Genetic mutation of Cep76 results in male infertility due to abnormal sperm tail composition.

The transition zone is a specialised gate at the base of cilia/flagella, which separates the ciliary compartment from the cytoplasm and strictly regulates protein entry. We identified a potential new regulator of the male germ cell transition zone, CEP76. We demonstrated that CEP76 was involved in the selective entry and incorporation of key proteins required for sperm function and fertility into the ciliary compartment and ultimately the sperm tail. In the mutant, sperm tails were shorter and immotile as a consequence of deficits in essential sperm motility proteins including DNAH2 and AKAP4, which accumulated at the sperm neck in the mutant. Severe annulus, fibrous sheath, and outer dense fibre abnormalities were also detected in sperm lacking CEP76. Finally, we identified that CEP76 dictates annulus positioning and structure. This study suggests CEP76 as a male germ cell transition zone protein and adds further evidence to the hypothesis that the spermatid transition zone and annulus are part of the same functional structure.

Enterovirus-Cardiomyocyte Interactions: Impact of Terminally Deleted Genomic RNAs on Viral and Host Functions.

Group B enteroviruses, including coxsackievirus B3 (CVB3), can persistently infect cardiac tissue and cause dilated cardiomyopathy. Persistence is linked to 5' terminal deletions of viral genomic RNAs that have been detected together with minor populations of full-length genomes in human infections. In this study, we explored the functions and interactions of the different viral RNA forms found in persistently infected patients and their putative role(s) in pathogenesis. Since enterovirus cardiac pathogenesis is linked to the viral proteinase 2A, we investigated the effect of different terminal genomic RNA deletions on 2A activity. We discovered that 5' terminal deletions in CVB3 genomic RNAs decreased the levels of 2A proteinase activity but could not abrogate it. Using newly generated viral reporters encoding nano-luciferase, we found that 5' terminal deletions resulted in decreased levels of viral protein and RNA synthesis in singly transfected cardiomyocyte cultures. Unexpectedly, when full-length and terminally deleted forms were cotransfected into cardiomyocytes, a cooperative interaction was observed, leading to increased viral RNA and protein production. However, when viral infections were carried out in cells harboring 5' terminally deleted CVB3 RNAs, a decrease in infectious particle production was observed. Our results provide a possible explanation for the necessity of full-length viral genomes during persistent infection, as they would stimulate efficient viral replication compared to that of the deleted genomes alone. To avoid high levels of viral particle production that would trigger cellular immune activation and host cell death, the terminally deleted RNA forms act to limit the production of viral particles, possibly as trans-dominant inhibitors. IMPORTANCE Enteroviruses like coxsackievirus B3 are able to initiate acute infections of cardiac tissue and, in some cases, to establish a long-term persistent infection that can lead to serious disease sequelae, including dilated cardiomyopathy. Previous studies have demonstrated the presence of 5' terminally deleted forms of enterovirus RNAs in heart tissues derived from patients with dilated cardiomyopathy. These deleted RNAs are found in association with very low levels of full-length enterovirus genomic RNAs, an interaction that may facilitate continued persistence while limiting virus particle production. Even in the absence of detectable infectious virus particle production, these deleted viral RNA forms express viral proteinases at levels capable of causing viral pathology. Our studies provide mechanistic insights into how full-length and deleted forms of enterovirus RNA cooperate to stimulate viral protein and RNA synthesis without stimulating infectious viral particle production. They also highlight the importance of targeting enteroviral proteinases to inhibit viral replication while at the same time limiting the long-term pathologies they trigger.

Structure of the PCBP2/stem-loop IV complex underlying translation initiation mediated by the poliovirus type I IRES.

The poliovirus type I IRES is able to recruit ribosomal machinery only in the presence of host factor PCBP2 that binds to stem-loop IV of the IRES. When PCBP2 is cleaved in its linker region by viral proteinase 3CD, translation initiation ceases allowing the next stage of replication to commence. Here, we investigate the interaction of PCBP2 with the apical region of stem-loop IV (SLIVm) of poliovirus RNA in its full-length and truncated form. CryoEM structure reconstruction of the full-length PCBP2 in complex with SLIVm solved to 6.1 Å resolution reveals a compact globular complex of PCBP2 interacting with the cruciform RNA via KH domains and featuring a prominent GNRA tetraloop. SEC-SAXS, SHAPE and hydroxyl-radical cleavage establish that PCBP2 stabilizes the SLIVm structure, but upon cleavage in the linker domain the complex becomes more flexible and base accessible. Limited proteolysis and REMSA demonstrate the accessibility of the linker region in the PCBP2/SLIVm complex and consequent loss of affinity of PCBP2 for the SLIVm upon cleavage. Together this study sheds light on the structural features of the PCBP2/SLIV complex vital for ribosomal docking, and the way in which this key functional interaction is regulated following translation of the poliovirus genome.

Exploitation of nuclear functions by human rhinovirus, a cytoplasmic RNA virus.

Protein production, genomic RNA replication, and virion assembly during infection by picornaviruses like human rhinovirus and poliovirus take place in the cytoplasm of infected human cells, making them the quintessential cytoplasmic pathogens. However, a growing body of evidence suggests that picornavirus replication is promoted by a number of host proteins localized normally within the host cell nucleus. To systematically identify such nuclear proteins, we focused on those that appear to re-equilibrate from the nucleus to the cytoplasm during infection of HeLa cells with human rhinovirus via quantitative protein mass spectrometry. Our analysis revealed a highly selective re-equilibration of proteins with known mRNA splicing and transport-related functions over nuclear proteins of all other functional classes. The multifunctional splicing factor proline and glutamine rich (SFPQ) was identified as one such protein. We found that SFPQ is targeted for proteolysis within the nucleus by viral proteinase 3CD/3C, and a fragment of SFPQ was shown to migrate to the cytoplasm at mid-to-late times of infection. Cells knocked down for SFPQ expression showed significantly reduced rhinovirus titers, viral protein production, and viral RNA accumulation, consistent with SFPQ being a pro-viral factor. The SFPQ fragment that moved into the cytoplasm was able to bind rhinovirus RNA either directly or indirectly. We propose that the truncated form of SFPQ promotes viral RNA stability or replication, or virion morphogenesis. More broadly, our findings reveal dramatic changes in protein compartmentalization during human rhinovirus infection, allowing the virus to systematically hijack the functions of proteins not normally found at its cytoplasmic site of replication.

Functional Consequences of RNA 5'-Terminal Deletions on Coxsackievirus B3 RNA Replication and Ribonucleoprotein Complex Formation.

Group B coxsackieviruses are responsible for chronic cardiac infections. However, the molecular mechanisms by which the virus can persist in the human heart long after the signs of acute myocarditis have abated are still not completely understood. Recently, coxsackievirus B3 strains with 5'-terminal deletions in genomic RNAs were isolated from a patient suffering from idiopathic dilated cardiomyopathy, suggesting that such mutant viruses may be the forms responsible for persistent infection. These deletions lacked portions of 5' stem-loop I, which is an RNA secondary structure required for viral RNA replication. In this study, we assessed the consequences of the genomic deletions observed in vivo for coxsackievirus B3 biology. Using cell extracts from HeLa cells, as well as transfection of luciferase replicons in two types of cardiomyocytes, we demonstrated that coxsackievirus RNAs harboring 5' deletions ranging from 7 to 49 nucleotides in length can be translated nearly as efficiently as those of wild-type virus. However, these 5' deletions greatly reduced the synthesis of viral RNA in vitro, which was detected only for the 7- and 21-nucleotide deletions. Since 5' stem-loop I RNA forms a ribonucleoprotein complex with cellular and viral proteins involved in viral RNA replication, we investigated the binding of the host cell protein PCBP2, as well as viral protein 3CDpro, to deleted positive-strand RNAs corresponding to the 5' end. We found that binding of these proteins was conserved but that ribonucleoprotein complex formation required higher PCBP2 and 3CDpro concentrations, depending on the size of the deletion. Overall, this study confirmed the characteristics of persistent CVB3 infection observed in heart tissues and provided a possible explanation for the low level of RNA replication observed for the 5'-deleted viral genomes-a less stable ribonucleoprotein complex formed with proteins involved in viral RNA replication.IMPORTANCE Dilated cardiomyopathy is the most common indication for heart transplantation worldwide, and coxsackie B viruses are detected in about one-third of idiopathic dilated cardiomyopathies. Terminal deletions at the 5' end of the viral genome involving an RNA secondary structure required for RNA replication have been recently reported as a possible mechanism of virus persistence in the human heart. These mutations are likely to disrupt the correct folding of an RNA secondary structure required for viral RNA replication. In this report, we demonstrate that transfected RNAs harboring 5'-terminal sequence deletions are able to direct the synthesis of viral proteins, but not genomic RNAs, in human and murine cardiomyocytes. Moreover, we show that the binding of cellular and viral replication factors to viral RNA is conserved despite genomic deletions but that the impaired RNA synthesis associated with terminally deleted viruses could be due to destabilization of the ribonucleoprotein complexes formed.

Divergent Requirement for a DNA Repair Enzyme during Enterovirus Infections.

UNLABELLED: Viruses of the Enterovirus genus of picornaviruses, including poliovirus, coxsackievirus B3 (CVB3), and human rhinovirus, commandeer the functions of host cell proteins to aid in the replication of their small viral genomic RNAs during infection. One of these host proteins is a cellular DNA repair enzyme known as 5' tyrosyl-DNA phosphodiesterase 2 (TDP2). TDP2 was previously demonstrated to mediate the cleavage of a unique covalent linkage between a viral protein (VPg) and the 5' end of picornavirus RNAs. Although VPg is absent from actively translating poliovirus mRNAs, the removal of VPg is not required for the in vitro translation and replication of the RNA. However, TDP2 appears to be excluded from replication and encapsidation sites during peak times of poliovirus infection of HeLa cells, suggesting a role for TDP2 during the viral replication cycle. Using a mouse embryonic fibroblast cell line lacking TDP2, we found that TDP2 is differentially required among enteroviruses. Our single-cycle viral growth analysis shows that CVB3 replication has a greater dependency on TDP2 than does poliovirus or human rhinovirus replication. During infection, CVB3 protein accumulation is undetectable (by Western blot analysis) in the absence of TDP2, whereas poliovirus protein accumulation is reduced but still detectable. Using an infectious CVB3 RNA with a reporter, CVB3 RNA could still be replicated in the absence of TDP2 following transfection, albeit at reduced levels. Overall, these results indicate that TDP2 potentiates viral replication during enterovirus infections of cultured cells, making TDP2 a potential target for antiviral development for picornavirus infections. IMPORTANCE: Picornaviruses are one of the most prevalent groups of viruses that infect humans and livestock worldwide. These viruses include the human pathogens belonging to the Enterovirus genus, such as poliovirus, coxsackievirus B3 (CVB3), and human rhinovirus. Diseases caused by enteroviruses pose a major problem for public health and have significant economic impact. Poliovirus can cause paralytic poliomyelitis. CVB3 can cause hand, foot, and mouth disease and myocarditis. Human rhinovirus is the causative agent of the common cold, which has a severe economic impact due to lost productivity and severe health consequences in individuals with respiratory dysfunction, such as asthma. By gaining a better understanding of the enterovirus replication cycle, antiviral drugs against enteroviruses may be developed. Here, we report that the absence of the cellular enzyme TDP2 can significantly decrease viral yields of poliovirus, CVB3, and human rhinovirus, making TDP2 a potential target for an antiviral against enterovirus infections.

Altered interactions between stem-loop IV within the 5' noncoding region of coxsackievirus RNA and poly(rC) binding protein 2: effects on IRES-mediated translation and viral infectivity.

Coxsackievirus B3 (CVB3) is a causative agent of viral myocarditis, meningitis, pancreatitis, and encephalitis. Much of what is known about the coxsackievirus intracellular replication cycle is based on the information already known from a well-studied and closely related virus, poliovirus. Like that of poliovirus, the 5' noncoding region (5' NCR) of CVB3 genomic RNA contains secondary structures that function in both viral RNA replication and cap-independent translation initiation. For poliovirus IRES-mediated translation, the interaction of the cellular protein PCBP2 with a major secondary structure element (stem-loop IV) is required for gene expression. Previously, the complete secondary structure of the coxsackievirus 5' NCR was determined by chemical structure probing and overall, many of the RNA secondary structures bear significant similarity to those of poliovirus; however, the functions of the coxsackievirus IRES stem-loop structures have not been determined. Here we report that a CVB3 RNA secondary structure, stem-loop IV, folds similarly to poliovirus stem-loop IV and like its enterovirus counterpart, coxsackievirus stem-loop IV interacts with PCBP2. We used RNase foot-printing to identify RNA sequences protected following PCBP2 binding to coxsackievirus stem-loop IV. When nucleotide substitutions were separately engineered at two sites in coxsackievirus stem-loop IV to reduce PCBP2 binding, inhibition of IRES-mediated translation was observed. Both of these nucleotide substitutions were engineered into full-length CVB3 RNA and upon transfection into HeLa cells, the specific infectivities of both constructs were reduced and the recovered viruses displayed small-plaque phenotypes and slower growth kinetics compared to wild type virus.

The linker domain of poly(rC) binding protein 2 is a major determinant in poliovirus cap-independent translation.

Poliovirus, a member of the enterovirus genus in the family Picornaviridae, is the causative agent of poliomyelitis. Translation of the viral genome is mediated through an internal ribosomal entry site (IRES) encoded within the 5' noncoding region (5' NCR). IRES elements are highly structured RNA sequences that facilitate the recruitment of ribosomes for translation. Previous studies have shown that binding of a cellular protein, poly(rC) binding protein 2 (PCBP2), to a major stem-loop structure in the genomic 5' NCR is necessary for the translation of picornaviruses containing type I IRES elements, including poliovirus, coxsackievirus, and human rhinovirus. PCBP1, an isoform that shares approximately 90% amino acid identity to PCBP2, cannot efficiently stimulate poliovirus IRES-mediated translation, most likely due to its reduced binding affinity to stem-loop IV within the poliovirus IRES. The primary differences between PCBP1 and PCBP2 are found in the so-called linker domain between the second and third K-homology (KH) domains of these proteins. We hypothesize that the linker region of PCBP2 augments binding to poliovirus stem-loop IV RNA. To test this hypothesis, we generated six PCBP1/PCBP2 chimeric proteins. The recombinant PCBP1/PCBP2 chimeric proteins were able to interact with poliovirus stem-loop I RNA and participate in protein-protein interactions. We demonstrated that the PCBP1/PCBP2 chimeric proteins with the PCBP2 linker, but not with the PCBP1 linker, were able to interact with poliovirus stem-loop IV RNA, and could subsequently stimulate poliovirus IRES-mediated translation. In addition, using a monoclonal anti-PCBP2 antibody (directed against the PCBP2 linker domain) in mobility shift assays, we showed that the PCBP2 linker domain modulates binding to poliovirus stem-loop IV RNA via a mechanism that is not inhibited by the antibody.

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-DeltaKH3, 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.

An authentic 3' noncoding region is necessary for efficient poliovirus replication.

Picornavirus RNA replication involves the specific synthesis of negative-strand intermediates followed by an accumulation of positive-strand viral RNA in the presence of a multitude of cellular mRNAs. Previously, in an effort to identify cis-acting elements required for initiation of negative-strand RNA synthesis, we deleted the entire 3' noncoding regions from human rhinovirus and poliovirus genomic RNAs. These deletion mutation transcripts displayed a severe delay in RNA accumulation following transfection of HeLa cells. Interestingly, in subsequent infection of HeLa cells, the deletion-mutant poliovirus displayed only a moderate deficiency in RNA synthesis. These data suggested that the delay in the production of cytopathic effects after transfection may have been due to an RNA replication defect overcome by the accumulation of a compensatory mutation(s) generated during initial rounds of RNA synthesis. In this study, we have sequenced the entire genome of the deletion-mutant virus and found only two nucleotide changes from the parental clone. Transfection analysis of these sequence variants revealed that the sequence changes did not provide compensatory functions for the 3' noncoding region deletion mutation replication defect. Further examination of the deletion mutant phenotype revealed that the severe replication defect following RNA transfection is due, in part, to nonviral terminal sequences present in the in vitro-derived deletion mutation transcripts. Our data suggest that poliovirus RNA harboring a complete 3' noncoding region deletion mutation is infectious (not merely quasi-infectious).

Functional interaction of heterogeneous nuclear ribonucleoprotein C with poliovirus RNA synthesis initiation complexes.

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, 3D(pol), as well as to P2 and P3, precursors to the nonstructural proteins. Truncation of the auxiliary domain in hnRNP C1 (C1DeltaC) diminished these protein-protein interactions. When GST/hnRNP C1DeltaC 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.

Functional conservation of the hydrophobic domain of polypeptide 3AB between human rhinovirus and poliovirus.

In this study we exchanged portions of the poliovirus type 1 (PV1) hydrophobic domain within the membrane-associated polypeptide 3AB for the analogous sequences from human rhinovirus 14 (HRV14). The sequence exchanges were based upon a previous report in which the 22 amino acid hydrophobic region was subdivided into two domains, I and II, the latter of which was shown to be required for membrane association (J. Biol. Chem. 271 (1996), 26810). Using these divisions, the HRV14 sequences were cloned into the complete poliovirus type 1 cDNA sequence. RNAs transcribed from these cDNAs were transfected into HeLa cell monolayers and used in HeLa cell-free translation/replication assays. The data indicated that 3AB sequences from PV1 and HRV14 are interchangeable; however, the substitutions cause a range of significant RNA replication defects, and in some cases, protein processing defects. Following transfection of RNAs encoding the domain substitutions into HeLa cell monolayers, virus isolates were harvested, and the corresponding viral RNAs were sequenced. The sequence data revealed that for the carboxy-terminal domain substitutions (domain II), multiple nucleotide changes were identified in the first, second, and third positions of different codons. In addition, the data indicated that for one of the PV1/HRV14 chimeras to replicate, compensatory mutations within poliovirus protein 2B may be required.