Bert L. Semler

Protein 3CD is the major poliovirus proteinase responsible for cleavage of the P1 capsid precursor

The rate and extent of polyprotein processing are the major steps controlling picornavirus gene expression. It is, therefore, important to determine the enzymes responsible for each proteolytic event. The poliovirus protein 3C has been identified as a proteinase which specifically cleaves between Q-G pairs. However, recent data have suggested that 3C precursor polypeptides containing 3C sequences may also have proteolytic capabilities. In this study we have analyzed the cleavage specificities of protein 3C and its precursor, 3CD. We have carried out in vitro translation of genetically altered poliovirus mRNAs to demonstrate that 3CD is required for efficient processing of the P1 capsid precursor to capsid proteins. In addition, we suggest 3CD and 3C process Q-G pairs in the P2 and P3 precursors with similar efficiencies.

Bridging IRES elements in mRNAs to the eukaryotic translation apparatus.

IRES elements are highly structured RNA sequences that function to recruit ribosomes for the initiation of translation. In contrast to the canonical cap-binding, ribosome-scanning model, the mechanism of IRES-mediated translation initiation is not well understood. IRES elements, first discovered in viral RNA genomes, were subsequently found in a subset of cellular RNAs as well. Interestingly, these cellular IRES-containing mRNAs appear to play important roles during conditions of cellular stress, development, and disease (e.g., cancer). It has been shown for viral IRESes that some require specific IRES trans-acting factors (ITAFs), while others require few if any additional proteins and can bind ribosomes directly. Current studies are aimed at elucidating the mechanism of IRES-mediated translation initiation and features that may be common or differ greatly among cellular and viral IRESes. This review will explore IRES elements as important RNA structures that function in both cellular and viral RNA translation and the significance of these structures in providing an alternative mechanism of eukaryotic translation initiation.

A nucleo‐cytoplasmic SR protein functions in viral IRES‐mediated translation initiation

A significant number of viral and cellular mRNAs utilize cap‐independent translation, employing mechanisms distinct from those of canonical translation initiation. Cap‐independent translation requires noncanonical, cellular RNA‐binding proteins; however, the roles of such proteins in ribosome recruitment and translation initiation are not fully understood. This work demonstrates that a nucleo‐cytoplasmic SR protein, SRp20, functions in internal ribosome entry site (IRES)‐mediated translation of a viral RNA. We found that SRp20 interacts with the cellular RNA‐binding protein, PCBP2, a protein that binds to IRES sequences within the genomic RNAs of certain picornaviruses and is required for viral translation. We utilized in vitro translation in HeLa cell extracts depleted of SRp20 to demonstrate that SRp20 is required for poliovirus translation initiation. Targeting SRp20 in HeLa cells with short interfering RNAs resulted in inhibition of SRp20 protein expression and a corresponding decrease in poliovirus translation. Our data have identified a previously unknown function of an SR protein (i.e., the stimulation of IRES‐mediated translation), further documenting the multifunctional nature of this important class of cellular RNA‐binding proteins.

Distinct Poly(rC) Binding Protein KH Domain Determinants for Poliovirus Translation Initiation and Viral RNA Replication

ABSTRACT The limited coding capacity of picornavirus genomic RNAs necessitates utilization of host cell factors in the completion of an infectious cycle. One host protein that plays a role in both translation initiation and viral RNA synthesis is poly(rC) binding protein 2 (PCBP2). For picornavirus RNAs containing type I internal ribosome entry site (IRES) elements, PCBP2 binds the major stem-loop structure (stem-loop IV) in the IRES and is essential for translation initiation. Additionally, the binding of PCBP2 to the 5′-terminal stem-loop structure (stem-loop I or cloverleaf) in concert with viral protein 3CD is required for initiation of RNA synthesis directed by poliovirus replication complexes. PCBP1, a highly homologous isoform of PCBP2, binds to poliovirus stem-loop I with an affinity similar to that of PCBP2; however, PCBP1 has reduced affinity for stem-loop IV. Using a dicistronic poliovirus RNA, we were able to functionally uncouple translation and RNA replication in PCBP-depleted extracts. Our results demonstrate that PCBP1 rescues RNA replication but is not able to rescue translation initiation. We have also generated mutated versions of PCBP2 containing site-directed lesions in each of the three RNA-binding domains. Specific defects in RNA binding to either stem-loop I and/or stem-loop IV suggest that these domains may have differential functions in translation and RNA replication. These predictions were confirmed in functional assays that allow separation of RNA replication activities from translation. Our data have implications for differential picornavirus template utilization during viral translation and RNA replication and suggest that specific PCBP2 domains may have distinct roles in these activities.

Cellular Protein Modification by Poliovirus: the Two Faces of Poly(rC)-Binding Protein

ABSTRACT During picornavirus infection, several cellular proteins are cleaved by virus-encoded proteinases. Such cleavage events are likely to be involved in the changing dynamics during the intracellular viral life cycle, from viral translation to host shutoff to RNA replication to virion assembly. For example, it has been proposed that there is an active switch from poliovirus translation to RNA replication mediated by changes in RNA-binding protein affinities. This switch could be a mechanism for controlling template selection for translation and negative-strand viral RNA synthesis, two processes that use the same positive-strand RNA as a template but proceed in opposing directions. The cellular protein poly(rC)-binding protein (PCBP) was identified as a primary candidate for regulating such a mechanism. Among the four different isoforms of PCBP in mammalian cells, PCBP2 is required for translation initiation on picornavirus genomes with type I internal ribosome entry site elements and also for RNA replication. Through its three K-homologous (KH) domains, PCPB2 forms functional protein-protein and RNA-protein complexes with components of the viral translation and replication machinery. We have found that the isoforms PCBP1 and -2 are cleaved during the mid-to-late phase of poliovirus infection. On the basis of in vitro cleavage assays, we determined that this cleavage event was mediated by the viral proteinases 3C/3CD. The primary cleavage occurs in the linker between the KH2 and KH3 domains, resulting in truncated PCBP2 lacking the KH3 domain. This cleaved protein, termed PCBP2-ΔKH3, is unable to function in translation but maintains its activity in viral RNA replication. We propose that through the loss of the KH3 domain, and therefore loss of its ability to function in translation, PCBP2 can mediate the switch from viral translation to RNA replication.

Defective Interfering RNA Viruses and the Host-Cell Response

This chapter will emphasize recently derived knowledge concerning the nature of defective interfering (DI) particles of RNA animal viruses, their biological origins and functions, and their involvement in long-term persistent infections. We will not attempt to review all of the DI literature, and we will confine ourselves to DI particles of RNA viruses. The previous review by Huang and Baltimore (1977) amply documents the occurrence and behavior of DI particles in a wide variety of DNA and RNA viruses and discusses their biological effects, and a very thorough recent review of rhabdovirus DI particles by Reichmann and Schnitzlein (1978) provides excellent in-depth coverage of many areas not covered by the present chapter as well as some alternate viewpoints of areas which are considered here. We will omit DNA virus DI particles from extensive consideration because of space limitations and because they are generally less well characterized at present.

Functional Interaction of Heterogeneous Nuclear Ribonucleoprotein C with Poliovirus RNA Synthesis Initiation Complexes

ABSTRACT We had previously demonstrated that a cellular protein specifically interacts with the 3′ end of poliovirus negative-strand RNA. We now report the identity of this protein as heterogeneous nuclear ribonucleoprotein (hnRNP) C1/C2. Formation of an RNP complex with poliovirus RNA was severely impaired by substitution of a lysine, highly conserved among vertebrates, with glutamine in the RNA recognition motif (RRM) of recombinant hnRNP C1, suggesting that the binding is mediated by the RRM in the protein. We have also shown that in a glutathione S-transferase (GST) pull-down assay, GST/hnRNP C1 binds to poliovirus polypeptide 3CD, a precursor to the viral RNA-dependent RNA polymerase, 3Dpol, as well as to P2 and P3, precursors to the nonstructural proteins. Truncation of the auxiliary domain in hnRNP C1 (C1ΔC) diminished these protein-protein interactions. When GST/hnRNP C1ΔC was added to in vitro replication reactions, a significant reduction in RNA synthesis was observed in contrast to reactions supplemented with wild-type fusion protein. Indirect functional depletion of hnRNP C from in vitro replication reactions, using poliovirus negative-strand cloverleaf RNA, led to a decrease in RNA synthesis. The addition of GST/hnRNP C1 to the reactions rescued RNA synthesis to near mock-depleted levels. Furthermore, we demonstrated that poliovirus positive-strand and negative-strand RNA present in cytoplasmic extracts prepared from infected HeLa cells coimmunoprecipitated with hnRNP C1/C2. Our findings suggest that hnRNP C1 has a role in positive-strand RNA synthesis in poliovirus-infected cells, possibly at the level of initiation.

Expression of a cloned gene segment of poliovirus in E. coli: Evidence for autocatalytic production of the viral proteinase

The poliovirus polyprotein is proteolytically processed predominantly by a virus-encoded proteinase (P3-7c) that cleaves glutamine-glycine amino acid pairs. The biosynthesis of the viral proteinase, itself a product of glutamine-glycine cleavages, was studied by constructing a bacterial expression plasmid that contained a cloned segment of the poliovirus genome slightly larger than the coding region for P3-7c. The induction of expression of this plasmid in E. coli produced several poliovirus-specific polypeptides. One polypeptide, an unstable protein called 3i, was the product of fortuitous in-phase initiation of translation within the coding region of P3-7c. Three other induced polypeptides were products of proteolytic cleavages, the smallest (polypeptide 3) having the properties (amino-terminal amino acids, carboxy-terminal amino acids, size, antigenicity) of P3-7c. Insertion of a DNA linker into the P3-7c coding region results in the loss of P3-7c-specific glutamine-glycine cleavage activity. We conclude that P3-7c was produced by autocatalytic cleavage.

Requirements for Assembly of Poliovirus Replication Complexes and Negative-Strand RNA Synthesis

ABSTRACT 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 3Dpol 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 protein-membrane 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.

SARS coronavirus nsp1 protein induces template-dependent endonucleolytic cleavage of mRNAs: viral mRNAs are resistant to nsp1-induced RNA cleavage.

SARS coronavirus (SCoV) nonstructural protein (nsp) 1, a potent inhibitor of host gene expression, possesses a unique mode of action: it binds to 40S ribosomes to inactivate their translation functions and induces host mRNA degradation. Our previous study demonstrated that nsp1 induces RNA modification near the 5′-end of a reporter mRNA having a short 5′ untranslated region and RNA cleavage in the encephalomyocarditis virus internal ribosome entry site (IRES) region of a dicistronic RNA template, but not in those IRES elements from hepatitis C or cricket paralysis viruses. By using primarily cell-free, in vitro translation systems, the present study revealed that the nsp1 induced endonucleolytic RNA cleavage mainly near the 5′ untranslated region of capped mRNA templates. Experiments using dicistronic mRNAs carrying different IRESes showed that nsp1 induced endonucleolytic RNA cleavage within the ribosome loading region of type I and type II picornavirus IRES elements, but not that of classical swine fever virus IRES, which is characterized as a hepatitis C virus-like IRES. The nsp1-induced RNA cleavage of template mRNAs exhibited no apparent preference for a specific nucleotide sequence at the RNA cleavage sites. Remarkably, SCoV mRNAs, which have a 5′ cap structure and 3′ poly A tail like those of typical host mRNAs, were not susceptible to nsp1-mediated RNA cleavage and importantly, the presence of the 5′-end leader sequence protected the SCoV mRNAs from nsp1-induced endonucleolytic RNA cleavage. The escape of viral mRNAs from nsp1-induced RNA cleavage may be an important strategy by which the virus circumvents the action of nsp1 leading to the efficient accumulation of viral mRNAs and viral proteins during infection.