Different domains of the RNA polymerase of infectious bursal disease virus contribute to virulence.

BACKGROUND: Infectious bursal disease virus (IBDV) is a pathogen of worldwide significance to the poultry industry. IBDV has a bi-segmented double-stranded RNA genome. Segments A and B encode the capsid, ribonucleoprotein and non-structural proteins, or the virus polymerase (RdRp), respectively. Since the late eighties, very virulent (vv) IBDV strains have emerged in Europe inducing up to 60% mortality. Although some progress has been made in understanding the molecular biology of IBDV, the molecular basis for the pathogenicity of vvIBDV is still not fully understood. METHODOLOGY, PRINCIPAL FINDINGS: Strain 88180 belongs to a lineage of pathogenic IBDV phylogenetically related to vvIBDV. By reverse genetics, we rescued a molecular clone (mc88180), as pathogenic as its parent strain. To study the molecular basis for 88180 pathogenicity, we constructed and characterized in vivo reassortant or mosaic recombinant viruses derived from the 88180 and the attenuated Cu-1 IBDV strains. The reassortant virus rescued from segments A of 88180 (A88) and B of Cu-1 (BCU1) was milder than mc88180 showing that segment B is involved in 88180 pathogenicity. Next, the exchange of different regions of BCU1 with their counterparts in B88 in association with A88 did not fully restore a virulence equivalent to mc88180. This demonstrated that several regions if not the whole B88 are essential for the in vivo pathogenicity of 88180. CONCLUSION, SIGNIFICANCE: The present results show that different domains of the RdRp, are essential for the in vivo pathogenicity of IBDV, independently of the replication efficiency of the mosaic viruses.

Molecular analysis of the role of IRES stem-loop V in replicative capacities and translation efficiencies of Coxsackievirus B3 mutants.

Coxsackievirus B3 (CVB3) is a principal viral cause of acute myocarditis in humans and has been implicated in the pathogenesis of dilated cardiomyopathy. The natural genetic determinants of cardiovirulence for CVB3 have not been identified, although using strains engineered in the laboratory, it has been demonstrated elsewhere that, for several wild-type CB3 strains, the primary molecular determinant of cardiovirulence phenotype localizes to the 5' nontranslated region (5'NTR) and capsid. Stable RNA tetraloop motifs are found frequently in biologically active RNAs. These motifs carry out a wide variety of functions in RNA folding, in RNA-RNA and RNA-protein interactions. A great deal of knowledge about the structures and functions of tetraloop motifs has accumulated largely due to intensive theoretical, biochemical, and biophysical studies on one most frequently occurring family of tetraloop sequences, namely, the GNRA sequence, especially the GNAA sequence conserved in all enteroviruses. Here in this study, through construction of CVB3 chimeric mutants, the predicted stem loop (SL) V within the 5'NTR has been identified as important in determining viral cardiovirulence. Replication assays in HeLa cell monolayers revealed that wild-type CVB3 virus and two of the six mutants constructed here grow efficiently, whereas other mutant viruses replicate poorly. Furthermore, the in vitro translation products from these mutants and wild-type CVB3, demonstrated that the two mutants who replicate efficiently, translated at relatively equivalent amount than the wild-type. However, other mutants demonstrated a low efficiency in their production of protein when translated in a Rabbit Reticulocytes Lysats.

Role of GNRA motif mutations within stem-loop V of internal ribosome entry segment in coxsackievirus B3 molecular attenuation.

The lengthy 5' nontranslated region of coxsackievirus B3 (CVB3) forms a highly ordered secondary structure containing an internal ribosome entry segment (IRES), which plays an important role in controlling viral translation and pathogenesis. The stem-loop V (SL-V) of this IRES contains a large lateral bulge loop which encompasses two conserved GNRA motifs. In this study, we analyzed the effects of point mutations within the GNRA motifs of the CVB3 IRES. We characterized in vitro virus production and translation efficiency and we tested in vivo virulence of two CVB3 mutants produced by site-directed mutagenesis. The GNAA1 and GNAA2 RNAs displayed decreased translation initiation efficiency when translated in rabbit reticulocyte lysates. This translation defect was correlated with reduced yields of infectious virus particles in HeLa cells in comparison with the wild type. When inoculated orally into Swiss mice, both mutant viruses were avirulent and caused neither inflammation nor necrosis in hearts. These results highlight the important role of the GNRA motifs within the SL-V of the IRES of CVB3, in directing translation initiation.

The substitution U475 --> C with Sabin3-like mutation within the IRES attenuate Coxsackievirus B3 cardiovirulence.

The Sabin3 mutation in the viral RNA plays an important role in directing attenuation phenotype of Sabin vaccine strain of poliovirus type 1 (PV1). We previously described that Sabin3-like mutation introduced in Coxsackievirus B3 (CVB3) genome led to a defective mutant. However, this mutation do not led to destruction of secondary structure motif C within the stem-loop V of CVB3 RNA because of the presence of one nucleotide difference (C --> U) in the region encompassing the Sabin3 mutation at nucleotides 471 of PV1 and 475 of CVB3 RNA. In order to reproduce the same sequence of PV1 sabin3 vaccine strain, we introduce in this study an additional mutation (U475 --> C) to CVB3 Sabin3-like mutant. Our results demonstrated that Sabin3-like+C mutant displayed a decreased translation initiation defects when translated in cell-free system. This translation initiation defect was correlated with reduced yields of infectious virus particles in HeLa cells in comparison with Sabin3-like mutant and wild-type CVB3 viruses. Inoculation of Swiss mice with mutant viruses resulted in no inflammatory heart disease when compared to heart of mice infected with wild-type. Theses findings indicate that the double mutant could be exploited for the development of a live attenuated vaccine against CVB3.

Rabies virus matrix protein interplay with eIF3, new insights into rabies virus pathogenesis.

Viral proteins are frequently multifunctional to accommodate the high density of information encoded in viral genomes. Matrix (M) protein of negative-stranded RNA viruses such as Rhabdoviridae is one such example. Its primary function is virus assembly/budding but it is also involved in the switch from viral transcription to replication and the concomitant down regulation of host gene expression. In this study we undertook a search for potential rabies virus (RV) M protein's cellular partners. In a yeast two-hybrid screen the eIF3h subunit was identified as an M-interacting cellular factor, and the interaction was validated by co-immunoprecipitation and surface plasmon resonance assays. Upon expression in mammalian cell cultures, RV M protein was localized in early small ribosomal subunit fractions. Further, M protein added in trans inhibited in vitro translation on mRNA encompassing classical (Kozak-like) 5'-UTRs. Interestingly, translation of hepatitis C virus IRES-containing mRNA, which recruits eIF3 via a different noncanonical mechanism, was unaffected. Together, the data suggest that, as a complement to its functions in virus assembly/budding and regulation of viral transcription, RV M protein plays a role in inhibiting translation in virus-infected cells through a protein-protein interaction with the cellular translation machinery.

Evidence that PTB does not stimulate HCV IRES-driven translation.

It is now well established that Hepatitis C Virus (HCV) translation is driven by an Internal Ribosome Entry Site (IRES) resulting in cap-independent translation. Such a mechanism usually occurs with the help of IRES Associated Factors (ITAFs). Moreover, an important translational feature is likely conserved from the model of classical mRNA circularisation (5'-3' cross-talk), involving the HCV RNA highly structured 3' extremity called the 3'X region. This could bind several cellular factors and modulate the translation efficacy, at least in Rabbit Reticulocyte Lysate (RRL). In particular, polypyrimidine-binding proteins have been proposed to be potential HCV ITAFs, such as Polypyrimidine Tract Binding protein (PTB). However, contradictions still exist as to the role of PTB: its ability to bind both the HCV IRES and the 3'X region leads to the hypothesis that it could positively modulate IRES-driven translation in the presence of the X structure. Results of translational and PTB-binding studies of X mutant sequences led us to discredit PTB as protagonist of 3'X region stimulation on HCV IRES-driven translation. Moreover, competition assays of X RNA in trans on IRES-driven translation demonstrate the involvement of at least two stimulating factors and led to the conclusion that this mechanism is more complex than initially thought. Although we did not identify these factors, it is no longer doubtful that there is effectively a stimulating functional interaction between the HCV IRES and the 3'X region in RRL.

Effects of the Sabin-like mutations in domain V of the internal ribosome entry segment on translational efficiency of the Coxsackievirus B3.

The domain V within the internal ribosome entry segment (IRES) of poliovirus (PV) is expected to be important in its own neurovirulence because it contains an attenuating mutation in each of the Sabin vaccine strains. In this study, we try to find out if the results observed in the case of Sabin vaccine strains of PV can be extrapolated to another virus belonging to the same genus of enteroviruses but with a different tropism. To test this hypothesis, we used the coxsackievirus B3 (CVB3), known to be the most common causal agent of viral myocarditis. The introduction of the three PV Sabin-like mutations in the equivalent positions (nucleotides 484, 485, and 473) to the domain V of the CVB3 IRES results in significant reduced viral titer of the Sabin3-like mutant (Sab3-like) but not on those of Sab1- and Sab2-like mutants. This low titer was correlated with poor translation efficiency in vitro when all mutants were translated in rabbit reticulocyte lysates. However, elucidation by biochemical probing of the secondary structure of the entire domain V of the IRES of Sabin-like mutants reveals no distinct profiles in comparison with the wild-type counterpart. Prediction of secondary structure by MFOLD program indicates a structural perturbation of the stem containing the Sab3-like mutation, suggesting that specific protein-viral RNA interactions are disrupted, preventing efficient viral translation.

Cumulative mutations in the genome of Echovirus 6 during establishment of a chronic infection in precursors of glial cells.

Although Enteroviruses are mainly described as responsible for acute diseases, their role in severe chronic pathology has been also established. Echovirus 6-like sequences have been detected by PCR analysis in central nervous system specimens from patients presenting with Amyotrophic Lateral Sclerosis. These findings suggested a persistent infection with viruses that underwent, genetic changes precluding viral particle release. To support this hypothesis, we developed a model system of Echovirus 6 chronic infection in precursors of glial cells. The nucleotide sequences of the 5'non-translated region (5'NTR), 2A and 3C regions of the virus developing persistent genome were analysed during establishment of the chronic phenotype. This study revealed that at day 160 of chronic infection, several mutations were observed: one mutation at nucleotide 108 upstream the domain II of the internal ribosome entry site (IRES) structure, one mutation at nucleotide 30 in the cloverleaf, and two mutations in the 2A region (translated in His48 to Tyr, and Ile 123 to Met). No mutations were detected in the 3C region. The impact of these mutations on viral replication have been analysed in a rabbit reticulocyte lysate (RRL) translation assay supplemented with HeLa cell lysate, and by plaque assay. Viruses with these mutations displayed a phenotype with a significant reduction of replication, while in vitro translation was not affected by the nucleotide 108 mutation. This model allowed the description of molecular changes observed in the genome of Echovirus 6 during the establishment of a chronic infection phenotype, and may be helpful for the understanding of the mechanisms leading Enteroviruses to develop chronic infections in man.

Effects of vaccine strain mutations in domain V of the internal ribosome entry segment compared in the wild type poliovirus type 1 context.

Initiation of poliovirus (PV) protein synthesis is governed by an internal ribosome entry segment structured into several domains including domain V, which is accepted to be important in PV neurovirulence because it harbors an attenuating mutation in each of the vaccine strains developed by A. Sabin. To better understand how these single point mutations exert their effects, we placed each of them into the same genomic context, that of PV type 1. Only the mutation equivalent to the Sabin type 3 strain mutation resulted in significantly reduced viral growth both in HeLa and neuroblastoma cells. This correlated with poor translation efficiency in vitro and could be explained by a structural perturbation of the domain V of the internal ribosome entry segment, as evidenced by RNA melting experiments. We demonstrated that reduced cell death observed during infection by this mutant is due to the absence of inhibition of host cell translation. We confirmed that this shut-off is correlated principally with cleavage of eIF4GII and not eIF4GI and that this cleavage is significantly impaired in the case of the defective mutant. These data support the previously reported conclusion that the 2A protease has markedly different affinities for the two eIF4G isoforms.

The role of mRNA 5'-noncoding and 3'-end sequences on 40S ribosomal subunit recruitment, and how RNA viruses successfully compete with cellular mRNAs to ensure their own protein synthesis.

Since the elaboration of the scanning model to explain eukaryotic translation initiation, alternative hypotheses have gained support. Cap and 5' end-independent recruitment of the 40S ribosomal subunit conferred by the presence of an internal ribosome entry segment (IRES) in the 5'UTR of the mRNA is widely accepted, and has been formally and definitively proven for a picornavirus. However, the mechanism of IRES function remains essentially a black box. Using the complex viral IRESes as model systems, approaches taken to shed light on the mystery include systematic comparisons and molecular genetic analyses. The hypothesis that actively translated mRNAs are circular, rather than linear, molecules is based on rather indirect evidence. This model has invoked a revision of the image of 40S ribosomal subunit recruitment, to include recycling from the mRNA 3'- to the 5'-end in addition to true de novo 5'-end directed entry. Biochemical and genetic studies are used to define the network of interactions necessary for efficient ribosome recruitment. This has lent weight to the concept of mRNA 5'-3' cross-talk and clarified the mechanics of how this enhances translation efficiency. These refinements and revisions to the model of translation initiation form the core of this review, with current knowledge being considered from the perspective on how host-cell translation could yield to selective viral translation via the phenomenon of translational shut-off.

Comparison of the capacity of different viral internal ribosome entry segments to direct translation initiation in poly(A)-dependent reticulocyte lysates.

Polyadenylation stimulates translation of capped eukaryotic mRNAs and those carrying picornaviral internal ribosome entry segments (IRESes) in vivo. Rabbit reticulocyte lysates (RRL) reproduce poly(A)-mediated translation stimulation in vitro after partial depletion of ribosomes and ribosome-associated factors. Here, we have evaluated the effects of varying different parameters (extent of extract depletion, cleavage of eIF4G, concentrations of KCl, MgCl(2) and programming mRNA) on IRES-driven translation efficiency and poly(A)-dependency in ribosome-depleted RRL. For comparison, the study included a standard capped, polyadenylated mRNA. Dramatic differences were observed in the abilities of the different IRESes to direct translation in ribosome-depleted extracts. While the hepatitis A virus IRES was incapable of driving translation in physiological conditions in depleted RRL, mRNAs carrying the foot-and-mouth disease virus and hepatitis C virus IRESes were translated significantly better than a standard cellular mRNA in the same conditions. Indeed, the capacities of these IRESes to direct translation in ribosome-depleted RRL were similar to those reported previously in certain cell lines. Both the abilities of the IRESes to drive translation and their individual salt optima in ribosome-depleted extracts suggest that these elements have dramatically different affinities for some component(s) of the canonical translation machinery. Finally, using poliovirus as an example, we show that the ribosome-depleted system is well suited to the study of the translational capacity of naturally occurring IRES variants.

Poliovirus internal ribosome entry segment structure alterations that specifically affect function in neuronal cells: molecular genetic analysis.

Translation of poliovirus RNA is driven by an internal ribosome entry segment (IRES) present in the 5' noncoding region of the genomic RNA. This IRES is structured into several domains, including domain V, which contains a large lateral bulge-loop whose predicted secondary structure is unclear. The primary sequence of this bulge-loop is strongly conserved within enteroviruses and rhinoviruses: it encompasses two GNAA motifs which could participate in intrabulge base pairing or (in one case) could be presented as a GNRA tetraloop. We have begun to address the question of the significance of the sequence conservation observed among enterovirus reference strains and field isolates by using a comprehensive site-directed mutagenesis program targeted to these two GNAA motifs. Mutants were analyzed functionally in terms of (i) viability and growth kinetics in both HeLa and neuronal cell lines, (ii) structural analyses by biochemical probing of the RNA, and (iii) translation initiation efficiencies in vitro in rabbit reticulocyte lysates supplemented with HeLa or neuronal cell extracts. Phenotypic analyses showed that only viruses with both GNAA motifs destroyed were significantly affected in their growth capacities, which correlated with in vitro translation defects. The phenotypic defects were strongly exacerbated in neuronal cells, where a temperature-sensitive phenotype could be revealed at between 37 and 39.5 degrees C. Biochemical probing of mutated domain V, compared to the wild type, demonstrated that such mutations lead to significant structural perturbations. Interestingly, revertant viruses possessed compensatory mutations which were distant from the primary mutations in terms of sequence and secondary structure, suggesting that intradomain tertiary interactions could exist within domain V of the IRES.

Free poly(A) stimulates capped mRNA translation in vitro through the eIF4G-poly(A)-binding protein interaction.

The 5' cap and 3' poly(A) tail of classical eukaryotic mRNAs functionally communicate to synergistically enhance translation initiation. Synergy has been proposed to result in part from facilitated ribosome recapture on circularized mRNAs. Here, we demonstrate that this is not the case. In poly(A)-dependent, ribosome-depleted rabbit reticulocyte lysates, the addition of exogenous poly(A) chains of physiological length dramatically stimulated translation of a capped, nonpolyadenylated mRNA. When the poly(A):RNA ratio approached 1, exogenous poly(A) stimulated translation to the same extent as the presence of a poly(A) tail at the mRNA 3' end. In addition, exogenous poly(A) significantly improved translation of capped mRNAs carrying short poly(A(50)) tails. Trans stimulation of translation by poly(A) required the eIF4G-poly(A)-binding protein interaction and resulted in increased affinity of eIF4E for the mRNA cap, exactly as we recently described for cap-poly(A) synergy. These results formally demonstrate that mRNA circularization per se is not the cause of cap-poly(A) synergy at least in vitro.

Quasispecies heterogeneity and constraints on the evolution of the 5' noncoding region of hepatitis C virus (HCV): relationship with HCV resistance to interferon-alpha therapy.

Hepatitis C virus (HCV) polyprotein translation depends on direct internal entry of the 40S ribosomal subunit mediated by an internal ribosome entry segment (IRES) located in the 5' noncoding (5'NC) region of the viral genome. HCV is genetically heterogeneous and is characterized by the existence of a quasispecies distribution of the virus population within a single infected individual. Cloning and sequencing strategies were used to characterize 5'NC quasispecies genetically. Similar to coding regions, the HCV 5'NC region was distributed as a quasispecies, but it appeared to be subjected to stronger conservatory constraints than other regions of the HCV genome, probably due to the need for structural (and functional) conservation of the IRES. Indeed, most of the quasispecies substitutions were in unpaired regions of the IRES or clustered such that base-pairing was maintained, whereas only 21% were expected to result in a loss of base-pairing. Quasispecies-related structural changes could be predicted in the core cruciform of IRES domain III composed of the RNA helices which extend from the four-way junction JIIIabc, mostly in minor variants, but sometimes in major ones. The results presented here suggest the simultaneous presence in infected patients of a mixture of genetically distinct but closely related IRES sequences that may have different structures. No significant genetic changes of 5'NC quasispecies were observed after interferon-alpha treatment, except in patients with mixed genotype infection who cleared one of the infecting strains during therapy, suggesting that the quasispecies distribution of IRES sequences does not play a role in HCV resistance to interferon-alpha therapy. In contrast, the overall quasispecies distribution of HCV genomes (including IRES sequences) might participate in regulation of hepatic and extrahepatic HCV replication.