No abstract

Poliovirus (PV) infection induces the rearrangement of intracellular membranes into characteristic vesicles

Hepatitis A virus (HAV) is distinguished from other picornaviruses by its slow and relatively poor, noncytopathic growth in cultures of mammalian cells. The 2C and 2BC proteins of HAV have been implicated in the determination of virus growth in cultured cells. The homologous proteins from other picornaviruses, such as poliovirus, have been demonstrated to exhibit multiple activities, such as RNA binding, nucleotide binding and NTPase, and membrane binding and reorganization. At least some of these activities are required for viral RNA replication. We report here that HAV 2C and 2BC proteins, like their poliovirus counterparts, can induce rearrangement of intracellular membranes and directly or indirectly interact with membranes. Therefore, the inefficient replication properties of HAV are not consequences of the inherent ability of 2C (2BC) to interact with membranes. The effect of 2C (2BC) protein sequences derived from a cell culture-adapted (cc) strain of HAV was compared with that of corresponding protein sequences from either a wild-type (wt) strain of HAV or a faster replicating cytopathic (cp) strain. The analysis demonstrated that mutations acquired in wt virus during adaptation to cell culture do not change dramatically either the ability of these proteins to associate with membranes and induce membrane alterations or the specific architecture of the induced membrane structures. On the other hand, 2C, but not 2BC, protein from the cp strain of HAV induced different membrane structures.

The amino acid sequence of the poliovirus 2C protein contains two highly conserved stretches, GSPGTGKS t36 and MDD 177, which correspond to the consensus 'A' and 'B' motifs (GXXXXGKS/T and DD/E, respectively) found in nucleoside triphosphatebinding proteins. To assess the functional importance of these amino acid sequences, we changed conserved and non-conserved amino acids. The replacement of the non-conserved Thr 133 residue with Ser or Ala did not markedly change the virus phenotype. Similarly, replacement of the non-conserved Pro TM residue by Ala did not abolish virus viability, but changes of this residue to Thr or Asn were not tolerated. No viable mutant could be isolated after transfection of cultured cells with transcripts mutated at the conserved Lys 135, Ser 136 or Asp ~77 residues. However, true revertants were selected from Arg 135 and Ser ~35 mutants, from Glu 177 and Gly 177 mutants, and from Ala 136 mutants. Thr ~ 36 mutants not only gave rise to true revertants, but also to two independent isolates of a suppressor mutant, Asn14°-~Tyr. All the lethal mutations resulted in severe inhibition of viral RNA synthesis in vivo, although no translational deficiency was detected in a cell-free system. This is the first direct evidence for the functional significance of the nucleoside triphosphatebinding pattern in the poliovirus 2C protein.

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...

Poliovirus proteins 3A and 3AB are small, membrane-binding proteins that play multiple roles in viral RNA replication complex formation and function. In the infected cell, these proteins associate with other viral and cellular proteins as part of a supramolecular complex whose structure and composition are unknown. We isolated viable viruses with three different epitope tags (FLAG, hemagglutinin [HA], and c-myc) inserted into the N-terminal region of protein 3A. These viruses exhibited growth properties and characteristics very similar to those of the wild-type, untagged virus. Extracts prepared from the infected cells were subjected to immunoaffinity purification of the tagged proteins by adsorption to commercial antibody-linked beads and examined after elution for cellular and other viral proteins that remained bound to 3A sequences during purification. Viral proteins 2C, 2BC, 3D, and 3CD were detected in all three immunopurified 3A samples. Among the cellular proteins previously reported to interact with 3A either directly or indirectly, neither LIS1 nor phosphoinositol-4 kinase (PI4K) were detected in any of the purified tagged 3A samples. However, the guanine nucleotide exchange factor GBF1, which is a key regulator of membrane trafficking in the cellular protein secretory pathway and which has been shown previously to bind enteroviral protein 3A and to be required for viral RNA replication, was readily recovered along with immunoaffinity-purified 3A-FLAG. Surprisingly, we failed to cocapture GBF1 with 3A-HA or 3A-myc proteins. A model for variable binding of these 3A mutant proteins to GBF1 based on amino acid sequence motifs and the resulting practical and functional consequences thereof are discussed.Poliovirus (PV) is a member of the human enterovirus C cluster in the Enterovirus genus of the virus family Picornaviridae. The PV genome encodes a single polyprotein that is proteolytically processed to generate a set of intermediate precursors and final cleavage products that are all required for virus replication. The N-terminal region of the polyprotein (P1) forms the viral capsid proteins, which are dispensable for viral RNA translation and replication but are needed for encapsidation and assembly of infectious particles. The remainder of the polyprotein (P2 and P3 regions) generates proteins that contribute catalytic and structural functions for viral RNA translation and replication, as well as for disruption and/or reorganization of numerous cellular processes and activities that could restrict or combat virus replication. All of the PV noncapsid proteins are essential for viral RNA replication; most manifest multiple activities during the virus replication cycle, and all at least partially localize to large, membraneassociated replication complexes that form from preexisting subcellular organelles after infection. Viral proteins 2B, 2C, and 3A contain hydrophobic trans-membrane regions or amphipathic helices, and these proteins as well as their larger precursor proteins bind membranes directly (7,12,...

HeLa cells were transfected with several plasmids that encoded all poliovirus (PV) nonstructural proteins. Viral RNAs were transcribed by T7 RNA polymerase expressed from recombinant vaccinia virus. All plasmids produced similar amounts of viral proteins that were processed identically; however, RNAs were designed either to serve as templates for replication or to contain mutations predicted to prevent RNA replication. The mutations included substitution of the entire PV 5 noncoding region (NCR) with the encephalomyocarditis virus (EMCV) internal ribosomal entry site, thereby deleting the 5-terminal cloverleaf-like structure, or insertion of three nucleotides in the 3D pol coding sequence. Production of viral proteins was sufficient to induce the characteristic reorganization of intracellular membranes into heterogeneous-sized vesicles, independent of RNA replication. The vesicles were stably associated with viral RNA only when RNA replication could occur. Nonreplicating RNAs localized to distinct, nonoverlapping regions in the cell, excluded from the viral proteinmembrane complexes. The absence of accumulation of positive-strand RNA from both mutated RNAs in transfected cells was documented. In addition, no minus-strand RNA was produced from the EMCV chimeric template RNA in vitro. These data show that the 5-terminal sequences of PV RNA are essential for initiation of minus-strand RNA synthesis at its 3 end.Recent studies of events associated with poliovirus (PV) RNA replication have contributed to new insights into this complex reaction. In vivo and in vitro studies have implicated interactions between cellular and viral proteins with distinct elements on the viral RNA (vRNA) in various steps leading to production of new vRNA strands. The viral protein 3D catalyzes primer-and template-dependent RNA synthesis and is the only protein required for elongation of RNA chains in vitro (36,52). It also catalyzes the uridylylation of VPg (viral protein 3B) in vitro, in the presence of poly(A) (42). A short unpaired nucleotide sequence in a highly conserved stem-loop formed by the RNA in the 2C coding region appears to be a component of the natural template for the 3D-catalyzed VPg uridylylation reaction; this reaction is stimulated greatly by uncleaved 3CD (41). Uridylylated VPg is thought to function as the primer for initiation of minus-strand RNA synthesis, and thus uridylylation represents the first step in vRNA replication. In infected cells, however, this reaction appears to require the integrity of a membranous replication complex (RC), which has been demonstrated to serve as the site for vRNA synthesis and is composed of heterogeneous-sized vesicles associated with viral nonstructural proteins and RNA (13). Although all of the viral nonstructural proteins, as well as several of their precursor forms (e.g., 2BC, 3AB, and 3CD), have been implicated in vRNA synthesis, their precise biochemical roles remain uncertain, and detailed analyses of their activities are complicated by the multiple functions manifested by ...

Poliovirus protein 2C is a 329-amino acid-protein that is essential for viral RNA synthesis and may perform multiple functions. In infected cells, it is associated with virus-specific membrane vesicles. Recombinant 2C protein expressed in transfected cells has been shown to associate with and induce rearrangement of the intracellular membrane network. This study was designed to map the determinants of membrane binding and rearrangement in the 2C protein. Computer-assisted analysis of the protein sequence led to a prediction that the protein folds into a structure composed of three domains. Expression plasmids that encode each or combinations of these predicted domains were used to examine the abilities of the partial protein sequences to associate with intracellular membranes and to induce rearrangement of these membranes in HeLa cells. Biochemical fractionation procedures suggested that the N-terminal region of the protein was required for membrane association. Electron microscopic and immunoelectron microscopic observation showed that both the N-and C-terminal regions, but not the central portion, of 2C protein interact with intracellular membranes and induce major changes in their morphology. The central portion, when fused to the N-terminal region, altered the specific membrane architecture induced by the N-terminal region, giving rise to vesicles resembling those observed during poliovirus infection.