Inherent instability of poliovirus genomes containing two internal ribosome entry site (IRES) elements supports a role for the IRES in encapsidation

Previous studies have described poliovirus genomes in which the internal ribosome entry (IRES) for encephalomyocarditis virus (EMCV) is positioned between the P1 and P2-P3 open reading frames of the poliovirus genome. Although these dicistronic poliovirus genomes were replication competent, most exhibited evidence of genetic instability, and the EMCV IRES was deleted upon serial passage. One possible reason for instability of the genome is that the dicistronic genome was at least 108% larger than the wild-type poliovirus genome, which could reduce the efficiency of encapsidation. To address this possibility, we have constructed dicistronic poliovirus replicons by substituting the EMCV IRES and the gene encoding luciferase in place of the poliovirus P1 region; the resulting dicistronic replicons are smaller than the wild-type poliovirus genome. One dicistronic genome was constructed in which the poliovirus 5' nontranslated region was fused to the gene encoding luciferase, followed by the complete EMCV IRES fused to the P2-P3 region of the poliovirus genome (PV-Luc-EMCV). A second dicistronic genome, EMCV-Luc-PV, was constructed with the first 108 nucleotides of the poliovirus genome fused to the EMCV IRES, followed by the gene encoding luciferase and the poliovirus IRES fused to the remaining P2-P3 region of the poliovirus genome. Both dicistronic replicons expressed abundant luciferase following transfection of in vitro-transcribed RNA into HeLa cells at 30, 33, or 37 degrees C. The luciferase activity detected from PV-Luc-EMCV increased rapidly during the first 4 h following transfection and then plateaued, peaking after approximately 24 h. In contrast, the luciferase activity detected from EMCV-Luc-PV increased for approximately 12 h following transfection; by 24 h posttransfection, the overall levels of luciferase activity were similar to that of PV-Luc-EMCV. To analyze encapsidation of the dicistronic replicons, we used a system in which the capsid protein (P1) is provided in trans from a recombinant vaccinia virus (VV-P1). The PV-Luc-EMCV replicon was unstable upon serial passage in the presence of VV-P1, with deletions of the EMCV IRES region detected even during the initial transfection at 37 degrees C. Following serial passage in the presence of VV-P1 at 33 or 30 degrees C, we detected deleted genomes in which the luciferase gene was fused with the P2-P3 genes of the poliovirus genome so as to maintain the translational reading frame. In contrast, the EMCV-Luc-PV replicon was genetically stable during passage with VV-P1 at 33 or 30 degrees C. The encapsidation of EMCV-Luc-PV was compared to that of monocistronic replicons encoding luciferase with either a poliovirus or EMCV IRES. Analysis of the encapsidated replicons after four serial passages with VV-P1 revealed that the dicistronic replicon was encapsidated more efficiently than the monocistronic replicon with the EMCV IRES but less efficiently than the monicistronic replicon with the poliovirus IRES. The results of this study suggest a genetic predisposition for picornavirus genomes to contain a single IRES region and are discussed with respect to a role of the IRES in encapsidation.     

Genetic analysis of a poliovirus/hepatitis C virus (HCV) chimera: interaction between the poliovirus cloverleaf and a sequence in the HCV 5' nontranslated region results in a replication phenotype

Internal ribosomal entry sites (IRESs) can function in foreign viral genomes or in artificial dicistronic mRNAs. We describe an interaction between the wild-type hepatitis C virus (HCV)-specific sequence and the poliovirus (PV) 5'-terminal cloverleaf in a PV/HCV chimeric virus (containing the HCV IRES), resulting in a replication phenotype. Either a point mutation at nucleotide (nt) 29 or a deletion up to nt 40 in the HCV 5' nontranslated region relieved the replication block, yielding PV/HCV variants replicating to high titers. Fortuitous yet crippling interactions between an IRES and surrounding heterologous RNA must be considered when IRES-based dicistronic expression vectors are being constructed.     

Genetic analysis of a poliovirus/hepatitis C virus chimera: new structure for domain II of the internal ribosomal entry site of hepatitis C virus

Internal ribosomal entry sites (IRESs) of certain plus-strand RNA viruses direct cap-independent initiation of protein synthesis both in vitro and in vivo, as can be shown with artificial dicistronic mRNAs or with chimeric viral genomes in which IRES elements were exchanged from one virus to another. Whereas IRESs of picornaviruses can be readily analyzed in the context of their cognate genome by genetics, the IRES of hepatitis C virus (HCV), a Hepacivirus belonging to Flaviviridae, cannot as yet be subjected to such analyses because of difficulties in propagating HCV in tissue culture or in experimental animals. This enigma has been overcome by constructing a poliovirus (PV) whose translation is controled by the HCV IRES. Within the PV/HCV chimera, the HCV IRES has been subjected to systematic 5' deletion analyses to yield a virus (P/H710-d40) whose replication kinetics match that of the parental poliovirus type 1 (Mahoney). Genetic analyses of the HCV IRES in P/H710-d40 have confirmed that the 5' border maps to domain II, thereby supporting the validity of the experimental approach applied here. Additional genetic experiments have provided evidence for a novel structural region within domain II. Arguments that the phenotypes observed with the mutant chimera relate solely to impaired genome replication rather than deficiencies in translation have been dispelled by constructing novel dicistronic poliovirus replicons with the gene order [PV]cloverleaf-[HCV]IRES-Deltacore-R-Luc-[PV]IRES-F-Luc-P2,3-3'NTR, which have allowed the measurement of HCV IRES-dependent translation independently from the replication of the replicon RNA.     

A segment of the 5'' nontranslated region of encephalomyocarditis virus RNA directs internal entry of ribosomes during in vitro translation.

Picornavirus RNAs are uncapped messengers and have unusually long 5' nontranslated regions (5'NTRs) which contain many noninitiating AUG triplets. The translational efficiency of different picornavirus RNAs varies between different cell-free extracts and even in the same extract, such as micrococcal nuclease-treated rabbit reticulocyte lysates. The effect of the poliovirus 5'NTR on in vitro translation was compared with that of the 5'NTR of encephalomyocarditis virus by the use of synthetic mRNAs, micrococcal nuclease-treated HeLa cell extracts, and rabbit reticulocyte lysates. Artificial mono- and dicistronic mRNAs synthesized with T7 RNA polymerase were used to investigate whether the 5'NTR of encephalomyocarditis virus RNA contains a potential internal ribosomal entry site. The sequence between nucleotides 260 and 484 in the 5'NTR of encephalomyocarditis RNA was found to play a critical role in the efficient translation in both mono- and dicistronic mRNAs. Our data suggest that an internal ribosomal entry site resides in this region.     

Vaccinia Virus Protein Synthesis Has a Low Requirement for the Intact Translation Initiation Factor eIF4F, the Cap-Binding Complex, within Infected Cells

The role of the cap-binding complex, eIF4F, in the translation of vaccinia virus mRNAs has been analyzed within infected cells. Plasmid DNAs, which express dicistronic mRNAs containing a picornavirus internal ribosome entry site, produced within vaccinia virus-infected cells both β-glucuronidase and a cell surface-targeted single-chain antibody (sFv). Cells expressing sFv were selected from nonexpressing cells, enabling analysis of protein synthesis specifically within the transfected cells. Coexpression of poliovirus 2A or foot-and-mouth disease virus Lb proteases, which cleaved translation initiation factor eIF4G, greatly inhibited cap-dependent protein (β-glucuronidase) synthesis. Under these conditions, internal ribosome entry site-directed expression of sFv continued and cell selection was maintained. Furthermore, vaccinia virus protein synthesis persisted in the selected cells containing cleaved eIF4G. Thus, late vaccinia virus protein synthesis has a low requirement for the intact cap-binding complex eIF4F. This may be attributed to the short unstructured 5′ noncoding regions of the vaccinia virus mRNAs, possibly aided by the presence of poly(A) at both 5′ and 3′ termini.     

Sequences within the poliovirus internal ribosome entry segment control viral RNA synthesis.

The 5' untranslated region of poliovirus RNA has been reported to possess two functional elements: (i) the 5' proximal 88 nucleotides form a cloverleaf structure implicated in positive-strand RNA synthesis during viral replication, and (ii) nucleotides 134 to at least 556 function as a highly structured internal ribosome entry segment (IRES) during cap-independent, internal initiation of translation. We show here that the IRES itself is bifunctional and contains sequences necessary for viral RNA synthesis per se. For this purpose, we used a dicistronic poliovirus RNA in which the translation of the viral non-structural (replication) proteins is uncoupled from the poliovirus IRES. In this system, RNA synthesis is readily detectable in transfected cells, even when the poliovirus IRES is inactivated by point mutation. However, deletion of the major part of the poliovirus IRES renders viral-specific RNA synthesis undetectable. Using the same system, we show that a three nucleotide deletion at position 500 in the 5' untranslated region drastically affects both translation efficiency and RNA synthesis. Furthermore, disruption of the secondary structure of the IRES around nucleotide 343 has minimal effects on IRES function, but dramatically reduces viral RNA replication. Taken together, these results provide direct evidence that sequences essential for viral RNA synthesis are located in the 3' region of the poliovirus IRES.     

Molecular analysis of virus-producing and non-producing clones derived from a defective SSPE virus Yamagata-1 strain.

Two virus clones were isolated from a defective SSPE virus, the Yamagata-1 strain, and designated as the YA and YF clones. The YA clone-infected cells produced neither cell-free virus nor cell-associated virus, whereas the YF clone-infected cells produced both cell-associated and cell-free virus. No difference of epitopes on structural proteins was observed between these two clones. Both clones had hemadsorption activity. Quantitation of structural protein by Western dot blots showed relatively a lower amount of M protein in the YA-infected cells than that in the YF-infected cells. The ratio, P plus M dicistronic/M monocistronic mRNA, in the YA-infected cells was about twice that in the YF-infected cells. Sequence analysis of cDNA corresponding to P plus M dicistronic mRNA revealed that the deduced M protein of the YF virus was smaller than that of the YA virus by five amino acids from the carboxy terminal. These results suggest that abundant production of P plus M dicistronic mRNA is responsible for the reduced amount of M protein in the non-productive YA clone.     

Human Papovavirus, BK Strain: Biological Studies Including Antigenic Relationship to Simian Virus 40

Some of the properties of a new human papovavirus, BK, have been examined. Host range studies of BK virus (BKV) showed human cells to be more sensitive to infection than monkey cells; human fetal brain cells appear to be highly sensitive to BKV, with the production of extensive cytopathology characterized by cytoplasmic vacuolization. The hemagglutinin of BKV is associated with the virion and is resistant to ether or heating at 56 C for 30 min. Fluorescent antibody as well as neutralization tests indicated antigenic similarities between simian virus 40 (SV40) and BKV. Cells undergoing lytic infection with BKV synthesized intranuclear T antigen(s) which reacted with SV40 T antibody demonstrable by immunofluorescence. However, BKV did not appear to induce SV40 transplantation antigens in transplantation-resistance tests. Evidence was obtained that BKV was present in humans prior to the widespread use of polio vaccines, thus ruling out the possibility that BKV is an SV40-related monkey virus, introduced into the human population by accidental contamination of poliovirus vaccines.     

Effect of a capsid-stabilizing pyridazinamine, R 78206, on the eclipse and intracellular location of poliovirus.

R 78206 (a pyridazinamine derivative) inhibits the formation of poliovirus eclipse particles. Its effect on the intracellular location of poliovirus was studied by separating subcellular fractions in iso-osmotic Nycodenz gradients. The compound did not inhibit internalization of intact virus into small lipid vesicles, but it did inhibit the release of virus from these vesicles and its entry into lysosomes.     

Guiding the Immune Response against Influenza Virus Hemagglutinin toward the Conserved Stalk Domain by Hyperglycosylation of the Globular Head Domain

Influenza virus hemagglutinin consists of a highly variable and immunodominant head domain and a more conserved but immunosubdominant stalk domain. We introduced seven N-linked glycosylation sites in the hemagglutinin head domain to shield the immunodominant antigenic sites. The hyperglycosylated hemagglutinin enhanced stalk-directed seroreactivity while dampening the head response in immunized mice. Upon influenza virus challenge, mice vaccinated with the hyperglycosylated hemagglutinin were better protected against morbidity and mortality than mice receiving the wild-type hemagglutinin.     

Survival of Virus in Chilled, Frozen, and Processed Oysters

Samples of whole and shucked Pacific and Olympia oysters, contaminated with 10(4)-plaque-forming units (PFU) of poliovirus Lsc-2ab per ml, were held refrigerated at two temperatures, 5 and - 17.5 C. To study the survival of virus in the oysters under these conditions, samples were assayed for virus content at weekly intervals for as long as 12 weeks. Results indicated that poliovirus would survive in refrigerated oysters for a period varying from 30 to 90 days, depending upon temperature. The survival rate varied from 10 to 13%. To study the extent of the hazard presented by oysters contaminated with virus, samples of whole and shucked Pacific oysters contaminated with 10(4) PFU of poliovirus Lsc-2ab per ml were heat processed in four ways: by stewing, frying, baking, and steaming. Results indicated that virus in oysters withstood these methods of processing. The survival rate varied from 7 to 10% and appeared dependent upon the processing method used. Heat penetration studies showed that the internal temperature in the oyster was not sufficient to inactivate all of the virus present. These results suggest that not only fresh but also refrigerated and cooked oysters can serve as vectors for the dissemination of virus disease if the shellfish are harvested from a polluted area.     

Genetic evidence for an interaction between a picornaviral cis-acting RNA replication element and 3CD protein

Internally located, cis-acting RNA replication elements, termed cres, are essential for replication of the genomes of picornaviruses such as human rhinovirus 14 (HRV-14) and poliovirus because they template uridylylation of the protein primer, VPg, by the polymerase 3D(pol). These cres form stem-loop structures sharing a common loop motif, and the HRV-14 cre can substitute functionally for the poliovirus cre in both uridylylation in vitro and RNA replication in vivo. We show, however, that the poliovirus cre is unable to support HRV-14 RNA replication. This lack of complementation maps to the stem of the poliovirus cre and was reversed by single nucleotide substitutions in the stem as well as the base of the loop. Replication-competent, revertant viruses rescued from dicistronic HRV-14 RNAs containing the poliovirus cre, or a chimeric cre containing the poliovirus stem, contained adaptive amino acid substitutions. These mapped to the surface of both the polymerase 3D(pol), at the tip of the "thumb" domain, and the protease 3C(pro), on the side opposing the active site and near the end of an extended strand segment implicated previously in RNA binding. These mutations substantially enhanced replication competence when introduced into HRV-14 RNAs containing the poliovirus cre, and they were additive in their effects. The data support a model in which 3CD or its derivatives 3C(pro) and 3D(pol) interact directly with the stem of the cre during uridylylation of VPg.     

The sequence context of the initiation codon in the encephalomyocarditis virus leader modulates efficiency of internal translation initiation.

Translation initiation on poliovirus and encephalomyocarditis virus (EMCV) mRNAs occurs by a cap-independent mechanism utilizing an internal ribosomal entry site (IRES). However, no unifying mechanism for AUG initiation site selection has been proposed. Analysis of initiation of mRNAs translated in vitro has suggested that initiation of poliovirus mRNA translation likely involves both internal binding of ribosomes and scanning to the first AUG which is in a favorable context for initiation. In contrast, internal initiation on EMCV mRNA may not utilize scanning, since ribosomes bind directly or very close to the initiation codon AUG-11. We have studied in vivo the sequence requirements for internal initiation around the EMCV initiation codon, both in monocistronic and in dicistronic mRNAs. Our studies show that the upstream AUG-10 is normally not used and that there is no specific sequence requirement for nucleotides between AUG-10 and AUG-11. However, the sequence context of AUG-11 does influence the efficiency of initiation at AUG-11. Efficient IRES-mediated internal initiation at AUG-11 exhibits a requirement for an adenine in the -3 position, similar to cap-dependent initiation. These results support a model for internal initiation on EMCV mRNA in which scanning starts at or near AUG-11. Although initiation primarily occurs at AUG-11, initiation at multiple downstream AUG codons can be detected. In addition, a poor sequence context around AUG-11 results in increased initiation at one or more downstream AUG codons, indicative of leaky scanning or jumping by the ribosome from AUG-11 mediated by the EMCV IRES.     

我国登革2/4型病毒株Pr-M-E基因的双表达质粒构建及在真核细胞中的共表达

将我国登革 2、4型病毒分离株PrM E基因通过RT PCR以病毒RNA为模板扩增我国登革 2型 4 3株和 4型B5株的PrM E基因 .并分别克隆至pGEM TEasy载体 ,然后亚克隆至双顺反子表达质粒的两个多克隆位点 ,获得同时含有登革 2型 4 3株和 4型B5株PrM E基因的双顺反子重组表达质粒pIDME2 4 .在用该重组质粒转染的BHK2 1细胞中 ,不但可检测到PrM E基因的转录产物 ,而且采用间接免疫荧光法还可观察到针对登革 2型 4型病毒的特异荧光 .研究结果表明 ,重组的双顺反子表达质粒在真核细胞中可共表达登革两个血清型PrM E基因 ,为双价登革核酸疫苗的研究奠定基础     

Attenuation of neurovirulence, biodistribution, and shedding of a poliovirus:rhinovirus chimera after intrathalamic inoculation in Macaca fascicularis

A dependence of poliovirus on an unorthodox translation initiation mode can be targeted selectively to drive viral protein synthesis and cytotoxicity in malignant cells. Transformed cells are naturally susceptible to poliovirus, due to widespread ectopic upregulation of the poliovirus receptor, Necl-5, in ectodermal/neuroectodermal cancers. Viral tumor cell killing and the host immunologic response it engenders produce potent, lasting antineoplastic effects in animal tumor models. Clinical application of this principle depends on unequivocal demonstration of safety in primate models for paralytic poliomyelitis. We conducted extensive dose-range-finding, toxicity, biodistribution, shedding, and neutralizing antibody studies of the prototype oncolytic poliovirus recombinant, PVS-RIPO, after intrathalamic inoculation in Macaca fascicularis. These studies suggest that intracerebral PVS-RIPO inoculation does not lead to viral propagation in the central nervous system (CNS), does not cause histopathological CNS lesions or neurological symptoms that can be attributed to the virus, is not associated with extraneural virus dissemination or replication and does not induce shedding of virus with stool. Intrathalamic PVS-RIPO inoculation induced neutralizing antibody responses against poliovirus serotype 1 in all animals studied.     

Improved distribution of antigenic site specificity of poliovirus-neutralizing antibodies induced by a protease-cleaved immunogen in mice.

Previous studies showed that the distribution of antigenic site specificity of neutralizing antibodies to type 3 poliovirus obtained with the inactivated poliovirus vaccine can be deficient as compared with that obtained following poliovirus infection. This observation was shown by the relatively low capacity of sera from inactivated-poliovirus-vaccine-immunized persons to neutralize poliovirus cleaved at antigenic site 1. We investigated possibilities for improving the situation in a mouse model. Balb/c mice were immunized with intact or trypsin-cleaved type 3 poliovirus (Saukett strain). Sera from mice immunized with the intact virus readily neutralized the intact virus but neutralized the cleaved virus only rarely. In contrast, cleaved-virus-immunized mice produced antibodies that were able to neutralize the cleaved virus as well as the intact one. Mice immunized with a 100-fold-higher dose of the intact virus produced significant levels of antibodies to the cleaved virus, too. Somewhat surprisingly, mice immunized with high doses of the cleaved virus produced antibodies specific for the intact loop between beta sheets B and C of VP1 (virion protein 1), which should be cleaved in the immunogen. This was shown by a higher titer of antibodies to intact Saukett virus than to the corresponding cleaved virus, as well as to a type 1/type 3 hybrid poliovirus in which only the BC loop amino acids were derived from type 3 poliovirus. The cleavage-induced enhanced availability of antigenic determinants residing outside the BC loop was also shown by increased neutralization titers of monoclonal antibodies specific for some of these other determinants. These results indicate that by using a trypsin-cleaved type 3 poliovirus as a parenteral immunogen, it is possible to change the distribution of antigenic site specificities of neutralizing antibodies to resemble that following poliovirus infection.