Search

The failure and/or toxicity of conventional therapies for many types of human cancers underscore the need for development of safe and effective alternative treatments. Toward this goal, we describe the direct oncolytic activity of RNA-based vectors derived from poliovirus, termed replicons, which are genetically incapable of producing infectious virus. These replicons are cytopathic in vitro for human tumor cells originating from brain, breast, lung, ovary, and skin (melanoma). The cytopathic effects in a malignant glioma cell line were associated with nuclear DNA condensation, indicative of cells undergoing apoptosis. Injection of replicons into established xenograft flank tumors in scid mice resulted in oncolytic activity and extended survival. Inoculation of replicons into established intracranial xenograft tumors in scid mice resulted in tumor infection within 8 h and extended survival. Histological analysis revealed that replicons had infected tumor cells at the site of inoculation and, most importantly, diffused to infect tumor cells that had metastasized from the initial site of implantation. The wide spectrum of cytopathic activity for human tumors combined with effective distribution after in vivo inoculation establishes the therapeutic potential of poliovirus replicons for a variety of cancers.

Poliovirus-based vectors (replicons) can be used for gene delivery to motor neurons of the CNS. In the current study, a replicon encoding green fluorescent protein (GFP) was encapsidated into authentic poliovirions, using established procedures. Intrathecal delivery of encapsidated replicons encoding GFP to the CNS of mice transgenic for the human poliovirus receptor did not result in any functional deficits as judged by behavioral testing. Histological analysis of the CNS of mice given a single intrathecal injection of poliovirus replicons encoding GFP revealed no obvious pathogenesis in neurons (or other cell types) within the CNS. The expression of GFP was confined to motor neurons throughout the neuroaxis; a time course of expression of GFP revealed that expression was detectable 24 hr postinoculation and returned to background levels by 120 hr postinoculation. A procedure was devised to allow repetitive inoculation of replicons within the same animal. Behavioral testing of animals that had received 6 to 13 independent inoculations of replicons revealed no functional deficits. Histological analysis of the CNS from animals that had received 6 to 13 sequential inoculations of replicons revealed no obvious abnormalities in neurons or other cell types in the CNS; expression of GFP was demonstrated in neurons 24 to 72 hr after the final inoculation of the replicon. Furthermore, there was no obvious inflammatory response in the CNS after the multiple inoculations. These studies establish the safety and efficacy of replicons for gene delivery to the CNS and are discussed with respect to use of replicons as new therapeutic strategies for spinal cord injuries and/or neurological diseases.

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.

Poliovirus replicons were constructed which contain the internal ribosome entry site (IRES) of encephalomyocarditis virus (EMCV) substituted for the poliovirus IRES. To monitor gene expression and encapsidation, the gene encoding firefly luciferase was substituted for the P1 gene. Replicons can be encapsidated following serial passage in the presence of a recombinant vaccinia virus, VV-P1, which expresses the poliovirus P1 protein following infection. Encapsidation of the wild-type replicon (PV-Luc) was accomplished at either 33 or 37 degrees C; the lower temperature actually resulted in greater amounts of encapsidated replicon. In contrast, the replicon with the EMCV IRES element (EMCV-Luc) was not efficiently encapsidated at 37 degrees C and, following serial passage with VV-P1 at 37 degrees C, was not amplified. EMCV-Luc was efficiently encapsidated, however, following serial passage with VV-P1 at 33 degrees C. Using the encapsidated EMCV-Luc obtained at 33 degrees C, we found that cells infected with EMCV-Luc at 33 or 37 degrees C produced similar amounts of luciferase. Encapsidated EMCV-Luc and PV-Luc had similar thermal stability at 33 and 37 degrees C. A single-round encapsidation analysis revealed that less EMCV-Luc was encapsidated at 37 than at 33 degrees C; less EMCV-Luc was encapsidated at 33 degrees C compared to PV-Luc at either 37 or 33 degrees C. The results of our studies suggest that in addition to influencing translation/replication, the IRES region of poliovirus can function to enhance encapsidation., (Copyright 2000 Academic Press.)

A hallmark of poliovirus is the propensity to infect and replicate in spinal cord neurons of the central nervous system. Previously, we characterized a poliovirus self-replicating RNA genome (replicon), which encodes firefly luciferase in place of the capsid genes. This replicon is encapsidated into an authentic poliovirion by providing the poliovirus capsid protein in trans. The amount of enzymatically active luciferase in cells infected with this replicon correlated with the infectious dose. To begin to characterize the in vivo infectious potential of replicons, we have inoculated mice transgenic for the human receptor for poliovirus (PVR), either intracranially or intraspinally, with the replicon encoding luciferase. Wild-type poliovirus delivered to PVR mice via intracranial or intraspinal routes resulted in paralysis and death. Replicon preparations were shown by a sensitive biological assay to be free of infectious poliovirus. Neither intracranial nor intraspinal inoculation of the replicon encoding luciferase resulted in any obvious paralysis or disease symptoms. Following intraspinal inoculation with replicons encoding luciferase, luciferase enzyme activity was detected at 4 h post-inoculation, with peak activity at approximately 8 h post-inoculation; by 48 - 72 h, the luciferase activity had returned to background levels. Luciferase activity was detected in spinal cord predominantly near the site of inoculation, although activity was detected anterior and posterior to the site of inoculation, indicating that replicons undergo limited movement within the CNS presumably via the cerebrospinal fluid. In stark contrast to poliovirus though, inoculation of replicons into the spinal cords of PVR mice did not result in noticeable pathogenesis. Using immunofluorescence with antibodies to double-stain for replicons and neurons, we determined that replicons exclusively infect the neurons of the spinal cord, with the expression of the luciferase and replicon proteins confined to the cytoplasm of the infected cells. Replicons, then, possess the identical capacity for infection of spinal cord neurons in vivo as poliovirus. The lack of discernible neuronal destruction following replicon inoculation into the spinal cord suggests that some of the pathogenesis observed during a poliovirus infection might not be due entirely to primary infection of neurons. Finally, the results of this study point to future use of replicons as a means to target recombinant protein expression to neurons in the spinal cord.

The development of a vaccine for Helicobacter pylori is a key strategy for reducing the worldwide prevalence of H. pylori infection. Although immunization with recombinant B subunit of H. pylori urease (ureB) has yielded promising results, for the most part, these studies relied on the use of strong adjuvant, cholera toxin, precluding the use in humans. Thus, the development of new vaccine strategies for H. pylori is essential. Previous studies from our laboratory have described a vaccine vector based on poliovirus in which foreign genes are substituted for the poliovirus capsid genes. The genomes encoding foreign proteins (replicons) are encapsidated into authentic poliovirions by providing the capsids in trans. To test the utility of replicons as a vaccine vector for H. pylori, a replicon was constructed which encodes ureB. Expression of ureB in cells from the replicon was demonstrated by metabolic labeling followed by immunoprecipitation with anti-urease antibodies. To investigate the immunogenicity of the replicons, mice containing the transgene for the receptor for poliovirus were immunized via the intramuscular route. Mice given three doses of replicons did not develop substantial antibodies to ureB as determined by Western blot analysis using lysates from H. pylori. In contrast, mice given two doses of replicon followed by a single injection of recombinant ureB developed serum antibodies to ureB which were predominately IgG2a. Splenic lymphocytes from mice immunized with replicons alone, or replicons plus recombinant ureB produced abundant interferon-gamma and no detectable interleukin-4 upon stimulation with recombinant ureB. These results establish that poliovirus replicons encoding H. pylori ureB are immunogenic and induce primarily a T helper 1 associated immune response.

Poliovirus genomes have been constructed in which the capsid genes have been substituted with the murine gene encoding interleukin-2 (IL-2) (referred to as replicons). One replicon contained the gene for IL-2 in place of the poliovirus capsid VP2 and VP3 genes, and a second replicon was constructed that contained the murine IL-2 substituted for the poliovirus VP3 and VP1 genes. The IL-2 genes were cloned into the replicon so as to maintain the translational reading frame with the remaining poliovirus proteins. Transfection of either replicon into cells resulted in the expression of replicon-encoded proteins and replication of replicon RNA. Using a procedure developed in this laboratory, we have encapsidated these replicons into authentic polio virions by passaging the replicons in the presence of a recombinant vaccinia virus, VVP1, which expresses the capsid precursor, P1, protein. Using a quantitative immunoassay, we determined that the majority of the IL-2 produced remained intracellular, with approximately 1%-2% released from the infected cells, and that the IL-2 was biologically active. The results of these studies demonstrate the utility of poliovirus replicons for expression of small bioactive molecules and are discussed with respect to future applications as immune adjuvants as well as potential new tumor therapies.

The specificity of poliovirus encapsidation has been studied using a novel chimeric genome in which the gene encoding firefly luciferase has been substituted for the VP2-VP3-VP1 genes of the poliovirus capsid (P1) gene. Transfection of RNA transcribed in vitro from this genome resulted in a VP4-luciferase fusion protein which retained luciferase enzyme activity. Since the detection of enzyme activity was dependent upon replication of the transfected RNA genome, we refer to these genomes as replicons. The replicon encoding luciferase was encapsidated upon transfection of the genomic RNA into cells previously infected with a recombinant vaccinia virus, VV-P1, which encodes the poliovirus type 1 capsid proteins (P1). Infection of cells with each serial passage, followed by analysis of luciferase enzyme activity, revealed that encapsidated replicons could be detected at the first passage with VV-P1. Amplification of the titer of encapsidated replicons occurred upon serial passage with VV-P1, as evidenced by the high expression levels of luciferase enzyme activity following infection. Serial passage of the luciferase replicons with poliovirus type 1, 2, or 3 resulted in the trans encapsidation into the type 1, 2, or 3 capsids, respectively. In contrast, serial passage with bovine enterovirus, Coxsackievirus A21 or B3, or enterovirus 70 did not result in trans encapsidation, even though co-infection of cells with the replicon and different enteroviruses resulted in high-level expression of luciferase. The results of this study highlight the specificity of poliovirus encapsidation and point to the use of encapsidated replicons encoding luciferase as a reagent for dissecting elements of replication and encapsidation.

In this study, we describe the construction of poliovirus genomes or "replicons" which contain the C fragment gene of tetanus toxin substituted for the poliovirus P1 capsid. Upon transfection of replicon RNA into cells, we immunoprecipitated a protein corresponding to the C-fragment of tetanus toxin using tetanus-specific antibodies. Using a recombinant vaccinia virus expressing poliovirus P1 capsid protein (VV-P1) to provide P1 protein, the replicon RNA was encapsidated; stocks of the replicons were generated by passage with VV-P1. The immunogenicity of the replicons was determined by immunization of transgenic mice which are susceptible to poliovirus. A serum antibody response to poliovirus and tetanus toxoid was detected in all of the immunized mice. Protection against a lethal dose of tetanus toxin generally correlated with the levels of serum anti-tetanus antibodies. To address whether pre-existing antibodies to poliovirus limit the effectiveness of the replicon as a vaccine vector, mice were first immunized with the inactivated poliovirus vaccine followed by immunization with the replicons expressing C-fragment protein. Anti-tetanus antibodies were detected in these mice after a single administration of the replicon; these antibodies conferred protection upon challenge with tetanus toxin. These results demonstrate the potential use of poliovirus replicons encoding foreign proteins to induce a protective antibody response, even in the presence of pre-existing antibodies to poliovirus.

The effectiveness of the poliovirus vaccines to induce both systemic and mucosal immunity has prompted the development of this virus as a vector in which to express foreign proteins. Our laboratory has previously reported on the construction and characterization of poliovirus genomes that encode HIV-1 proteins (Porter DC, et al.: J Virol 1996;70:2643-2649). To develop this system further, we have constructed poliovirus genomes, referred to as replicons, which encode the SIVmac239 Gag or Env SU in place of the poliovirus capsid gene (P1). Since the replicons do not encode capsid proteins, they are encapsidated into poliovirus by passage with a recombinant vaccinia virus, VVP1, which provides the poliovirus capsid proteins in trans. Using this system, we have derived stocks of the encapsidated replicons which encode the SIVmac239 or Env SU protein. Infection of cells with the replicon that encodes SIVmac239 Gag resulted in the expression of a 55-kDa protein that was released from the infected cells. Analysis of the sedimentation of the released proteins by sucrose density gradient centrifugation revealed that the protein was released from the cell in the form of a virus-like particle. Infection of cells with the replicons encoding the SIVmac239 Env SU resulted in the expression of a 63-kDa protein, corresponding to the molecular mass predicted for the nonglycosylated SIVmac239 SU protein. A second protein with a molecular mass greater than 160 kDa was also immunoprecipitated. After enzymatic deglycosylation, this protein migrated at a molecular mass consistent with that for an Env SU dimer. Analysis of the medium from cells infected with the replicon encoding SIVmac239 Env SU revealed the presence of a protein of molecular mass 85-90 kDa, possibly representing a fragment of the SIVmac239 or Env SU protein. To determine the immunogenicity of the replicons encoding SIVmac239 Gag or Env SU, transgenic mice that express the human receptor for poliovirus, and are thus susceptible to poliovirus, were immunized via the intramuscular route. A serum antibody response to SIV envelope was detected following booster immunization, establishing that the encapsidated replicon was immunogenic. Finally, we demonstrate that the replicons have the capacity to infect peripheral blood mononuclear monocytes/macrophages, suggesting that this cell is a possible target for in vivo infection. The results of our studies, then, lend further support for the development and application of recombinant poliovirus replicons in a vaccine strategy.

The effectiveness of attenuated poliovirus vaccines when given orally to induce both systemic and mucosal immune responses against poliovirus has resulted in an effort to develop poliovirus-based vectors to express foreign proteins. We have previously described the construction of poliovirus genomes (referred to as replicons) in which the complete human immunodeficiency virus type 1 (HIV-1) gag gene was substituted for the capsid gene (P1) (D.C. Porter, D.C. Ansardi, and C.D. Morrow, J. Virol. 69:1548-1555, 1995). Infection of cells with encapsidated replicons resulted in the expression of a 55-kDa protein. To further characterize the biological features of the HIV-1 Gag proteins expressed in cells infected with encapsidated replicons, we utilized biochemical analysis and electron microscopy. Expression of the 55-kDa protein in cells infected with encapsidated replicons resulted in myristylation of the Pr55gag protein. The Gag precursor protein was released from infected cells; analysis on sucrose density gradients revealed that the precursor sedimented at a density consistent with that of an HIV-1 virus-like particle. Analysis of replicon-infected cells by electron microscopy demonstrated the presence of condensed structures at the plasma membrane and the release of virus-like particles. These studies demonstrate that poliovirus-based vectors can be used to express foreign proteins which require posttranslational modifications, such as myristylation, and assemble into higher-order structures, providing a foundation for the future use of poliovirus replicons as vaccine vectors.

The poliovirus RNA-dependent RNA polymerase (3Dpol) contains a region of homology centered around the amino acid motif YGDD (amino acids 326 to 329), which has been postulated to be involved in the catalytic activity of the enzyme. Previous studies from this laboratory have used oligonucleotide site-directed mutagenesis to substitute the tyrosine amino acid at this motif with other amino acids (S. A. Jablonski and C. D. Morrow, J. Virol. 67:373-381, 1993). The viruses recovered with 3Dpol genes with a methionine mutation also contained a second mutation at amino acid 108 resulting in a glutamic acid-to-aspartic acid change (3D-E-108 to 3D-D-108) in the poliovirus RNA polymerase. On the basis of these results, we suggested that the amino acid at position 108 might interact with the YGDD region of the poliovirus polymerase. To further investigate this possibility, we have constructed a series of constructs in which the poliovirus RNA polymerases contained a mutation at amino acid 108 (3D-E-108 to 3D-D-108) as well as a mutation in which the tyrosine amino acid (3D-Y-326) was substituted with cysteine (3D-C-326) or serine (3D-S-326). The mutant 3Dpol polymerases were expressed in Escherichia coli, and in vitro enzyme activity was analyzed. Enzymes containing the 3D-D-108 mutation with the wild-type amino acid (3D-Y-326) demonstrated in vitro enzyme activity similar to that of the wild-type enzyme containing 3D-E-108. In contrast, enzymes with the 3D-C-326 or 3D-S-326 mutation had less in vitro activity than the wild type. The inclusion of the second mutation at amino acid 3D-D-108 did not significantly affect the in vitro activity of the polymerases containing 3D-C-326 or 3D-S-326 mutation. Transfections of poliovirus cDNAs containing the substitution at amino acid 326 with or without the second mutation at amino acid 108 were performed. Consistent with previous findings, we found that transfection of poliovirus cDNAs containing the 3D-C-326 or 3D-S-326 mutation in 3Dpol did not result in the production of virus. Surprisingly, transfection of the poliovirus cDNAs containing the 3D-D-108/C-326 double mutation, but not the 3D-D-108/S-326 mutation, resulted in the production of virus. The virus obtained from transfection of polio-virus cDNAs containing 3D-D-108/C-326 mutation replicated with kinetics similar to that of the wild-type virus. RNA sequence analysis of the region of the 3Dpol containing the 3D-C-326 mutation revealed that the codon for cysteine (UGC) reverted to the codon for tyrosine (UAC). The results of these studies establish that under the appropriate conditions, poliovirus has the capacity to revert mutations within the YGDD amino acid motif of the poliovirus 3Dpol gene and further strengthen the idea that interaction between amino acid 108 and the YGDD region of 3Dpol is required for viral replication.

Several viruses have been exploited for the development of recombinant vaccine vectors in which to express foreign proteins. Recently, we have described a system utilizing the RNA virus, poliovirus. We have constructed poliovirus genomes in which regions of the capsid have been substituted with gene fragments of the HIV gag and env genes. A complementation system has been designed to encapsidate defective genomes by providing the capsid protein in trans from a recombinant vaccinia virus (VV-P1). Serial passage in the presence of VV-P1 resulted in the generation of stocks of these encapsidated replicons. Infection of cells with these encapsidated replicons resulted in the expression of the recombinant protein as a fusion protein with the poliovirus capsid proteins VP4 and VP1. In this study, we have utilized encapsidated replicons which express the HIV-1-gag capsid protein (p24) as well as 1.5 kb of the HIV-1 env gene. Stocks of these encapsidated replicons were obtained by 20 serial passages in the presence of VV-P1. In addition, passage of the encapsidated replicons in the presence of poliovirus type 2 Lansing resulted in the encapsidation of the replicons by the capsid proteins provided by poliovirus. The administration of the type 2 Lansing/encapsidated replicons expressing HIV-1 gag in BALB/c mice by intramuscular, intrarectal, or intragastric routes resulted in the generation of antibodies in the serum and secretions against both poliovirus and HIV-1 gag. To prove that the replicons alone are immunogenic, we administered replicons expressing either HIV-1 gag or env to transgenic mice which expressed the receptor for poliovirus type 1. Immunization of these mice by the intramuscular route resulted in the generation of serum antibodies specific for poliovirus as well as for HIV-1 antigens. The results obtained led us to the conclusion that the replicons are immunogenic when given alone or in the presence of poliovirus. These results are important for the use of the poliovirus replicons as a recombinant vaccine vector.

The assembly of infectious poliovirus virions requires a proteolytic cleavage between an asparagine-serine amino acid pair (the maturation cleavage site) in VP0 after encapsidation of the genomic RNA. In this study, we have investigated the effects that mutations in the maturation cleavage site have on P1 polyprotein processing, assembly of subviral intermediates, and encapsidation of the viral genomic RNA. We have made mutations in the maturation cleavage site which change the asparagine-serine amino acid pair to either glutamine-glycine or threonine-serine. The mutations were created by site-directed mutagenesis of P1 cDNAs which were recombined into wild-type vaccinia virus to generate recombinant vaccinia viruses. The P1 polyproteins expressed from the recombinant vaccinia viruses were analyzed for proteolytic processing and assembly defects in cells coinfected with a recombinant vaccinia virus (VV-P3) that expresses the poliovirus 3CD protease. A trans complementation system using a defective poliovirus genome was utilized to assess the capacity of the mutant P1 proteins to encapsidate genomic RNA (D. C. Ansardi, D. C. Porter, and C. D. Morrow, J. Virol. 67:3684-3690, 1993). The mutant P1 proteins containing the glutamine-glycine amino acid pair (VP4-QG) and the threonine-serine pair (VP4-TS) were processed by 3CD provided in trans from VV-P3. The processed capsid proteins VP0, VP3, and VP1 derived from the mutant precursor VP4-QG were unstable and failed to assemble into subviral structures in cells coinfected with VV-P3. However, the capsid proteins derived from VP4-QG did assemble into empty-capsid-like structures in the presence of the defective poliovirus genome. In contrast, the capsid proteins derived from processing of the VP4-TS mutant assembled into subviral intermediates both in the presence and in the absence of the defective genome RNA. By a sedimentation analysis, we determined that the capsid proteins derived from the VP4-TS precursor encapsidated the defective genome RNA. However, the cleavage of VP0 to VP4 and VP2 was delayed, resulting in the accumulation of provirions. The maturation cleavage of the VP0 protein containing the VP4-TS mutation was accelerated by incubation of the provirions at 37 degrees C. The results of these studies demonstrate that mutations in the maturation cleavage site have profound effects on the subsequent capability of the capsid proteins to assemble and provide evidence for the existence of the provirion as an assembly intermediate.

Poliovirus genomes which contain small regions of the human immunodeficiency virus type 1 (HIV-1) gag, pol, and env genes substituted in frame for the P1 capsid region replicate and express HIV-1 proteins as fusion proteins with the P1 capsid precursor protein upon transfection into cells (W. S. Choi, R. Pal-Ghosh, and C. D. Morrow, J. Virol. 65:2875-2883, 1991). Since these genomes, referred to as replicons, do not express capsid proteins, a complementation system was developed to encapsidate the genomes by providing P1 capsid proteins in trans from a recombinant vaccinia virus, VV-P1. Virus stocks of encapsidated replicons were generated after serial passage of the replicon genomes into cells previously infected with VV-P1 (D. C. Porter, D. C. Ansardi, W. S. Choi, and C. D. Morrow, J. Virol. 67:3712-3719, 1993). Using this system, we have further defined the role of the P1 region in viral protein expression and RNA encapsidation. In the present study, we constructed poliovirus replicons which contain the complete 1,492-bp gag gene of HIV-1 substituted for the entire P1 region of poliovirus. To investigate whether the VP4 coding region was required for the replication and encapsidation of poliovirus RNA, a second replicon in which the complete gag gene was substituted for the VP2, VP3, and VP1 capsid sequences was constructed. Transfection of replicon RNA with and without the VP4 coding region into cells resulted in similar levels of expression of the HIV-1 Gag protein and poliovirus 3CD protein, as indicated by immunoprecipitation using specific antibodies. Northern (RNA) blot analysis of RNA from transfected cells demonstrated comparable levels of RNA replication for each replicon. Transfection of the replicon genomes into cells infected with VV-P1 resulted in the encapsidation of the genomes; serial passage in the presence of VV-P1 resulted in the generation of virus stocks of encapsidated replicons. Analysis of the levels of protein expression and encapsidated replicon RNA from virus stocks after 21 serial passages of the replicon genomes with VV-P1 indicated that the replicon which contained the VP4 coding region was present at a higher level than the replicon which contained a complete substitution of the P1 capsid sequences. These differences in encapsidation, though, were not detected after only two serial passages of the replicons with VV-P1 or upon coinfection and serial passage with type 1 Sabin poliovirus.(ABSTRACT TRUNCATED AT 400 WORDS)

The poliovirus RNA-dependent RNA polymerase, 3Dpol, is known to share a region of sequence homology with all RNA polymerases centered at the GDD amino acid motif. The two aspartic acids have been postulated to be involved in the catalytic activity and metal ion coordination of the enzyme. To test this hypothesis, we have utilized oligonucleotide site-directed mutagenesis to generate defined mutations in the aspartic acids of the GDD motif of the 3Dpol gene. The codon for the first aspartate (3D-D-328 [D refers to the single amino acid change, and the number refers to its position in the polymerase]) was changed to that for glutamic acid, histidine, asparagine, or glutamine; the codons for both aspartic acids were simultaneously changed to those for glutamic acids; and the codon for the second aspartic acid (3D-D-329) was changed to that for glutamic acid or asparagine. The mutant enzymes were expressed in Escherichia coli, and the in vitro poly(U) polymerase activity was characterized. All of the mutant 3Dpol enzymes were enzymatically inactive in vitro when tested over a range of Mg2+ concentrations. However, when Mn2+ was substituted for Mg2+ in the in vitro assays, the mutant that substituted the second aspartic acid for asparagine (3D-N-329) was active. To further substantiate this finding, a series of different transition metal ions were substituted for Mg2+ in the poly(U) polymerase assay. The wild-type enzyme was active with all metals except Ca2+, while the 3D-N-329 mutant was active only when FeC6H7O5 was used in the reaction. To determine the effects of the mutations on poliovirus replication, the mutant 3Dpol genes were subcloned into an infectious cDNA of poliovirus. The cDNAs containing the mutant 3Dpol genes did not produce infectious virus when transfected into tissue culture cells under standard conditions. Because of the activity of the 3D-N-329 mutant in the presence of Fe2+ and Mn2+, transfections were also performed in the presence of the different metal ions. Surprisingly, the transfection of the cDNA containing the 3D-N-329 mutation resulted in the production of virus at a low frequency in the presence of FeSO4 or CoCl2. The virus derived from transfection in the presence of FeSO4 grew slowly, while the viruses recovered from transfection in CoCl2 grew at a rate which was similar to that of the wild-type poliovirus.(ABSTRACT TRUNCATED AT 400 WORDS)

The multiple roles of the viral proteinase 2A in poliovirus replication have been difficult to assess because, to date, it has not been possible to isolate and characterize a viral genome with an inactive 2Apro. We have previously reported that a poliovirus replicon containing an inactive 2Apro by virtue of a change at amino acid 109 from a cysteine to a serine (C109S) was replication competent when transfected into cells previously infected with vaccinia virus (R. Pal-Ghosh and C. D. Morrow, J. Virol. 67:4621-4629, 1993). To further develop this system, we have used a poliovirus replicon which contains the human immunodeficiency virus type 1 (HIV-1) gag gene positioned between nucleotides 1174 and 2470 of the poliovirus genome and have engineered a second mutation within this replicon to change the codon for amino acid 109 of the 2Apro from cysteine to serine (2AC109S). Transfection of this replicon into cells previously infected with vaccinia virus results in the replication and expression of a protein with a molecular mass consistent with that of a P1-HIV-1 Gag-2A fusion protein. Using a recently described complementation system which relies on the capacity of a recombinant vaccinia virus (VV-P1) to provide the capsid precursor (P1) in trans (D. C. Ansardi, D. C. Porter, and C. D. Morrow, J. Virol. 67:3684-3690, 1993; and D. C. Porter, D. C. Ansardi, W. S. Choi, and C. D. Morrow, J. Virol. 67:3712-3719, 1993), we have encapsidated this replicon containing the 2AC109S mutation. By using reverse transcription PCR, we demonstrated that after 15 serial passages the encapsidated replicon still contained the 2AC109S mutation. Infection of cells with a stock of encapsidated replicon, either in the presence or in the absence of vaccinia virus, resulted in the expression of the P1-HIV-1 Gag-2A fusion protein. Expression of the P1-HIV-1 Gag fusion protein in cells infected with the encapsidated replicon containing the 2AC109S mutation was reduced compared with the expression of P1-HIV-1 Gag in those cells infected with a replicon containing a wild type 2A gene. The protein expression and replication of the replicon RNA in cells containing the 2AC109S mutation was maintained for a longer period of time than for the replicons containing the wild-type 2A gene, possibly because of a reduced cytopathic effect.(ABSTRACT TRUNCATED AT 400 WORDS)

Recombinant vaccines hold great promise for the prevention and therapy of infections diseases and cancer. We have explored the use of poliovirus as a recombinant vector to deliver genes into cells for the purpose of vaccination. For our studies, we have chosen to express the gene-encoding carcinoembryonic antigen (CEA) using a novel poliovirus vector. We have constructed a recombinant CEA-poliovirus replicon in which the CEA gene was substituted for the poliovirus capsid gene. Following in vitro transcription, the RNA was transfected into cells to demonstrate CEA expression. We found that a genome in which the region encoding the signal sequence of the CEA protein (amino acids 1-34) was removed was replication competent (i.e., referred to as a replicon). We encapsidated the CEA-poliovirus replicon by transfecting this RNA into cells previously infected with a recombinant vaccinia virus (VV-P1) which expresses the poliovirus capsid protein (P1). Serial passage in the presence of VV-P1 resulted in the generation of stocks of these encapsidated replicons. Infection of cells with the encapsidated replicon containing the CEA-poliovirus genome resulted in expression of the CEA protein. To test immunogenicity, mice susceptible to poliovirus were given three doses of the encapsidated replicons via the i.m. route. By the third administration, a CEA-specific antibody response was detected. Potential future use of the poliovirus replicon system as both a parenteral and oral vaccine vector is discussed.

To begin to identify poliovirus capsid protein determinants required for assembly and RNA encapsidation, we have addressed the functional significance of three arginine residues of the poliovirus capsid in virus assembly and encapsidation of genomic RNA. These studies were conducted by using a recently described system in which recombinant vaccinia viruses are used to supply poliovirus capsid proteins in trans to a poliovirus subgenomic replicon [D. C. Ansardi, D. C. Porter, and C. D. Morrow (1993) J. Virol. 67, 3684-3690]. Two of the arginine residues, located at position 34 of VP4 (VP4-R034) and position 129 of VP1 (VP1-R129), are located within a cavity on the poliovirus capsid interior, whereas the third arginine, residue 223 of VP3 (VP3-R223), is located at a promoter-protomer interface. Five mutants were constructed by site-directed mutagenesis of poliovirus P1 capsid precursor cDNA to separately encode lysine or glutamine substitutions at VP4-R034 (VP4-R034K, VP4-R034Q), lysine or glutamine substitutions at residue 129 of VP1 (VP1-R129K, VP1-R129Q), or a lysine substitution at residue 223 of VP3 (VP3-R223K). Processed capsid proteins derived from the VP3-R223K, VP1-R129K, and VP1-R129Q mutant precursors were unstable and failed to assemble subviral particles or virions at 37 degrees. The assembly defect for cleavage products of the VP3-R223K precursor was partially overcome at 33 degrees, as empty capsids, but not mature virions, assembled from the mutant capsid subunits at the lower temperature. With regard to the third arginine residue analyzed, VP4-R034, processed capsid proteins derived from both the VP4-R034K and the VP4-R034Q mutant precursors assembled 155S virions at 37 degrees; however, capsid proteins derived from the VP4-R034Q precursor were temperature-sensitive for virion formation at 39.5 degrees. The reduced virion formation at 39.5 degrees was apparently a reflection of a defect in forming assembly competent subunits which also prevented accumulation of surplus VP4-R034Q subunits as empty capsids. By using graphics to display the poliovirus three-dimensional structure, the locations of these residues on the poliovirus capsid interior and their interactions with adjacent amino acids were visualized to provide structural explanations for the observed assembly defects which highlight the important role these residues play in capsid assembly and RNA encapsidation.

It is apparent that a safe and effective HIV vaccine is an important component in the development of rational approaches for the control and prevention of HIV transmission. Given the fact that the virus most often encounters a mucosal surface during sexual transmission, a vaccine designed to stimulate both the systemic and mucosal immune systems is essential. Poliovirus is attractive as a delivery system because of several biological features inherent to the virus. First, the pathogenesis of the virus has been well studied, and important features have been identified. The virus is naturally transmitted by a fecal-oral route and is stable in the harsh conditions of the gastrointestinal tract. Second, previous studies using attenuated vaccine strains of poliovirus showed that a long-lasting systemic and mucosal immunity is generated after administration of the vaccines. Studies have demonstrated the presence of circulating T cells that proliferate to whole inactive poliovirus or peptides corresponding to amino acids of the VP1 proteins in previously immunized individuals. These results established that immunization with poliovirus stimulates both the humoral and cell-mediated components of the immune system. Third, the attenuated strains of poliovirus are safe for humans and are given to infants as early as 6 months of age. The incorporation of foreign genes into the attenuated strains would be an attractive feature that should pose no more of a health risk than that associated with administration of the attenuated vaccines. Finally, studies from this laboratory, as well as others, have established the feasibility of incorporating foreign genes into the poliovirus cDNA.(ABSTRACT TRUNCATED AT 250 WORDS)

Assembly of poliovirus virions requires proteolytic cleavage of the P1 capsid precursor polyprotein between two separate glutamine-glycine (QG) amino acid pairs by the viral protease 3CD. In this study, we have investigated the effects on P1 polyprotein processing and subsequent assembly of processed capsid proteins caused by substitution of the glycine residue at the individual QG cleavage sites with valine (QG-->QV). P1 cDNAs encoding the valine substitutions were created by site-directed mutagenesis and were recombined into wild-type vaccinia virus to generate recombinant vaccinia viruses which expressed the mutant P1 precursors. The recombinant vaccinia virus-expressed mutant P1 polyproteins were analyzed for proteolytic processing defects in cells coinfected with a recombinant vaccinia virus (VVP3) that expresses the poliovirus 3CD protease and for processing and assembly defects by using a trans complementation system in which P1-expressing recombinant vaccinia viruses provide capsid precursor to a defective poliovirus genome that does not express functional capsid proteins (D. C. Ansardi, D. C. Porter, and C. D. Morrow, J. Virol. 67:3684-3690, 1993). The QV-substituted precursors were proteolytically processed at the altered sites both in cells coinfected with VVP3 and in cells coinfected with defective poliovirus, although the kinetics of cleavage at the altered sites were slower than those of cleavage at the wild-type QG site in the precursor. Completely processed capsid proteins VP0, VP3, and VP1 derived from the mutant precursor containing a valine at the amino terminus of VP3 (VP3-G001V) were unstable and failed to assemble stable subviral structures in cells coinfected with defective poliovirus. In contrast, capsid proteins derived from the P1 precursor with a valine substitution at the amino terminus of VP1 (VP1-G001V) assembled empty capsid particles but were deficient in assembling RNA-containing virions. The assembly characteristics of the VP1-G001V mutant were compared with those of a previously described VP3-VP1 cleavage site mutant (K. Kirkegaard and B. Nelsen, J. Virol. 64:185-194, 1990) which contained a deletion of the first four amino-terminal residues of VP1 (VP1-delta 1-4) and which was reconstructed for our studies into the recombinant vaccinia virus system. Complete proteolytic processing of the VP1-delta 1-4 precursor also occurred more slowly than complete cleavage of the wild-type precursor, and formation of virions was delayed; however, capsid proteins derived from the VP1-G001V mutant assembled RNA-containing virions less efficiently than those derived from the VP1-delta 1-4 precursor.(ABSTRACT TRUNCATED AT 400 WORDS)

The expression of the poliovirus genome occurs by the translation of a single open reading frame to generate a long polyprotein which is subsequently processed by viral encoded proteases. The initial proteolytic cleavages result in the production of a P1 polyprotein which contains the capsid proteins, and the P2 and P3 polyproteins which contain proteins required for replication. The P3 polyprotein consists of the 3AB protein (containing the viral genome-linked protein, VPg), the viral protease, 3Cpro, and RNA polymerase, 3Dpol. To further study the expression and proteolytic processing of poliovirus P3 proteins in vivo, we have utilized recombinant vaccinia virus vectors to express nucleotides 5240-7400 containing the P3 region proteins of poliovirus. The P3 protein expressed from the recombinant vaccinia virus VV-P3 exhibited in vivo proteolytic activity as evident by processing of the polyprotein to generate the 3CD protein, consisting of a fusion between the 3Cpro and 3Dpol proteins. Further processing of the 3CD protein to 3Cpro and 3Dpol, however, was not detected in cells infected with VV-P3. Subcellular fractionation of VV-P3-infected cells demonstrated that the 3CD protein was present in both the soluble and membrane fractions. Finally, the 3CD protein expressed from VV-P3 was stable in cells co-infected with VV-P3 and poliovirus and no further processing to 3Dpol was detected. These results are discussed with regards to in vivo studies which suggest that the 3CD polyprotein is not a precursor to 3Dpol in poliovirus-infected cells.

It has been difficult to evaluate the role of individual viral proteins in poliovirus replication because a suitable complementation system has not yet been developed. To approach this problem, we constructed a chimeric human immunodeficiency virus type 2 (HIV-2)-gag-poliovirus minireplicon in which regions of the gag gene of HIV-2 were inserted in the poliovirus genome between nucleotides 1174 and 2470. Transfection of this chimeric RNA into HeLa cells results in the replication of the minireplicon and expression of an HIV-2-gag-P1 fusion protein which can be immunoprecipitated with antibodies to HIV-2-gag. Expression of the HIV-2-gag-P1 fusion protein was dependent on replication of the chimeric RNA genome. Although the chimeric HIV-2-gag-poliovirus RNA genome replicated in poliovirus-infected cells, transfection of the chimeric HIV-2-gag-poliovirus genome into vaccinia virus-infected cells resulted in increased replication as measured by analysis of chimeric RNA. The increase in replication correlated with an increase in the expression of the HIV-2-gag-P1 fusion protein in vaccinia virus-infected cells. To characterize this system, we constructed a mutation in the 2A gene to change a cysteine at amino acid 109 to a serine. Expression of the HIV-2-gag-P1 fusion protein was not detected when the HIV-2-gag-poliovirus genome containing the 2A mutation was transfected into HeLa cells, demonstrating the mutation was lethal for replication. When the chimeric genome was transfected into poliovirus-infected cells, no RNA replication or expression of the HIV-2-gag-P1 fusion protein was observed. In contrast, transfection of this genome into vaccinia virus-infected cells resulted in replication of the chimeric RNA and expression of two proteins with larger molecular masses than the HIV-2-gag-P1 proteins, possibly representing HIV-2-gag-P1-2A and HIV-2-gag-P1-2ABC fusion proteins. The transfection of the chimeric HIV-2-gag-poliovirus genome containing the 2A mutation into poliovirus-vaccinia virus coinfected cells resulted in the expression and partial processing of the two larger HIV-2-gag-P1 fusion proteins to give the correct molecular mass for the HIV-2-gag-P1 fusion protein. The 2A mutation was reconstructed back into the full-length infectious cDNA of poliovirus. Transfection of this cDNA into vaccinia virus-infected cells followed by immunoprecipitation with anticapsid antibodies demonstrated the presence of two proteins with molecular masses larger than P1, possibly P1-2A and P1-2ABC fusion proteins.(ABSTRACT TRUNCATED AT 400 WORDS)

The use of recombinant viruses for the expression of a wide array of foreign proteins has become commonplace during the last few years. Recently, we have described the construction and characterization of chimeric human immunodeficiency virus type 1 (HIV-1)-poliovirus genomes in which the gag and pol genes of HIV-1 have been substituted for the VP2 and VP3 capsid genes of the P1 capsid precursor region of poliovirus. Transfection of these RNAs into tissue culture cells results in replication of the RNA genome and expression of HIV-1-P1 fusion proteins (W. S. Choi, R. Pal-Ghosh, and C. D. Morrow, J. Virol. 65:2875-2883, 1991). Here we report on the encapsidation and amplification of the minireplicons to obtain sufficient quantities for biological characterization. To do this, HIV-1-poliovirus minireplicon genomes containing the gag or pol gene were transfected into cells previously infected with a recombinant vaccinia virus (VV-P1) which expresses the poliovirus capsid precursor protein, P1 (D. C. Ansardi, D. C. Porter, and C. D. Morrow, J. Virol. 65:2088-2092, 1991). The chimeric minireplicons replicated and expressed the appropriate HIV-1-P1 fusion proteins as determined by immunoprecipitation with HIV-1-specific antibodies. The encapsidated genomes were isolated by ultracentrifugation. Reinfection of cells with the encapsidated chimeric RNA genomes resulted in expression of the HIV-1-Gag-P1 or HIV-1-Pol-P1 fusion protein. Serial passaging of the encapsidated chimeric HIV-1-poliovirus genomes was accomplished by coinfecting cells with the encapsidated minireplicons and VV-P1, resulting in stocks of the encapsidated minireplicons. Northern (RNA) blot analysis of passaged material revealed that no detectable deletions of the chimeric genomes occurred during 14 serial passages. Infection of cells by the encapsidated minireplicons was blocked by antipoliovirus antibodies. Coinfection of cells with encapsidated minireplicons and type 1 Sabin poliovirus resulted in encapsidation of the chimeric genomes by wild-type poliovirus as measured by immunoprecipitation of the HIV-1-P1 fusion proteins with HIV-1-specific antibodies. The results of this study demonstrate the encapsidation of poliovirus minireplicons which express foreign proteins and point to the future use of this system as a potential vaccine vector.

Defective interfering (DI) RNA genomes of poliovirus which contain in-frame deletions in the P1 capsid protein-encoding region have been described. DI genomes are capable of replication and can be encapsidated by capsid proteins provided in trans from wild-type poliovirus. In this report, we demonstrate that a previously described poliovirus DI genome (K. Hagino-Yamagishi and A. Nomoto, J. Virol. 63:5386-5392, 1989) can be complemented by a recombinant vaccinia virus, VVP1 (D. C. Ansardi, D. C. Porter, and C. D. Morrow, J. Virol. 65:2088-2092, 1991), which expresses the poliovirus capsid precursor polyprotein, P1. Stocks of defective polioviruses were generated by transfecting in vitro-transcribed defective genome RNA derived from plasmid pSM1(T7)1 into HeLa cells infected with VVP1 and were maintained by serial passage in the presence of VVP1. Encapsidation of the defective poliovirus genome was demonstrated by characterizing poliovirus-specific protein expression in cells infected with preparations of defective poliovirus and by Northern (RNA) blot analysis of poliovirus-specific RNA incorporated into defective poliovirus particles. Cells infected with preparations of defective poliovirus expressed poliovirus protein 3CD but did not express capsid proteins derived from a full-length P1 precursor. Poliovirus-specific RNA encapsidated in viral particles generated in cells coinfected with VVP1 and defective poliovirus migrated slightly faster on formaldehyde-agarose gels than wild-type poliovirus RNA, demonstrating maintenance of the genomic deletion. By metabolic radiolabeling with [35S]methionine-cysteine, the defective poliovirus particles were shown to contain appropriate mature-virion proteins. This is the first report of the generation of a pure population of defective polioviruses free of contaminating wild-type poliovirus. We demonstrate the use of this recombinant vaccinia virus-defective poliovirus genome complementation system for studying the effects of a defined mutation in the P1 capsid precursor on virus assembly. Following removal of residual VVP1 from defective poliovirus preparations, processing and assembly of poliovirus capsid proteins derived from a nonmyristylated P1 precursor expressed by a recombinant vaccinia virus, VVP1 myr- (D. C. Ansardi, D. C. Porter, and C. D. Morrow, J. Virol. 66:4556-4563, 1992), in cells coinfected with defective poliovirus were analyzed. Capsid proteins generated from nonmyristylated P1 did not assemble detectable levels of mature virions but did assemble, at low levels, into empty capsids.(ABSTRACT TRUNCATED AT 400 WORDS)

The poliovirus RNA-dependent RNA polymerase (3Dpol) shares a region of homology with all RNA polymerases, centered around the amino acid motif YGDD, which has been postulated to be involved in the catalytic activity of the enzyme. Using oligonucleotide site-directed mutagenesis, we substituted the tyrosine at this motif of the poliovirus RNA-dependent RNA polymerase with cysteine, histidine, isoleucine, methionine, phenylalanine, or serine. The enzymes were expressed in Escherichia coli, and in vitro enzyme activity was tested. The phenylalanine and methionine substitutions resulted in enzymes with activity equal to that of the wild-type enzyme. The cysteine substitution resulted in an enzyme with approximately 50% of the wild-type activity, while the serine substitution resulted in an enzyme with approximately 10% of the wild-type activity; the isoleucine and histidine substitutions resulted in background levels of enzyme activity. To assess the effects of the mutants in viral replication, the mutant polymerase genes were subcloned into the infectious cDNA clone of poliovirus. Transfection of poliovirus cDNA containing the phenylalanine mutation in 3Dpol gave rise to virus in all of the transfection trials, while cDNA containing the methionine mutation resulted in virus in only 3 of 40 transfections. Transfection of cDNAs containing the other substitutions at the tyrosine residue did not result in infectious virus. The recovered viruses demonstrated kinetics of replication similar to those of the wild-type virus, as measured by [3H]uridine incorporation at either 37 or 39 degrees C. RNA sequence analysis of the 3Dpol gene of both viruses demonstrated that the tyrosine-to-phenylalanine or tyrosine-to-methionine mutation was still present. No other differences in the 3Dpol gene between the wild-type and phenylalanine-containing virus were found. The virus containing the methionine mutation also contained two other nucleotide changes from the wild-type 3Dpol sequence; one resulted in a glutamic acid-to-aspartic acid change at amino acid 108 of the polymerase, and the other resulted in a C-to-T base change at nucleotide 6724, which did not result in an amino acid change. To confirm that the second amino acid mutation found in the 3Dpol gene of the methionine-substituted virus allowed for replication ability, a mutation corresponding to the glutamic acid-to-aspartic acid change was made in the polymerase containing the methionine substitution, and this double-mutant polymerase was expressed in E. coli. The double-mutant enzyme was as active as the wild-type enzyme under in vitro assay conditions.(ABSTRACT TRUNCATED AT 400 WORDS)

The poliovirus capsid precursor polyprotein, P1, is cotranslationally modified by the addition of myristic acid. We have examined the importance of myristylation of the P1 capsid precursor during the poliovirus assembly process by using a recently described recombinant vaccinia virus expression system which allows the independent production of the poliovirus P1 protein and the poliovirus 3CD proteinase (D. C. Ansardi, D. C. Porter, and C. D. Morrow, J. Virol. 65:2088-2092, 1991). We constructed a site-directed mutation in the poliovirus cDNA encoding an alanine at the second amino acid position of P1 in place of the glycine residue required for the myristic acid addition and isolated a recombinant vaccinia virus (VVP1myr-) that expressed a nonmyristylated form of the P1 capsid precursor. The 3CD proteinase expressed by a coinfecting vaccinia virus, VVP3, proteolytically processed the nonmyristylated precursor P1 expressed by VVP1myr-. However, the processed capsid proteins, VP0, VP3, and VP1, did not assemble into 14S or 75S subviral particles, in contrast to the VP0, VP3, and VP1 proteins derived from the myristylated P1 precursor. When cells were coinfected with VVP1myr- and poliovirus type 1, the nonmyristylated P1 precursor expressed by VVP1myr- was processed by 3CD expressed by poliovirus, and the nonmyristylated VP0-VP3-VP1 (VP0-3-1) protomers were incorporated into capsid particles and virions which sedimented through a 30% sucrose cushion. Thus, the nonmyristylated P1 precursor and VP0-3-1 protomers were not excluded from sites of virion assembly, and the assembly defects observed for the nonmyristylated protomers were overcome in the presence of myristylated capsid protomers expressed by poliovirus. We conclude that myristylation of the poliovirus P1 capsid precursor plays an important role during poliovirus assembly by facilitating the appropriate interactions required between 5S protomer subunits to form stable 14S pentamers. The results of these studies demonstrate that the independent expression of the poliovirus P1 and 3CD proteins by using recombinant vaccinia viruses provides a unique experimental tool for analyzing the dynamics of the poliovirus assembly process.

RNA-dependent RNA polymerases contain a highly conserved region of amino acids with a core segment composed of the amino acids YGDD which have been hypothesized to be at or near the catalytic active site of the molecule. Six mutations in this conserved YGDD region of the poliovirus RNA-dependent RNA polymerase were made by using oligonucleotide site-directed DNA mutagenesis of the poliovirus cDNA to substitute A, C, M, P, S, or V for the amino acid G. The mutant polymerase genes were expressed in Escherichia coli, and the purified RNA polymerases were tested for in vitro enzyme activity. Two of the mutant RNA polymerases (those in which the glycine residue was replaced with alanine or serine) exhibited in vitro enzymatic activity ranging from 5 to 20% of wild-type activity, while the remaining mutant RNA polymerases were inactive. Alterations in the in vitro reaction conditions by modification of temperature, metal ion concentration, or pH resulted in no significant differences in the activities of the mutant RNA polymerases relative to that of the wild-type enzyme. An antipeptide antibody directed against the wild-type core amino acid segment containing the YGDD region of the poliovirus polymerase reacted with the wild-type recombinant RNA polymerase and to a limited extent with the two enzymatically active mutant polymerases; the antipeptide antibody did not react with the mutant RNA polymerases which did not have in vitro enzyme activity. These results are discussed in the context of secondary-structure predictions for the core segment containing the conserved YGDD amino acids in the poliovirus RNA polymerase.

The assembly process of poliovirus occurs via an ordered proteolytic processing of the capsid precursor protein, P1, by the virus-encoded proteinase 3CD. To further delineate this process, we have isolated a recombinant vaccinia virus which expresses, upon infection, the poliovirus P1 capsid precursor polyprotein with an authentic carboxy terminus. Coinfection of HeLa cells with the P1-expressing vaccinia virus and with a second recombinant vaccinia virus which expresses the poliovirus proteinase 3CD resulted in the correct processing of P1 to yield the three individual capsid proteins VP0, VP3, and VP1. When extracts from coinfected cells were fractionated on sucrose density gradients, the VP0, VP3, and VP1 capsid proteins were immunoprecipitated with type 1 poliovirus antisera from fractions corresponding to a sedimentation consistent for poliovirus 75S procapsids. Examination of these fractions by electron microscopy revealed structures which lacked electron-dense cores and which corresponded in size and shape to those expected for poliovirus empty capsids. We conclude that the expression of the two poliovirus proteins P1 and 3CD in coinfected cells is sufficient for the correct processing of the capsid precursor to VP0, VP3, and VP1 as well as for the assembly of poliovirus empty capsid-like structures.

The poliovirus genome is replicated by a virus-encoded RNA-dependent RNA polymerase (RNA polymerase). The RNA polymerase is first synthesized as a larger precursor polypeptide, which is subsequently processed by a viral proteinase, 3Cpro, to give the mature polymerase molecule, 3Dpol. To further characterize the poliovirus RNA polymerase, we have constructed plasmids that expressed this protein in Escherichia coli. The plasmids consisted of fusions between the E. coli DNA encoding the first 13 amino acids of the trp operon leader protein and viral genes encoding the 3Cpro and 3Dpol polypeptides. E. coli harboring such plasmids gave significant, inducible levels of enzymatically active RNA polymerase as determined by the poly(A).oligo(U) poly(U) polymerase assay. The purified RNA polymerase activity from E. coli corresponded to a protein with the approximate molecular weight of the mature 3Dpol protein. The availability of a recombinant, enzymatically active poliovirus RNA polymerase provides a system in which we can precisely delineate the role this enzyme plays in the regulation of poliovirus replication.

Poliovirus replicase- and host factor-catalyzed copying of 3'-terminal polyadenylic acid [poly(A)] of poliovirion RNA was studied. Host factor-stimulated synthesis of polyuridylic acid [poly(U)] by the replicase required ATP in addition to UTP. ATP was not required for the oligouridylic acid-primed copying of 3'-terminal poly(A) of virion RNA. GTP, CTP, and AMP-PCP (5'-adenylyl beta-gamma methylenediphosphate, an ATP analog) could not replace ATP in host factor-stimulated synthesis of poly(U). Antibodies to poliovirus genome-linked protein (VPg) specifically precipitated in vitro-synthesized poly(U) from a host factor-stimulated reaction. The poly(U) synthesized in a host factor-stimulated reaction was shown to be attached to VPg precursor polypeptide(s) via a tyrosine-phosphate bond as found in poliovirion VPg-RNA.

The HeLa cell protein (host factor) required for in vitro replication of poliovirus has been identified as a 67,000 dalton phosphoprotein. The purified protein displays three activities in vitro: stimulation of poliovirus RNA synthesis in the presence of poliovirus replicase, apparent self-phosphorylation, and phosphorylation of the alpha-subunit of eukaryotic protein synthesis initiation factor 2 (eIF-2). All three activities can be removed or inhibited by an antibody to host factor. Partially purified preparations of reticulocyte eIF-2 contain a similar phosphoprotein and display host factor activity in the viral RNA synthesis assay in vitro. In vitro phosphorylation of the 67 kd protein can be stimulated by low concentrations of double-stranded RNA. Addition of phosphorylated host factor in an in vitro RNA synthesis assay significantly changes the kinetics of viral RNA synthesis, indicating that protein phosphorylation may play an important role in viral RNA replication.

A synthetic nonapeptide corresponding to the N-terminal sequence of poliovirus genome-linked protein (VPg) was linked to bovine serum albumin and used to raise antibodies in rabbits. The antipeptide antibodies specifically precipitated the nonapeptide, native VPg, and VPg-linked poliovirion RNA. The antipeptide antibodies inhibited host factor-stimulated, poliovirus replicase-catalyzed in vitro synthesis of full-length (35S) RNA in response to virion RNA. Oligouridylic acid-stimulated RNA synthesis was not affected by the antipeptide antibodies. Preincubation of the antibodies with excess nonapeptide reversed the antipeptide antibody-mediated inhibition of host factor-stimulated RNA synthesis by the poliovirus replicase. A role for VPg in the in vitro replication of poliovirus RNA genome is discussed.