Inhibition of poliovirus-induced cleavage of cellular protein PCBP2 reduces the levels of viral RNA replication.

UNLABELLED: Due to their small genome size, picornaviruses must utilize host proteins to mediate cap-independent translation and viral RNA replication. The host RNA-binding protein poly(rC) binding protein 2 (PCBP2) is involved in both processes in poliovirus infected cells. It has been shown that the viral proteinase 3CD cleaves PCBP2 and contributes to viral translation inhibition. However, cleaved PCBP2 remains active in viral RNA replication. This would suggest that both cleaved and intact forms of PCBP2 have a role in the viral RNA replication cycle. The picornavirus genome must act as a template for both translation and RNA replication. However, a template that is actively being translated cannot function as a template for RNA replication, suggesting that there is a switch in template usage from translation to RNA replication. We demonstrate that the cleavage of PCBP2 by the poliovirus 3CD proteinase is a necessary step for efficient viral RNA replication and, as such, may be important for mediating a switch in template usage from translation to RNA replication. IMPORTANCE: Poliovirus, like all positive-strand RNA viruses that replicate in the cytoplasm of eukaryotic cells, uses its genomic RNA as a template for both viral protein synthesis and RNA replication. Given that these processes cannot occur simultaneously on the same template, poliovirus has evolved a mechanism(s) to facilitate the switch from using templates for translation to using them for RNA synthesis. This study explores one possible scenario for how the virus alters the functions of a host cell RNA binding protein to mediate, in part, this important transition

Inhibition of poliovirus-induced cleavage of cellular protein PCBP2 reduces the levels of viral RNA replication.

UnlabelledDue to their small genome size, picornaviruses must utilize host proteins to mediate cap-independent translation and viral RNA replication. The host RNA-binding protein poly(rC) binding protein 2 (PCBP2) is involved in both processes in poliovirus infected cells. It has been shown that the viral proteinase 3CD cleaves PCBP2 and contributes to viral translation inhibition. However, cleaved PCBP2 remains active in viral RNA replication. This would suggest that both cleaved and intact forms of PCBP2 have a role in the viral RNA replication cycle. The picornavirus genome must act as a template for both translation and RNA replication. However, a template that is actively being translated cannot function as a template for RNA replication, suggesting that there is a switch in template usage from translation to RNA replication. We demonstrate that the cleavage of PCBP2 by the poliovirus 3CD proteinase is a necessary step for efficient viral RNA replication and, as such, may be important for mediating a switch in template usage from translation to RNA replication.ImportancePoliovirus, like all positive-strand RNA viruses that replicate in the cytoplasm of eukaryotic cells, uses its genomic RNA as a template for both viral protein synthesis and RNA replication. Given that these processes cannot occur simultaneously on the same template, poliovirus has evolved a mechanism(s) to facilitate the switch from using templates for translation to using them for RNA synthesis. This study explores one possible scenario for how the virus alters the functions of a host cell RNA binding protein to mediate, in part, this important transition

Inhibition of poliovirus-induced cleavage of cellular protein PCBP2 reduces the levels of viral RNA replication.

UnlabelledDue to their small genome size, picornaviruses must utilize host proteins to mediate cap-independent translation and viral RNA replication. The host RNA-binding protein poly(rC) binding protein 2 (PCBP2) is involved in both processes in poliovirus infected cells. It has been shown that the viral proteinase 3CD cleaves PCBP2 and contributes to viral translation inhibition. However, cleaved PCBP2 remains active in viral RNA replication. This would suggest that both cleaved and intact forms of PCBP2 have a role in the viral RNA replication cycle. The picornavirus genome must act as a template for both translation and RNA replication. However, a template that is actively being translated cannot function as a template for RNA replication, suggesting that there is a switch in template usage from translation to RNA replication. We demonstrate that the cleavage of PCBP2 by the poliovirus 3CD proteinase is a necessary step for efficient viral RNA replication and, as such, may be important for mediating a switch in template usage from translation to RNA replication.ImportancePoliovirus, like all positive-strand RNA viruses that replicate in the cytoplasm of eukaryotic cells, uses its genomic RNA as a template for both viral protein synthesis and RNA replication. Given that these processes cannot occur simultaneously on the same template, poliovirus has evolved a mechanism(s) to facilitate the switch from using templates for translation to using them for RNA synthesis. This study explores one possible scenario for how the virus alters the functions of a host cell RNA binding protein to mediate, in part, this important transition

Atomic force microscopy analysis of icosahedral virus RNA.

Single-stranded genomic RNAs from four icosahedral viruses (poliovirus, turnip yellow mosaic virus (TYMV), brome mosaic virus (BMV), and satellite tobacco mosaic virus (STMV)) along with the RNA from the helical tobacco mosaic virus (TMV) were extracted using phenol/chloroform. The RNAs were imaged using atomic force microscopy (AFM) under dynamic conditions in which the RNA was observed to unfold. RNAs from the four icosahedral viruses initially exhibited highly condensed, uniform spherical shapes with diameters consistent with those expected from the interiors of their respective capsids. Upon incubation at 26 degrees C, poliovirus RNA gradually transformed into chains of globular domains having the appearance of thick, irregularly segmented fibers. These ultimately unwound further to reveal segmented portions of the fibers connected by single strands of RNA of 0.5-1 nm thickness. Virtually the same transformations were shown by TYMV and BMV RNA, and with heating, the RNA from STMV. Upon cooling, the chains of domains of poliovirus RNA and STMV RNA condensed and re-formed their original spherical shapes. TMV RNAs initially appeared as single-stranded threads of 0.5-1.0 nm diameter but took on the structure of the multidomain chains upon further incubation at room temperature. These ultimately condensed into short, thick chains of larger domains. Our observations suggest that classical extraction of RNA from icosahedral virions produces little effect on overall conformation. As tertiary structure is lost however, it is evident that secondary structural elements are arranged in a sequential, linear fashion along the polynucleotide chain. At least in the case of poliovirus and STMV, the process of tertiary structure re-formation from the linear chain of secondary structural domains proceeds in the absence of protein. RNA base sequence, therefore, may be sufficient to encode the conformation of the encapsidated RNA even in the absence of coat proteins

Atomic force microscopy analysis of icosahedral virus RNA.

Single-stranded genomic RNAs from four icosahedral viruses (poliovirus, turnip yellow mosaic virus (TYMV), brome mosaic virus (BMV), and satellite tobacco mosaic virus (STMV)) along with the RNA from the helical tobacco mosaic virus (TMV) were extracted using phenol/chloroform. The RNAs were imaged using atomic force microscopy (AFM) under dynamic conditions in which the RNA was observed to unfold. RNAs from the four icosahedral viruses initially exhibited highly condensed, uniform spherical shapes with diameters consistent with those expected from the interiors of their respective capsids. Upon incubation at 26 degrees C, poliovirus RNA gradually transformed into chains of globular domains having the appearance of thick, irregularly segmented fibers. These ultimately unwound further to reveal segmented portions of the fibers connected by single strands of RNA of 0.5-1 nm thickness. Virtually the same transformations were shown by TYMV and BMV RNA, and with heating, the RNA from STMV. Upon cooling, the chains of domains of poliovirus RNA and STMV RNA condensed and re-formed their original spherical shapes. TMV RNAs initially appeared as single-stranded threads of 0.5-1.0 nm diameter but took on the structure of the multidomain chains upon further incubation at room temperature. These ultimately condensed into short, thick chains of larger domains. Our observations suggest that classical extraction of RNA from icosahedral virions produces little effect on overall conformation. As tertiary structure is lost however, it is evident that secondary structural elements are arranged in a sequential, linear fashion along the polynucleotide chain. At least in the case of poliovirus and STMV, the process of tertiary structure re-formation from the linear chain of secondary structural domains proceeds in the absence of protein. RNA base sequence, therefore, may be sufficient to encode the conformation of the encapsidated RNA even in the absence of coat proteins

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

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

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