HRV14) was constructed. The chimeric virus exhibited defects in viral RNA replication and produced minute plaques on HeLa cell monolayers. Large plaque variants that contained mutations within the 2C-encoding region were generated upon subsequent passage. However, the majority of viruses that emerged with improved growth properties contained no changes in the region encoding 2C. Sequence analysis and reconstruction of genomes with individual mutations revealed changes in 3A or 2B sequences that compensated for the HRV14 amphipathic helix in the polio 2C-containing proteins, implying functional interactions among these proteins during the replication process. Direct binding between these viral proteins was confirmed by mammalian cell twohybrid analysis.Poliovirus (PV) RNA replication takes place in replication complexes that form de novo in cultured cells after virus infection. Synthesis of viral proteins induces extensive rearrangement of intracellular membrane structures that produce perinuclear foci of vesicle-associated viral proteins and RNA (reference 11 and references therein). These coalesce into large clusters of vesicles engaged in viral RNA synthesis, accompanied by the loss of preexisting Golgi stacks and endoplasmic reticulum (ER). All viral nonstructural proteins, derived from the P2 and P3 polyprotein regions of the single open reading frame in the viral genome, are found associated with the membranous replication complexes and have been implicated by genetic analysis as playing essential roles in the process of viral RNA replication. Proteins containing 2B, 2C, or 3A sequences manifest inherent membrane-binding properties. It is not known how the other viral proteins are recruited to and/or retained in the replication complexes. They may enter or induce formation of the complexes as larger precursor proteins prior to cleavage and maintain their associations via protein-protein or protein-RNA interactions, a hypothesis supported by the observation that complementation of defective proteins by expression of individual functional gene products does not occur readily in infected cells (32) and requires expression of whole P2 or P3 precursor proteins in vitro (16,35).The precise biochemical roles in viral RNA synthesis played by each of the nonstructural proteins are poorly defined (summarized in reference 21). From the P3 region, protein 3D catalyzes polynucleotide chain elongation as well as uridylylation of VPg (protein 3B) to form a primer for RNA chain initiation. Protein 3C is the protease responsible for the majority of polyprotein cleavages, both in cis and in trans. Protein 3CD, in addition to serving a proteinase function for generation of capsid proteins from P1 precursors, binds and sti...

The N-terminal region of the picornaviral 2C protein is predicted to fold into an amphipathic alpha-helix that is responsible for the protein's association with membranes in the viral RNA replication complex. We have identified a similar sequence in the N-terminal region of NS5A of hepaciviruses that was recently shown to form an amphipathic alpha-helix. The conservation of the N-terminal region in two apparently unrelated proteins of two different RNA virus families suggested that this helix might represent an independent module. To test this hypothesis, we constructed chimeric poliovirus (PV) genomes in which the sequence encoding the N-terminal 2C amphipathic helix was replaced by orthologous sequences from other picornaviral genomes or a similar sequence from NS5A of HCV. Effects of the mutations were assessed by measuring the accumulation of viable virus and viral RNA in HeLa cells after transfection, examining membrane morphology in cells expressing chimeric proteins and by in vitro analysis of RNA translation, protein processing and negative strand RNA synthesis in HeLa cell extracts. The chimeras manifested a wide range of growth and RNA synthesis phenotypes. The results are compatible with our hypothesis, although they demonstrate that helix exchangeability may be restricted due to requirements for interactions with other viral components involved in virus replication.

Hematopoietic progenitor cells die by apoptosis after removal of the appropriate colony-stimulating factor (CSF). Recent pharmacologic data have implicated protein kinase C (PKC) in the suppression of apoptosis in interleukin-3 (IL-3) and granulocyte-macrophage (GM)-CSF–dependent human myeloid cells. Because IL-3 and GM-CSF induce increases in diacylglycerol without mobilizing intracellular Ca++, it seemed that one of the novel Ca++ independent isoforms of PKC was involved. We report here that overexpression of PKC in factor-dependent human TF-1 cells extends cell survival in the absence of cytokine. Overexpression of PKCδ does not have this effect. By 72 to 96 hours after cytokine withdrawal, the PKC transfectants remain distributed in all phases of the cell cycle, as shown by fluorescence-activated cell sorting (FACS) analysis, while little intact cellular DNA is detectable in vector or PKCδ transfectants. PKC induces bcl-2 protein expression fivefold to sixfold over the levels in empty vector transfectants, whereas the levels in PKCδ transfectants are similar to those in vector controls.