Substitution in the poliovirus replicase gene determines actinomycin D sensitivity of viral replication at elevated temperature

A series of ts+ revertants and recombinants derived from a temperature-sensitive plurimutant of poliovirus type 1 showed identical plaquing efficiencies at 37 degrees C and at 39 degrees C and exhibited similar yields and plaque morphology to wild-type virus. However, these viruses were characterized by clear inhibition of viral RNA synthesis at 39 degrees C, as measured by uridine incorporation in the presence of actinomycin D. Similarly, virus yields were decreased by one log in the presence of actinomycin D during infection at 39 degrees C. All the ts+ recombinants formed between temperature-sensitive mutants of poliovirus that were inhibited by actinomycin D carried a glutamine----histidine modification at residue 170 of their viral replicase (polypeptide 3D), due to a G----U substitution at nucleotide 6496. Inhibition of viral growth was increased by pretreatment of cells with actinomycin D for 3 h prior to infection, suggesting that actinomycin D sensitivity could reflect an increased dependence of viral RNA replication on host factor(s).     

The herpes simplex virus UL33 gene product is required for the assembly of full capsids.

Phenotypic analysis of the herpes simplex virus type 1 temperature-sensitive DNA-positive mutant, ts1233, revealed that the mutant had a structural defect at the nonpermissive temperature (NPT). Cells infected with ts1233 at the NPT contained large numbers of intermediate capsids, lacking dense cores but possessing some internal structure. No full capsids or enveloped virus particles were detected. In contrast to the defect in another packaging-deficient mutant ts1201, the block in the formation of dense-cored, DNA-containing capsids in ts1233-infected cells at the NPT could not be reversed by transferring the cells to the permissive temperature in the presence of a protein synthesis inhibitor. Furthermore, the capsids produced by ts1233 at the NPT had more compact internal structures than those of the gene UL26 mutant ts1201. Southern blot analysis of viral DNA in ts1233-infected cells confirmed that the mutant DNA was not encapsidated at the NPT and showed that the unpackaged DNA was not cleaved into genome-length molecules. The ts1233 mutation was mapped by marker rescue to the vicinity of genes UL32 and UL33. Sequence analysis of the DNA in this region from the mutant and two independently isolated revertants for growth revealed that ts1233 had a single base-pair change at the amino-terminal end of UL33, resulting in the substitution of an isoleucine with an asparagine. The nucleotide sequence of the revertants in this part of the genome was identical to that of wild-type virus.     

Temperature sensitive shut-off of alphavirus minus strand RNA synthesis maps to a nonstructural protein, nsP4

Minus strand RNA synthesis by the positive strand alphaviruses, Sindbis and Semliki Forest viruses, normally occurs early in infection, is coupled to synthesis of viral nonstructural proteins and to formation of viral replication complexes, and terminates and does not occur late in infection. Previously, ts24 of the A complementation group of Sindbis virus RNA-negative mutants was found to possess, among its other temperature sensitive defects, a temperature sensitivity in the normal cessation of minus strand synthesis which enabled minus strands to be synthesized late in infection at 40 degrees in the absence of protein synthesis. Revertants of ts24 (ts24R1, ts24R2) retained the defect in the shutoff of minus strand synthesis, indicating the lesion was not conditionally lethal and could map outside the A cistron. The studies reported here used an infectious clone of Sindbis virus to identify the mutation responsible for this phenotype. Hybrid viruses were prepared from constructs containing restriction fragments of the cDNA of ts24R1 in place of the corresponding fragments in the infectious SIN HR clone and screened for their ability to synthesize minus strands at 40 degrees in the presence of cycloheximide. A unique base change of an A for a C residue at nt 6339, predicting a change from glutamine to lysine at amino acid 195 in nsP4, was found in genomes of ts24, ts24R1, and ts24R2. Other nucleotide changes present at the 5' and 3' termini did not affect minus strand synthesis. The substitution of the parental Sindbis virus sequence that encompassed nt 6339 in an infectious clone of the ts24R1 revertant eliminated the mutant phenotype. We conclude that the ability to continue minus strand synthesis at 40 degrees exhibited by ts24 and its revertants is caused by an alteration in nsP4, which is the alphavirus replicase or an essential component of the replicase. We hypothesize that this domain of nsP4 functions to fix the minus strand as the stable template of alphavirus replication complexes.     

Multiple mutations involved in the phenotype of a temperature-sensitive small-plaque mutant of poliovirus

A temperature-sensitive small-plaque mutant of poliovirus type 1, ts247, has been analyzed previously. Several mutations were detected in the P3 region of the genome by analysis of proteins and by T1 oligonucleotide mapping of viral RNA. We have now studied spontaneous reversion of ts247 to the wild-type phenotype. This was found to be a two-step event, reversion to a ts+ phenotype (revertant R247-51) being distinct from acquisition of normal plaque size (revertant R247-12). The mutation responsible for the ts phenotype of ts247, implicated also in virus aggregation and heat lability, could not be detected by biochemical studies. Analysis of homotypic recombinants obtained by crossing ts247 with a guanidine-resistant derivative of a temperature-sensitive replicase mutant mapped this mutation to the P1 region or to the 5' end of the P2 region of the genome. The small-plaque phenotype of ts247 and R247-51 was correlated with an abnormality in polypeptide 3C (protease); direct sequencing of viral RNA revealed a U to C change at nucleotide 5658, which altered an isoleucine to threonine in the protease of ts247 and R247-51 but not of R247-12. Two other mutations were present in the region of the genome coding for polypeptide 3D of ts247 and of both classes of revertants. They thus seemed to play no role in the phenotype of ts247. One mutation, an A to G change at nucleotide 7135, was silent at the protein level, whereas the other, an A to G change at nucleotide 6264, determined a major amino acid change from glutamate to glycine in the viral replicase.     

Avian retrovirus integration protein: structure-functional analysis of viable mutants

A replication-competent avian retrovirus mutant, containing a single amino acid substitution at amino acid residue 115 in the 3' endonuclease (IN) region of the polymerase (pol) gene, was characterized. DNA transfection experiments demonstrated that the mutant virus exhibited a delayed growth phenotype at 41 degrees while replicating efficiently at 35 degrees. Examination of virus-infected cells at the molecular level demonstrated that the mutant virus at either temperature was capable of synthesizing viral DNA as efficiently as wild-type Rous sarcoma virus, strain Prague A. This result suggested that the same mutation, which was also present in the IN moeity of the polymerase beta polypeptide, did not affect DNA synthesis. Further analyses demonstrated that at either temperature the mutant virus integrated its DNA at about 10-20% of wild-type level, although possibly less efficiently at 41 degrees than at 35 degrees. The mutation at residue 115 (Pro to Ser) appeared to lower the ability of IN to function in the integration of viral DNA relative to wild-type virus. No definitive conclusion could be made as to whether IN in this mutant possessed a temperature-sensitive lesion which caused the observed replication defect at 41 degrees.     

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.     

The herpes simplex virus type 1 temperature-sensitive mutant ts1222 has a single base pair deletion in the small subunit of ribonucleotide reductase

The herpes simplex virus type 1 (HSV-1) temperature-sensitive (ts) mutant, ts1222, has a defect within the gene specifying the small subunit of ribonucleotide reductase. Sequence determination of the lesion revealed that the mutant DNA had a single base pair deletion at the 3' end of the gene. The mutation altered the translational reading frame such that the codons of all but one of the last 15 amino acids of the protein were changed and the termination codon removed. Although ts1222 did not induce detectable amounts of enzyme activity at both 31 degrees and 39.5 degrees, it replicated as well as wild-type virus at 31 degrees in exponentially growing tissue culture cells under one step growth conditions. At 39.5 degrees, however, ts1222 behaved as a ts mutant. These findings suggest that at low temperatures the virus-coded enzyme is dispensable for virus growth in actively dividing tissue culture cells but at high temperatures the enzyme is essential for virus replication. Under these conditions altered properties of the host cell contribute to the ts phenotype of the mutant. In the presence of hydroxyurea, which inactivates both the cellular and virus ribonucleotide reductases, growth of the mutant at 31 degrees was inhibited more than wild-type virus replication. Growth of the mutant at the permissive temperature was also sensitive to high concentrations of thymidine whereas wild-type virus multiplication was resistant to the nucleoside. It is therefore likely that ts1222 is dependent on the cellular ribonucleotide reductase for growth at this temperature. In serum-starved cells, growth of the mutant virus at 31 degrees was severely impaired. Thus, like thymidine kinase, the HSV-coded ribonucleotide reductase is required for virus multiplication in resting tissue culture cells.     

Effect of temperature-sensitive, replication-defective mutations on RNA synthesis of cowpea chlorotic mottle virus

The RNA synthesis of replication-deficient, temperature-sensitive mutants of cowpea chlorotic mottle virus (CCMV) with mutations on either RNA 1 or RNA 3 was examined in temperature shift experiments. Viral RNA synthesis by the mutants at 25 degrees was similar to that of wild-type CCMV, but upon shift to 35 degrees , synthesis of mutant virus RNAs declined over a 16-hr period in contrast to continued synthesis by wild-type CCMV. Continued RNA synthesis, though at a reduced level, by the mutants during the initial periods following the shift to 35 degrees demonstrated that the enzymes involved in viral RNA synthesis of the mutants continued activity after the shift to the nonpermissive temperature. There was no clear correlation between a specific, defect in viral RNA synthesis and whether the mutation occurred on either RNA 1 or RNA 3. New replicative complexes appeared not to be produced at 35 degrees suggesting that gene products from both RNA 1 and RNA 3 may function coordinately in the replication complex. One mutant produced a reduced ratio of RNA 3 that was exaggerated upon shift to the restrictive temperature.     

A mutant of sindbis virus with a host-dependent defect in maturation associated with hyperglycosylation of E2

Following serial passage of Sindbis virus (SV) on Aedes albopictus mosquito cells a mutant (SVap15/21) was isolated which in chick cells produced small plaques and was temperature sensitive (ts). At 34.5 degrees this mutant replicated normally in mosquito cells, but only poorly in chick or BHK cells. In the vertebrate cells SVap15/21 was RNA+ at both 34.5 and 40 degrees and on the basis of complementation tests carried out at 40 degrees, was assigned to complementation group E. The block in the replication of this mutant, like that of ts20, the prototype mutant of complementation group E, was at the level of nucleocapsid envelopment. The PE2 and E2 glycoproteins of SVap15/21 were found to be hyperglycosylated relative to the corresponding glycoproteins of the parent virus (SVstd). Analysis of revertants of SVap15/21 suggests a causal relationship between PE2 and E2 hyperglycosylation and the host-specific defect in virus maturation. The association of a host-specific defect in virion assembly with hyperglycosylation of a viral structural protein points to the potential importance of host-specific glycosylation patterns in the determination of viral host range.     

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.     

The effect of loss of regulation of minus-strand RNA synthesis on Sindbis virus replication

During the replication cycle of Sindbis virus minus-strand synthesis stops normally at the time that plus-strand synthesis reaches a maximum rate. We have isolated and characterized revertants of ts24, a member of the A complementation group of Sindbis HR mutants, that we had demonstrated previously to have a temperature-sensitive defect in the regulation of minus-strand synthesis. These revertants of ts24 replicated efficiently at 40 degrees but nevertheless retained the temperature sensitive defect in the regulation of minus-strand synthesis: they continued to synthesize minus strands late in the replication cycle at 40 degrees but not at 30 degrees and in the presence or absence of protein synthesis. Although failure to regulate the synthesis of minus strands resulted in continuous minus-strand synthesis and in the accumulation of minus strands, the rate of plus-strand synthesis was not increased concertedly. Minus strands synthesized after the rate of plus-strand synthesis had become constant were demonstrated to be utilized as templates for 26 S mRNA synthesis. Thus, the change from an increasing to a constant rate of plus-strand synthesis during the alphavirus replication cycle cannot be governed solely by the number of minus strands that accumulate in infected cells. We present a model for the preferential utilization of minus strands as a mechanism for the cessation of minus-strand synthesis that occurs normally during alphavirus replication.     

Spontaneous reversion of a C/T transition mutation in the adenovirus endoproteinase gene

A temperature-sensitive mutation (H2ts1) that abolishes the viral endoproteinase activity at the nonpermissive temperature has been mapped by marker rescue between map coordinates 59.8 and 61.9 on the adenovirus type 2 genome. The mutation has been identified by sequencing to be a C/T transition at coordinate 61.1 changing a proline residue to a leucine residue and eliminating a HaeIII restriction enzyme cleavage site (L. Yeh-Kai, G. Akusjarvi, P. Alestrom, U. Pettersson, M. Tremblay, and J. Weber, 1983, J. Mol. Biol. 167, 217-222). This feature of the mutation offered a convenient assay to distinguish between true revertants and suppressor mutations among phenotypic revertants of H2ts1. Seventeen spontaneous revertants were isolated in three independent experiments by picking plaques at 39 degrees after three passages of H2ts1 at 39 degrees in HEp2 cells. All revertants grew like wild-type virus and regained normal endoproteinase activity. The Ncol fragment encompassing the H2ts1 region was terminally labeled and subcleaved with HaeIII to determine the presence or absence of the HaeIII site at 61.1 for each revertant. All revertants had regained the HaeIII site by true reversion. We conclude that the H2ts1 mutation probably lies in a critical domain of the enzyme and is therefore not suppressible, and that the C/T transition at coordinate 61.1 is the sole cause of the H2ts1 phenotype.     

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.     

Characterization of an internal element in turnip crinkle virus RNA involved in both coat protein binding and replication.

The major coat-protein-binding element of turnip crinkle virus RNA was previously mapped in the region of the UAG termination codon in the viral polymerase gene. This region encompasses two of the high-affinity coat-protein-binding sites (Fa and Ff) that we suggested were physically associated in a stem-loop in a ribonucleoprotein complex involved in assembly initiation (Wei, Heaton, Morris, and Harrison, J. Mol. Biol. 214, 85-95, 1990). We have also demonstrated that this RNA element was capable of specific coat protein binding in vitro (Wei and Morris, J. Mol. Biol. 222, 437-443, 1991). We now provide physical evidence, by in vitro chemical and enzymatic probing of the viral RNA, that support the suggestion that the two coat-protein-binding sites base pair to form a stem structure (A/F stem) surrounding the UAG terminator in wild-type RNA. We have shown here that a mutant with seven conservative nucleotide substitutions in Fa does not accumulate to detectable levels in plants or protoplasts and that the A/F stem structure is drastically altered in this mutant. We suggest that the primary effect of this mutation is on replication rather than on a reduction in RNA stability resulting from a defect in encapsidation of the virion RNA because previous results have shown that encapsidation-deficient mutants have little or no effect on viral RNA replication (Hacker, Petty, Wei, and Morris, Virology 186, 1-8, 1992). The analysis of the A/F stem was extended by construction and characterization of a series of mutants and revertants that displayed variable levels of replication deficiency but minimal concomitant defect in encapsidation efficiency. The extent of the replication defect correlated with the predicted destabilization of the A/F stem structure. We conclude from these results that this RNA element is involved in viral replication, and we tentatively suggest that the A/F stem structure may be functionally involved in the readthrough translation of the viral polymerase.     

Analysis of the nonviral antigens immunoprecipitable by SV40 T antibody from SV40-transformed human/mouse hybrid cell lines

A temperature-sensitive (ts) mutant of herpes simplex virus type 1 (HSV-1), tsJ12, is able to undergo one cycle of replication at the nonpermissive temperature (39°) yielding wild-type quantities of enveloped virus particles. These particles contain viral DNA which is as infectious as wild-type viral DNA; however, they are not infectious. Analysis of [¹⁴C]glucosamine-labeled mutant-infected cell extracts by one- and two-dimensional polyacrylamide gel electrophoresis demonstrated that at 39° tsJ12 fails to induce the synthesis of both the mature gB glycoprotein and its dimeric form which are normal constituents of the virion envelope. Polyethylene glycol, an agent which promotes membrane fusion, enhances the infectivity of tsJ12 virions by greater than 1000-fold following adsorption of virus to susceptible cells demonstrating that mutant virions are able to attach to cells but not penetrate. Consistent with a defect in the virion envelope, tsJ12 is able to interfere with the production of infectious wild-type virus, presumably by the formation of pseudotypic virions composed of wild-type viral genomes in gB-deficient envelopes. Physical mapping of the is defect in this mutant demonstrates that it lies within the limits of the DNA sequence which specifies gB on the physical map of the genome. A ts⁺ revertant of tsJ12 is as infectious as wild-type virus and synthesizes a gB glycoprotein which is indistinguishable from that of wild-type virus. Thus, biological and biochemical studies of tsJ12 and of a ts+ revertant of this mutant (1) demonstrate that glycoprotein gB is essential for infectivity at the level of penetration and (2) further define the physical map location of the gene for this glycoprotein.