Poliovirus temperature-sensitive mutant containing a single nucleotide deletion in the 5'-noncoding region of the viral RNA

The effect on viral replication of deleting nucleotide 10 of the poliovirus RNA genome was determined. This deletion, which removes a base pair from a predicted hairpin structure in the viral RNA, was introduced into full-length cDNA. Virus recovered after transfection of HeLa cells with the mutated cDNA contained the expected deletion and was temperature sensitive for plaque formation. Analysis of viral replication by one-step growth experiments indicated that mutant virus production at the nonpermissive temperature was at least 100 times less than that of wild type virus, and release of virus from mutant-infected cells was delayed. The synthesis of positive- and negative-strand viral RNA in mutant virus-infected cells was temperature sensitive. Virus-specific protein synthesis in mutant virus-infected cells was not temperature sensitive but occurred at a slower rate than that of wild type virus at permissive and nonpermissive temperatures. Replication of the mutant virus was sensitive to actinomycin D, in contrast to the wild type parent virus, which was resistant to the drug. Mutant virus stocks contained a small percentage of ts+ viruses that were able to form plaques at the nonpermissive temperature. Nucleotide sequence analysis of genomic RNA from these ts+ viruses revealed a single base change at position 34 from a G to U. In the positive RNA strand, the effect of this mutation is to restore to the hairpin structure the single base pair whose formation was prevented by the original deletion. The ts+ pseudorevertants replicated to similar titers as wild type virus at 33 and 38.5 degrees and were partially sensitive to actinomycin D.     

A poliovirus mutant defective for self-cleavage at the COOH-terminus of the 3C protease exhibits secondary processing defects

By in vitro recombination between the wild-type full-length infectious cDNA of poliovirus and a clone generated by the construction of a cDNA bank from a chemically derived temperature-sensitive plurimutant, we obtained a mutant cDNA with a T to C change at nucleotide 5658. This mutation replaces the isoleucine at residue 74 of the viral protease 3C by a threonine. The mutant virus recovered after transfection exhibited a small-plaque phenotype, and was deficient for viral RNA synthesis. Both these defects were more marked at 39 than at 37 degrees. The mutation was introduced into a bacterial plasmid which expresses the 3C protease along with its flanking autocatalytic cleavage sites. Analysis of the cleavage products expressed in Escherichia coli provided direct evidence that the modification impaired cleavage at the COOH-terminus of 3C. Cleavage at this same site was partially defective in mutant virus-infected HeLa cells, reducing the production of mature 3C and the viral replicase, 3D. Cleavage of P1, the precursor to the capsid polypeptides, was apparently unaffected by this defect, whereas cleavage events within the P2 region of the genome occurred inefficiently. This is indicative of differential strategies for 3C-specific cleavage events in vivo.     

E1 protein of bovine papillomavirus 1 is not required for the maintenance of viral plasmid DNA replication

Derivatives of bovine papillomavirus 1 (BPV1) with temperature-sensitive and dominant-negative mutation the E1 gene were used to determine the requirement for E1 in the maintenance of viral plasmid DNA replication. The abilities of these mutant BPV1 genomes to replicate as nuclear plasmids were monitored at permissive (32 degrees C) and nonpermissive (37 degrees C) temperatures in mouse C127 cells. We found that the temperature-sensitive E1 mutant BPV1 genomes replicate as nuclear plasmids as efficiently as does wild-type BPV1 in C127 cells after shifting to the nonpermissive temperature. These findings indicate that BPV1 does not require E1 for the maintenance of viral plasmids.     

Replication of eastern equine encephalitis viruses (New Jersey and Louisiana strains and the Ets-4 mutant) in rabbit kidney cells

Summary Virus yield, viral RNA synthesis, viral protein synthesis, cytopathology, and virus-induced shutoff of host protein synthesis were examined in rabbit kidney (RK) cells infected with eastern equine encephalitis (EEE) viruses. The New Jersey (NJ) strain replicated most rapidly, and exhibited a distinct pattern in the synthesis of structural and non-structural viral proteins. The Louisiana (La) strain and the Ets-4 mutant derived from it replicated more slowly than NJ, and synthesized most of their viral proteins simultaneously. The properties of the Ets-4 mutant appeared to be similar in RK cells and chick embryo fibroblasts. Although the viral peptides comprising the viral RNA polymerase could not be conclusively identified, two non-structural proteins of molecular weights 105,000 and 85,000 were temporally associated with increasing rates of polymerase activity in NJ-infected RK cells and were found in increased amounts in Ets-4-in-fected cells compared to La-infected cells.With 6 FiguresThis work was supported in part by NIH Grant 5 R 22-AI-02686.     

Efficient rescue of infectious bursal disease virus from cloned cDNA: evidence for involvement of the 3'-terminal sequence in genome replication

To study the mechanism of replication of infectious bursal disease virus (IBDV), and to determine factors on the IBDV RNA which are involved in viral replication, we used cloned full-length cDNA of both the A- and B-segments to generate infectious IBDV. Infectious IBDV was rescued from plasmids that contained full-length IBDV cDNA behind a T7 promoter, by transfecting these plasmids into cells which were infected with a recombinant Fowlpox virus that expressed T7 RNA polymerase. By using the cDNA transfection system we evaluated the effect of the length of the 3' terminus of the A-segment plus strand of IBDV. Although wild-type IBDV predominantly contains four cytosines at the 3' terminus, no difference in virus yield was found when virus was rescued from cDNAs containing three to six adjacent cytosines. When the 3' terminus was shorter than three cytosines the efficiency to generate infectious IBDV from cDNA was reduced, but IBDV could still be recovered reproducibly. The rescued viruses from cDNAs containing 3'-terminal deletions appeared to have a restored 3'-terminal sequence. The missing nucleotides are probably restored by using complementary bases of a stem-loop structure as template.     

Human rhinovirus serotype 2: in vitro synthesis of an infectious RNA

A complete cDNA copy of human rhinovirus serotype 2 RNA was placed under the control of a T7 RNA polymerase promoter. An in vitro transcribed RNA containing two extra G residues at the 5' end gave rise to plaques on transfection into HeLa cells. The efficiency was approximately half that obtained with viral RNA. On the contrary, an in vitro synthesized RNA containing 16 additional nucleotides at the 5' end was not infectious. This ability to make an infectious in vitro transcribed RNA will be useful in studying the characteristics of viruses using the human rhinoviral minor group receptor.     

Mutations in conserved domains IV and VI of the large (L) subunit of the sendai virus RNA polymerase give a spectrum of defective RNA synthesis phenotypes

The Sendai virus RNA polymerase is a complex of two virus-encoded proteins, the phosphoprotein (P) and the large (L) protein. When aligned with amino acid sequences of L proteins from other negative-sense RNA viruses, the Sendai L protein contains six regions of good conservation, designated domains I-VI, which have been postulated to be important for the various enzymatic activities of the polymerase. To directly address the roles of domains IV and VI, 14 site-directed mutations were constructed either by changing clustered charged amino acids to ala or by substituting selected Sendai L amino acids with the corresponding sequence from measles virus L. Each mutant L protein was tested for its ability to transcribe and replicate the Sendai genome. The series of mutations created a spectrum of phenotypes, from those with significant, near wild-type, activity to those being completely defective for all RNA synthesis. The inactive L proteins, however, were still able to bind P protein and form a polymerase capable of binding the nucleocapsid template. The remainder of the mutations reduced, but did not abolish, enzymatic activity and included one mutant with a specific defect in the synthesis of the leader RNA compared with mRNA, and three mutants that replicated genome RNA much more efficiently in vivo than in vitro. Together, these data suggest that even within a domain, the function of the Sendai L protein is likely to be very complex. In addition, SS3 and SS10 L in domain IV and SS13 L in domain VI were shown to be temperature-sensitive. Both SS3 and SS10 gave significant, although not wild-type, activity at 32 degrees C; however, each was completely inactivated for all RNA synthesis at 37 and 39.6 degrees C. SS13 was completely inactive only when synthesized at the higher temperature. Each polymerase synthesized at 32 degrees C could only be partially heat inactivated in vitro at 39.6 degrees C, suggesting that inactivation involves both thermal lability of the protein and temperature sensitivity for its synthesis.     

Immunological analysis of 140-kDa adenovirus-encoded DNA polymerase in adenovirus type 2-infected HeLa cells using antibodies raised against the protein expressed in Escherichia Coli

The E2B region of adenovirus genome contains a long open reading frame (ORF) extending from 24 to 14.2 map units which encodes most of the 140-kDa DNA polymerase. It was cloned at the polylinker region of pUC18 vector with Escherichia coli JM109 as the host. A clone was serendipitously isolated that expressed in E. coli a protein of approximately 120 kDa in size at high levels. DNA sequence analysis of this clone showed the presence of an in-frame fusion of a region, encoding 13 amino acids located upstream, to the first ATG of the ORF. Polyclonal antibodies raised against this protein purified from E. coli were used for immunological analysis. The antibodies were able to detect a 140- and a 66-kDa polypeptide from the adenovirus type 2-infected HeLa cells on Western blots. In addition, the antibodies showed evidence of cross-reactivity with partially purified DNA polymerase alpha from uninfected HeLa cells. The subcellular localization of the viral polymerase in the infected HeLa cells by using indirect immunofluorescence showed that the viral protein is associated with globular structures in the nucleus. The replicating viral DNA and the polymerase were colocalized in these globular sites. Furthermore, HeLa cells infected with Ad5ts149, a temperature-sensitive mutant defective in DNA replication, showed the presence of these globular sites only at the permissive temperature, suggesting that these sites are probably involved in viral DNA replication.     

Characterization of a foot-and-mouth disease mutant temperature-sensitive for viral RNA synthesis

Summary A temperature-sensitive (ts) mutant of foot-and-mouth disease virus (FMDV) did not produce RNA polymerase activity nor synthesize viral RNA when incubated in cells solely at the nonpermissive temperature (38.5° C). Infected cells initially incubated at 38.5° C and then shifted down to 33° C synthesized increased amounts of viral RNA at earlier times compared to infected cells kept at 33° C throughout, indicating that RNA polymerase precursors were synthesized at 38.5° C. In cells shifted up to 38.5° C from 33° C, the total amount of viral RNA synthesized after infection increased sharply for about 15 minutes and then rapidly decreased over the next 2 hours. RNA polymerase activity presented a similar pattern in its initial twofold increase and subsequent rapid decrease. Pulse labeling experiments showed that mutant viral RNA synthesis continued at a diminishing rate for 2 hours in cells shifted up to 38.5° C. The data from temperature shift experiments indicated that essentially only the mutant RNA formed after shift-up was degraded. The FMDVts mutant is apparently additionally defective in being unable to protect viral RNA synthesized after shift-up to 38.5° C.With 5 FiguresMention of a trademark of proprietary product does not constitute a guarantee or warranty of the product by the U.S. Department of Agriculture and does not imply its approval to the exclusion of other products that may also be suitable.     

我国登革2、3、4型病毒全长cDNA体外RNA转录体的感染性研究

目的 研究我国登革2、3、4型病毒全长cDNA体外RNA转录体的感染性，为构建登革病毒的感染性克隆、深入阐明病毒的致病机理奠定基础。方法 以我国登革2、3、4型病毒基因组全长cDNA为模板，用SP6 RNA聚合酶系统制备体外RNA转录体，经电穿孔转染细胞，观察其致细胞病变效应。从病变细胞培养上清中提取总RNA，用病毒特异引物进行RT-PCR扩增，以证实细胞病变为转录体RNA的恢复病毒所致。结果 我国登革2、3、4型病毒全长cDNA的体外RNA转录体均可使细胞产生病变，从细胞培养上清中可扩增出登革病毒特异的片段。结论 构建的我国登革2、3、4型病毒全长cDNA的体外RNA转录体具有感染性．可在蚊传代细胞内恢复为登革病毒颗粒。     

Expression and characterization of poliovirus proteins 3BVPg, 3Cpro, and 3Dpol in recombinant baculovirus-infected Spodoptera frugiperda cells.

As an initial step toward investigating the roles of poliovirus proteins in viral RNA replication, a baculovirus expression system was used to produce poliovirus proteins from the P3 region. Spodoptera frugiperda (Sf9) cells were infected with a recombinant baculovirus, vETL-PoV3A*BCD, which contains cDNA coding for poliovirus proteins 3D, 3C, 3B, and a portion of 3A protein sequence. Immunofluorescence microscopy revealed that the majority of 3D (polymerase) was in the cytoplasm of recombinant baculovirus-infected Sf9 cells. In the same cells, the 3C (protease) and 3B (VPg) proteins appeared to be located in distinct subcellular regions, possibly membrane structures, suggesting that the expressed polyprotein was cleaved to generate mature proteins. Processing of the polypeptide was confirmed by immunoblot analysis which demonstrated that 3Cpro sequences were active in cleavage of the polyproteins 3A*BCD and 3CD. Over 95% of the 3D sequences accumulated in the form of mature 3Dpol, with only low levels of 3CD remaining. The majority of 3Dpol remained in the supernatant after low speed centrifugation of sonicated cells. The 3Dpol had RNA-dependent RNA polymerase activity as measured by elongation of an oligo(U) primer using a poly(A) template. The protein 3CDpro was active in cleaving P1 protein. The yield and activities of the poliovirus proteins expressed will facilitate future biochemical studies.     

A site on the influenza A virus NS1 protein mediates both inhibition of PKR activation and temporal regulation of viral RNA synthesis

It is not known how influenza A viruses, important human pathogens, counter PKR activation, a crucial host antiviral response. Here we elucidate this mechanism. We show that the direct binding of PKR to the NS1 protein in vitro that results in inhibition of PKR activation requires the NS1 123-127 amino acid sequence. To establish whether such direct binding of PKR to the NS1 protein is responsible for inhibiting PKR activation in infected cells, we generated recombinant influenza A/Udorn/72 viruses expressing NS1 proteins in which amino acids 123/124 or 126/127 are changed to alanines. In cells infected with these mutant viruses, PKR is activated, eIF-2alpha is phosphorylated and viral protein synthesis is inhibited, indicating that direct binding of PKR to the 123-127 sequence of the NS1 protein is necessary and sufficient to block PKR activation in influenza A virus-infected cells. Unexpectedly, the 123/124 mutant virus is not attenuated because reduced viral protein synthesis is offset by enhanced viral RNA synthesis at very early times of infection. These early viral RNAs include those synthesized predominantly at later times during wild-type virus infection, demonstrating that wild-type temporal regulation of viral RNA synthesis is absent in 123/124 virus-infected cells. Enhanced early viral RNA synthesis after 123/124 virus infection also occurs in mouse PKR-/- cells, demonstrating that PKR activation and deregulation of the time course of viral RNA synthesis are not coupled. These results indicate that the 123/124 site of the NS1A protein most likely functionally interacts with the viral polymerase to mediate temporal regulation of viral RNA synthesis. This interaction would occur in the nucleus, whereas PKR would bind to NS1A proteins in the cytoplasm prior to their import into the nucleus.     

An efficient helper-virus-free method for rescue of recombinant paramyxoviruses and rhadoviruses from a cell line suitable for vaccine development

Recovery of recombinant, negative-strand, nonsegmented RNA viruses from a genomic cDNA clone requires a rescue system that promotes de novo assembly of a functional ribonucleoprotein (RNP) complex in the cell cytoplasm. This is accomplished typically by cotransfecting permissive cells with multiple plasmids that encode the positive-sense genomic RNA, the nucleocapsid protein (N or NP), and the two subunits of the viral RNA-dependent RNA polymerase (L and P). The transfected plasmids are transcribed in the cell cytoplasm by phage T7 RNA polymerase (T7 RNAP), which usually is supplied by infection with a recombinant vaccinia virus or through use of a stable cell line that expresses the polymerase. Although both methods of providing T7 RNAP are effective neither is ideal for viral vaccine development for a number of reasons. Therefore, it was necessary to modify existing technology to make it possible to routinely rescue a variety of recombinant viruses when T7 RNAP was provided by a cotransfected expression plasmid. Development of a broadly applicable procedure required optimization of the helper-virus-free methodology, which resulted in several modifications that improved rescue efficiency such as inclusion of plasmids encoding viral glycoproteins and matrix protein, heat shock treatment, and use of electroporation. The combined effect of these enhancements produced several important benefits including: (1) a helper-virus-free methodology capable of rescuing a diverse variety of paramyxoviruses and recombinant vesicular stomatitis virus (rVSV); (2) methodology that functioned effectively when using Vero cells, a suitable substrate for vaccine production; and (3) a method that enabled rescue of highly attenuated recombinant viruses, which had proven refractory to rescue using published procedures.