Virus Temperature-sensitive Mutants Research Articles

Vaccinia Virus B1 Kinase Is Required for Postreplicative Stages of the Viral Life Cycle in a BAF-Independent Manner in U2OS Cells.

The vaccinia virus B1R gene encodes a highly conserved protein kinase that is essential for the poxviral life cycle. As demonstrated in many cell types, B1 plays a critical role during viral DNA replication when it inactivates the cellular host defense effector barrier to autointegration factor (BAF or BANF1). To better understand the role of B1 during infection, we have characterized the growth of a B1-deficient temperature-sensitive mutant virus (Cts2 virus) in U2OS osteosarcoma cells. In contrast to all other cell lines tested to date, we found that in U2OS cells, Cts2 viral DNA replication is unimpaired at the nonpermissive temperature. However, the Cts2 viral yield in these cells was reduced more than 10-fold, thus indicating that B1 is required at another stage of the vaccinia virus life cycle. Our results further suggest that the host defense function of endogenous BAF may be absent in U2OS cells but can be recovered through either overexpression of BAF or fusion of U2OS cells with mouse cells in which the antiviral function of BAF is active. Interestingly, examination of late viral proteins during Cts2 virus infection demonstrated that B1 is required for optimal processing of the L4 protein. Finally, execution point analyses as well as electron microscopy studies uncovered a role for B1 during maturation of poxviral virions. Overall, this work demonstrates that U2OS cells are a novel model system for studying the cell type-specific regulation of BAF and reveals a role for B1 beyond DNA replication during the late stages of the viral life cycle. The most well characterized role for the vaccinia virus B1 kinase is to facilitate viral DNA replication by phosphorylating and inactivating BAF, a cellular host defense responsive to foreign DNA. Additional roles for B1 later in the viral life cycle have been postulated for decades but are difficult to examine directly due to the importance of B1 during DNA replication. Here, we demonstrate that in U2OS cells, a B1 mutant virus escapes the block in DNA replication observed in other cell types and, instead, this mutant virus exhibits impaired late protein accumulation and incomplete maturation of new virions. These data provide the clearest evidence to date that B1 is needed for multiple critical junctures in the poxviral life cycle in a manner that is both dependent on and independent of BAF.

Studies of an Influenza A Virus Temperature-Sensitive Mutant Identify a Late Role for NP in the Formation of Infectious Virions

The influenza A virus nucleoprotein (NP) is a single-stranded RNA-binding protein that encapsidates the virus genome and has essential functions in viral-RNA synthesis. Here, we report the characterization of a temperature-sensitive (ts) NP mutant (US3) originally generated in fowl plague virus (A/chicken/Rostock/34). Sequence analysis revealed a single mutation, M239L, in NP, consistent with earlier mapping studies assigning the ts lesion to segment 5. Introduction of this mutation into A/PR/8/34 virus by reverse genetics produced a ts phenotype, confirming the identity of the lesion. Despite an approximately 100-fold drop in the viral titer at the nonpermissive temperature, the mutant US3 polypeptide supported wild-type (WT) levels of genome transcription, replication, and protein synthesis, indicating a late-stage defect in function of the NP polypeptide. Nucleocytoplasmic trafficking of the US3 NP was also normal, and the virus actually assembled and released around sixfold more virus particles than the WT virus, with normal viral-RNA content. However, the particle/PFU ratio of these virions was 50-fold higher than that of WT virus, and many particles exhibited an abnormal morphology. Reverse-genetics studies in which A/PR/8/34 segment 7 was swapped with sequences from other strains of virus revealed a profound incompatibility between the M239L mutation and the A/Udorn/72 M1 gene, suggesting that the ts mutation affects M1-NP interactions. Thus, we have identified a late-acting defect in NP that, separate from its function in RNA synthesis, indicates a role for the polypeptide in virion assembly, most likely involving M1 as a partner.

Evaluation of a virus derived from MDCK cells infected persistently with influenza A virus as a potential live‐attenuated vaccine candidate in the mouse model

A temperature-sensitive mutant virus unable to replicate at 38 degrees C was recovered from passage 189 (IVpi-189) of Madin-Darby canine kidney cells infected persistently with influenza A. Immunofluorescent staining of the IVpi-189 virus-infected cells revealed disrupted transport of the matrix (M) 1 protein into the nucleus at non-permissive temperatures, resulting in retention of the nucleoprotein (NP) in the nucleus. Upon comparison with the parental influenza A E61-24-P15 strain used to establish persistent infection, amino acid exchanges were found in the M1 protein of IVpi-189 virus; arginine to glutamine at position 72 and threonine to alanine at position 139. When mice were inoculated intranasally with IVpi-189 virus, virus growth in the lungs was restrained and terminated rapidly. Prior intranasal inoculation with only a small dose of IVpi-189 virus induced humoral and cellular immune responses and protected mice against subsequent virulent virus challenge. These results indicate that IVpi-189 virus, an avirulent temperature-sensitive mutant, is a promising candidate for use as a live-attenuated vaccine.

Marker rescue mapping of the combined Condit/Dales collection of temperature-sensitive vaccinia virus mutants

Complementation analysis of the combined Condit/Dales collection of vaccinia virus temperature-sensitive mutants has been reported (Lackner, C.A., D'Costa, S.M., Buck, C., Condit, R.C., 2003. Complementation analysis of the Dales collection of vaccinia virus temperature-sensitive mutants. Virology 305, 240–259), however not all complementation groups have previously been assigned to single genes on the viral genome. We have used marker rescue to map at least one representative of each complementation group to a unique viral gene. The final combined collection contains 124 temperature-sensitive mutants affecting 38 viral genes, plus five double mutants.

Temperature-Sensitive Mutations in the Putative Herpes Simplex Virus Type 1 Terminase Subunits pU L 15 and pU L 33 Preclude Viral DNA Cleavage/Packaging and Interaction with pU L 28 at the Nonpermissive Temperature

Terminases comprise essential components of molecular motors required to package viral DNA into capsids in a variety of DNA virus systems. Previous studies indicated that the herpes simplex virus type 1 U(L)15 protein (pU(L)15) interacts with the pU(L)28 moiety of a pU(L)28-pU(L)33 complex to form the likely viral terminase. In the current study, a novel temperature-sensitive mutant virus was shown to contain a mutation in U(L)33 codon 61 predicted to change threonine to proline. At the nonpermissive temperature, this virus, designated ts8-22, replicated viral DNA and produced capsids that became enveloped at the inner nuclear membrane but failed to form plaques or to cleave or package viral DNA. Incubation at the nonpermissive temperature also precluded coimmunoprecipitation of U(L)33 protein with its normal interaction partners encoded by U(L)28 and U(L)15 in ts8-22-infected cells and with pU(L)28 in transient-expression assays. Moreover, a temperature-sensitive mutation in U(L)15 precluded coimmunoprecipitation of pU(L)15 with the U(L)28 and U(L)33 proteins at the nonpermissive temperature. We conclude that interactions between putative terminase components are tightly linked to successful viral DNA cleavage and packaging.

The vaccinia virus E8R gene product is required for formation of transcriptionally active virions

Two vaccinia virus temperature-sensitive mutants were mapped to the E8R gene and subjected to phenotypic characterization. Dts23 contains a missense mutation in the coding region of E8R (L81F), and in Cts19 the initiating methionine codon is changed from ATG to ATA (M1I). The two ts mutants display normal patterns of gene expression and DNA replication during infection. The E8 protein is synthesized exclusively late during infection and packaged into virion cores Western blot analysis revealed that E8 synthesis is reduced in Dts23 infected cells at permissive (31 °C) and non-permissive temperature (39.7 °C) and absent in Cts19 infection under both conditions. Dts23 virions produced at 39.7 °C were indistinguishable in appearance from wt virions. Cts19 fails to produce identifiable viral structures when incubated at 39.7 °C. Purified Dts23 virions produced at 39.7 °C contain reduced amounts of E8 and have a high particle to infectivity ratio; purified Cts19 virions grown at 31 °C also show reduced infectivity and do not contain detectable E8. Dts23 grown at 39.7 °C could enter cells but failed to synthesize early mRNA or produce CPE. Soluble extracts from mutant virions were active in a promoter dependent in vitro transcription assay, however intact mutant cores were defective in transcription. We suggest that E8 plays a subtle role in virion core structure that impacts directly or indirectly on core transcription.

Vaccinia virus temperature-sensitive mutants in the A28 gene produce non-infectious virions that bind to cells but are defective in entry

The vaccinia virus temperature-sensitive mutations Cts6 and Cts9 were mapped by marker rescue and DNA sequencing to the A28 gene. Cts6 and Cts9 contain an identical 2-bp deletion truncating the A28 protein and removing the fourth conserved cysteine near the C-terminus. Cts9 mutant virions produced at 40 °C were non-infectious and unable to cause cytopathic effect. However, the mutant A28 protein localized to purified mature virions (MV) at 31 °C and 40 °C. MV of Cts9 produced at 40 °C bound to cells but did not enter cells. Low pH treatment of Cts9-infected cells at 18 h p.i. failed to produce fusion from within at 40 °C, but gave fusion at 31 °C. Adsorption of Cts9 mutant virions to cells followed by low pH treatment showed a defect in fusion from without. The Cts9 phenotype suggests that the A28 protein is involved in both virus entry and cell–cell fusion, and supports the linkage between the two processes.

Enzymatic Defects of the nsP2 Proteins of Semliki Forest Virus Temperature-Sensitive Mutants

We have analyzed the biochemical consequences of mutations that affect viral RNA synthesis in Semliki Forest virus temperature-sensitive (ts) mutants. Of the six mutations mapping in the multifunctional replicase protein nsP2, three were located in the N-terminal helicase region and three were in the C-terminal protease domain. Wild-type and mutant nsP2s were expressed, purified, and assayed for nucleotide triphosphatase (NTPase), RNA triphosphatase (RTPase), and protease activities in vitro at 24 degrees C and 35 degrees C. The protease domain mutants (ts4, ts6, and ts11) had reduced protease activity at 35 degrees C but displayed normal NTPase and RTPase. The helicase domain mutation ts1 did not have enzymatic consequences, whereas ts13a and ts9 reduced both NTPase and protease activities but in different and mutant-specific ways. The effects of these helicase domain mutants on protease function suggest interdomain interactions within nsP2. NTPase activity was not directly required for protease activity. The similarities of the NTPase and RTPase results, as well as competition experiments, suggest that these two reactions utilize the same active site. The mutations were also studied in recombinant viruses first cultivated at the permissive temperature and then shifted up to the restrictive temperature. Processing of the nonstructural polyprotein was generally retarded in cells infected with viruses carrying the ts4, ts6, ts11, and ts13a mutations, and a specific defect appeared in ts9. All mutations except ts13a were associated with a large reduction in the production of the subgenomic 26S mRNA, indicating that both protease and helicase domains influence the recognition of the subgenomic promoter during virus replication.

Analysis of vaccinia virus temperature-sensitive I7L mutants reveals two potential functional domains

As an approach to initiating a structure-function analysis of the vaccinia virus I7L core protein proteinase, a collection of conditional-lethal mutants in which the mutation had been mapped to the I7L locus were subjected to genomic sequencing and phenotypic analyses. Mutations in six vaccinia virus I7L temperature sensitive mutants fall into two groups: changes at three positions at the N-terminal end between amino acids 29 and 37 and two different substitutions at amino acid 344, near the catalytic domain. Regardless of the position of the mutation, mutants at the non-permissive temperature failed to cleave core protein precursors and had their development arrested prior to core condensation. Thus it appears that the two clusters of mutations may affect two different functional domains required for proteinase activity.

Attenuation and immunogenicity in mice of temperature-sensitive influenza viruses expressing truncated NS1 proteins

It was previously shown that two mutant influenza A viruses expressing C-terminally truncated forms of the NS1 protein (NS1-81 and NS1-110) were temperature sensitive in vitro. These viruses contain HA, NA and M genes derived from influenza A/WSN/33 H1N1 virus (mouse-adapted), and the remaining five genes from human influenza A/Victoria/3/75 virus. Mice intranasally infected with the NS1 mutant viruses showed undetectable levels of virus in lungs at day 3, whereas those infected with the NS1 wild-type control virus still had detectable levels of virus at this time. Nevertheless, the temperature-sensitive mutant viruses induced specific cellular and humoral immune responses similar to those induced by the wild-type virus. Mice immunized with the NS1 mutant viruses were protected against a lethal challenge with influenza A/WSN/33 virus. These results indicate that truncations in the NS1 protein resulting in temperature-sensitive phenotypes in vitro correlate with attenuation in vivo without compromising viral immunogenicity, an ideal characteristic for live attenuated viral vaccines.

Effects of a Temperature Sensitivity Mutation in the J1R Protein Component of a Complex Required for Vaccinia Virus Assembly

Vaccinia virus J1R protein is required for virion morphogenesis (W. L. Chiu and W. Chang, J. Virol. 76:9575-9587, 2002). In this work, we further characterized the J1R protein of wild-type vaccinia virus and compared it with the protein encoded by the temperature-sensitive mutant virus Cts45. The mutant Cts45 was found to contain a Pro-to-Ser substitution at residue 132 of the J1R open reading frame, which is responsible for a loss-of-function phenotype. The half-life of the J1R-P132S mutant protein was comparable at both 31 and 39 degrees C, indicating that the P132S mutation did not affect the stability of the J1R protein. We also showed that the J1R protein interacts with itself in the virus-infected cells. The N-terminal region of the J1R protein, amino acids (aa) 1 to 77, interacted with the C-terminal region, aa 84 to 153, and the P132 mutation did not abolish this interaction, as determined by two-hybrid analysis. Furthermore, we demonstrated that J1R protein is part of a viral complex containing the A30L, G7L, and F10L proteins in virus-infected cells. In immunofluorescence analyses, wild-type J1R protein colocalized with the A30L, G7L, and F10L proteins in virus-infected cells but the loss-of-function P132 mutant did not. Furthermore, without a functional J1R protein, rapid degradation of A30L and the 15-kDa forms of the G7L and F10L proteins was observed in cells infected with Cts45 at 39 degrees C. This study thus demonstrated the importance of the J1R protein in the formation of a viral assembly complex required for morphogenesis.

Temperature-sensitive mutants in the vaccinia virus 4b virion structural protein assemble malformed, transcriptionally inactive intracellular mature virions

Two noncomplementing vaccinia virus temperature-sensitive mutants, Cts8 and Cts26, were mapped to the A3L gene, which encodes the major virion structural protein, 4b. The two ts mutants display normal patterns of gene expression, DNA replication, telomere resolution, and protein processing during infection. Morphogenesis during mutant infections is normal through formation of immature virions with nucleoids (IVN) but appears to be defective in the transition from IVN to intracellular mature virus (IMV). In mutant infections, aberrant particles that have the appearance of malformed IMV accumulate. The mutant particles are wrapped in Golgi-derived membranes and exported from cells. Purified mutant particles are indistinguishable from wt particles in protein and DNA composition; however, they are defective in a permeabilized-virion-directed transcription reaction despite containing significant (Cts8) or even normal (Cts26) levels of specific transcription enzymes. These results indicate that the 4b protein is required for proper metamorphosis of IMV from IVN and that proper organization of the IMV structure is required to produce a transcriptionally active virion particle.

Immediate and delayed learning

THE LANCET • Vol 363 • June 26, 2004 • www.thelancet.com 2127 live attenuated respiratory syncytial virus (RSV) temperature-sensitive mutant vaccines and vaccinia virus recombinants that express the surface glycoproteins of RSV. Vaccine 1993; 11: 1395–1404. 23 Weiss RC, Scott FW. Antibody-mediated enhancement of disease in feline infectious peritonitis: comparisons with dengue hemorrhagic fever. Comp Immunol Microbiol Infect Dis 1981; 4: 175–89. 24 Fouchier RA, Kuiken T, Schutten M, et al. Aetiology: Koch’s postulates fulfilled for SARS virus. Nature 2003; 423: 240. 25 Martina BE, Haagmans BL, Kuiken T, et al. Virology: SARS virus infection of cats and ferrets. Nature 2003; 425: 915. 26 Clements ML, Belshe RB, King J, et al. Evaluation of bovine, coldadapted human, and wild-type human parainfluenza type 3 viruses in adult volunteers and in chimpanzees. J Clin Microbiol 1991; 29: 1175–82. 27 Karron RA, Wright PF, Newman FK, et al. A live human parainfluenza type 3 virus vaccine is attenuated and immunogenic in healthy infants and children. J Infect Dis 1995; 172: 1445–50. 28 Tao T, Skiadopoulos MH, Davoodi F, Riggs JM, Collins PL, Murphy BR. Replacement of the ectodomains of the hemagglutininneuraminidase and fusion glycoproteins of recombinant parainfluenza virus type 3 (PIV3) with their counterparts from PIV2 yields attenuated PIV2 vaccine candidates. J Virol 2000; 74: 6448–58. 29 Tao T, Durbin AP, Whitehead SS, Davoodi F, Collins PL, Murphy BR. Recovery of a fully viable chimeric human parainfluenza virus (PIV) type 3 in which the hemagglutininneuraminidase and fusion glycoproteins have been replaced by those of PIV type 1. J Virol 1998; 72: 2955–61.

Intracellular topology and epitope shielding of poliovirus 3A protein.

The poliovirus RNA replication complex comprises multiple viral and possibly cellular proteins assembled on the cytoplasmic surface of rearranged intracellular membranes. Viral proteins 3A and 3AB perform several functions during the poliovirus replicative cycle, including significant roles in rearranging membranes, anchoring the viral polymerase to these membranes, inhibiting host protein secretion, and possibly providing the 3B protein primer for RNA synthesis. During poliovirus infection, the immunofluorescence signal of an amino-terminal epitope of 3A-containing proteins is markedly shielded compared to 3A protein expressed in the absence of other poliovirus proteins. This is not due to luminal orientation of all or a subset of the 3A-containing polypeptides, as shown by immunofluorescence following differential permeabilization and proteolysis experiments. Shielding of the 3A epitope is more pronounced in cells infected with wild-type poliovirus than in cells with temperature-sensitive mutant virus that contains a mutation in the 3D polymerase coding region adjacent to the 3AB binding site. Therefore, it is likely that direct binding of the poliovirus RNA-dependent RNA polymerase occludes the amino terminus of 3A-containing polypeptides in the RNA replication complex.

Unique temperature-sensitive defect in vaccinia virus morphogenesis maps to a single nucleotide substitution in the A30L gene.

Marker rescue experiments demonstrated that the genetic lesion of a previously isolated vaccinia virus temperature-sensitive mutant which forms multilayered envelope structures with lucent interiors and foci of viroplasm with dense centers mapped to the A30L open reading frame. A single base change, resulting in a nonconservative Ser-to-Phe substitution at residue 17, was associated with degradation of the A30L protein at elevated temperatures.

Packaging-competent capsids of a herpes simplex virus temperature-sensitive mutant have properties similar to those of in vitro-assembled procapsids.

Newcomb and coworkers (W. W. Newcomb, F. L. Homa, D. R. Thomsen, F. P. Booy, B. L. Trus, A. C. Steven, J. V. Spencer, and J. C. Brown, J. Mol. Biol. 263:432-446, 1996; W. W. Newcomb, F. L. Homa, D. R. Thomsen, Z. Ye, and J. C. Brown, J. Virol. 68:6059-6063, 1994) have recently described an in vitro herpes simplex virus (HSV) capsid assembly product which, because of certain parallels between its properties and those of bacteriophage proheads, they have designated the procapsid. As in their bacteriophage counterparts, there are marked differences between the structures of the two types of particle, and conversion from the procapsid to the capsid form requires extensive reconfiguration of the subunits. This reconfiguration occurs spontaneously upon extended in vitro incubation. One of the distinctive features of the HSV procapsids is that, unlike mature capsids, they are unstable and disassemble upon storage at 2 degrees C. Using a mutant of HSV type 1 (ts1201), which has a lesion in the protease responsible for maturational cleavage of the scaffolding protein, we have demonstrated that capsids present within cells infected at nonpermissive temperatures are also cryosensitive and disappear if the cells are incubated at 0 degrees C. This suggests that ts1201 capsids may resemble procapsids in structure. However, ts1201 capsids remain cryosensitive following extended incubation at an elevated temperature and, therefore, do not appear to undergo the spontaneous reconfiguration seen with in vitro-assembled procapsids. The lesion in ts1201 is reversible, and capsids formed at the nonpermissive temperature can undergo maturational cleavage and go on to form infectious virions following downshift to permissive temperatures. The sensitivity of ts1201 capsids to low temperatures is closely correlated with the cleavage status of the scaffolding protein, suggesting that proteolysis may act to trigger their conversion to the stable form. The experiments described here provide the firmest evidence yet that the procapsid has a biologically relevant role in the virus life cycle.

Influenza Virus Inhibits Cleavage of the HSP70 Pre-mRNAs at the Polyadenylation Site

Influenza virus infection is known to shut off the expression of host genes. To study the mechanism, we examined the effects of influenza A/Udorn/72 virus infection on the heat induction of a major heat shock protein, HSP70, in Madin–Darby canine kidney cells. The induction of HSP70 protein synthesis was progressively suppressed with postinfection time when heat shock was applied. Northern hybridization analysis revealed the appearance of longer, heterogeneous HSP70 transcripts in the range of 2.7 to 30 kb with a concomitant decrease in the amount of the mature 2.7-kb mRNAs in the nucleus of the infected cells. Such longer β-actin transcripts were also observed but with much less intensity. The longer HSP70 transcripts contained the downstream sequence of the polyadenylation site, as demonstrated by RNase protection with an antisense RNA probe containing the sequence through the polyadenylation sites. This clearly proved that influenza virus infection inhibits the polyadenylation-site cleavage of the pre-mRNAs by the host cleavage and polyadenylation machinery. One temperature-sensitive mutant virus carrying a temperature-sensitive mutation on the NS1gene failed to inhibit the cleavage at the nonpermissive temperature, indicating that the NS1protein is involved in the inhibition of the pre-mRNA cleavage. This is the first report of the down-regulation of cellular mRNA maturation at the point of polyadenylation-site cleavage by virus infection and identifies a new mechanism by which the influenza virus shuts off host gene expression.

Ovarian steroids reduced apoptotic death in SV40 temperature-sensitive mutant virus transformed uterine epithelial cells

Ovarian steroids have been shown to inhibit uterine cell death in vivo. In this study, we investigated whether ovarian steroids regulated cell death in an uterine epithelial cell line transformed with SV40 temperature-sensitive (ts) mutant virus. To assess cell death rate, cells were grown at permissive temperature (34 °C) and pulsed with 3H-thymidine. The retention of incorporated radioactivity was then examined after a temperature shift to nonpermissive temperature (40 °C) in the absence or presence of estradiol and progesterone. When cells were continuously cultured at 34 °C, cell number increased rapidly and most of radioactivity was retained in the attached cells. However, the temperature shift from 34 °C to 40 °C resulted in a decrease in cell number and radioactivity in attached cells. Estradiol and progesterone attenuated this temperature shift-induced cell death. Morphological examination with Hoechst 33258 staining revealed that the temperature shift increased the percentage of apoptotic death. The treatment of ovarian steroids reduced the extent of apoptotic death. Our studies demonstrated that ovarian steroids could act directly on uterine epithelial cells to reduce apoptotic death in culture.

Influenza virus NS1 protein interacts with viral transcription-replication complexes in vivo.

The interaction of influenza virus NS1 protein with other viral products in the infected cell was analysed by co-immunoprecipitation studies. The three subunits of the polymerase and the nucleoprotein, but not M1 protein, were co-immunoprecipitated by NS1-specific serum but not when control serum was used. Such co-immunoprecipitation was not sensitive to RNase treatment of the immunoprecipitates. Co-immunoprecipitation was also obtained when the viral transcription-replication system was reconstituted in vivo by transfection of cDNAs and model vRNA template into vaccinia virus-T7-infected cells. Analysis of the RNA pulled-down in the NS1-specific precipitates indicated the presence of both vRNA and mRNA. These results are discussed in the context of the phenotype of virus temperature-sensitive mutants affected in the NS1 gene.

Map locations of mouse hepatitis virus temperature-sensitive mutants: confirmation of variable rates of recombination.

Using standard genetic recombination techniques, studies in our laboratory suggest that recombination rates are very high and vary in different portions of the mouse hepatitis virus (MHV) genome. To determine the actual recombination frequencies in the MHV genome and localize the nucleotide boundaries of individual viral genes, we have sequenced temperature-sensitive and revertant viruses to identify the location of specific mutant alleles. Complementation group F RNA+ ts mutants (LA7, NC6, and NC16) each contained a unique mutation which was tightly linked to the ts phenotype and resulted in a conservative or nonconservative amino acid change in the MHV S glycoprotein gene. In agreement with previous recombination mapping studies, the mutation in LA7 and NC6 mapped within the S1 domain while NC16 mapped within the S2 domain. To determine the map coordinates of the MHV polymerase genes, several RNA- mutants and their revertants belonging to complementation groups C (NC3 and LA9) and E (LA18 and NC4) were also sequenced. Mutations were identified in each virus that were tightly linked to the ts phenotype and resulted in either a conservative or nonconservative amino acid change. The group C allele spanned the ORF 1a/ORF 1b junction, while the group E mutants mapped at the C terminus of ORF 1b about 20 to 22 kb from the 5' end of the genome. Mutation rates, calculated from the reversion frequencies of plaque-purified ts viruses requiring a single nucleotide alteration for reversion, approached 1.32 (+/- 0.89) x 10(-4) substitutions per nucleotide site per round of template copying. Detailed recombination mapping studies across known distances between these different ts alleles has confirmed that homologous recombination rates approached 25% and varied within different portions of the MHV genome.