Analysis of an influenza A virus mutant with a deletion in the NS segment.

The influenza virus host range mutant CR43-3, derived by recombination from the A/Alaska/6/77 and the cold-adapted and temperature-sensitive A/Ann Arbor/6/60 viruses, has previously been shown to possess a defect in the NS gene. To characterize this defect, nucleotide sequence data were obtained from cloned cDNAs. The CR43-3 NS gene was found to be 854 nucleotides long and to derive from the NS gene of the A/Alaska/6/77 parent virus by an internal deletion of 36 nucleotides. Direct sequencing of RNA 8 of CR43-3 virus confirmed that the deletion in the NS1-coding region was not an artifact that was generated during the cloning procedure. Protein analysis indicated that the NS1 protein of CR43-3 virus was synthesized in equal amounts in the restrictive (MDCK) cells as well as in the permissive (PCK) host cells. Also, indirect immunofluorescence studies of virus-infected cells showed that the NS1 protein of CR43-3 virus, like that of the parent viruses, accumulates in the nuclei of both cell systems. Although no differences in synthesis or localization of the NS1 protein could be detected, a consistent reduction in M1 protein was noted in CR43-3 virus-infected, nonpermissive cells as compared with that of the permissive host. Since analysis of the CR43-3 virus required us to obtain the NS nucleotide sequence of the 1977 isolate A/Alaska/6/77, we were able to compare this sequence with those of corresponding genes of earlier strains. The result of this analysis supports the idea of a common lineage of human influenza A viruses isolated over a 43-year period.     

Characterization of a ts mutant of BALB/3T3 cells and correction of the defect by in vitro addition of extracts from wild-type cells.

ts20 is a temperature-sensitive mutant cell line derived from BALB/3T3 cells. DNA synthesis in the mutant decreased progressively after an initial increase during the first 3 h at the restrictive temperature. RNA and protein synthesis increased for 20 h and remained at a high level for 40 h. Cells were arrested in S phase as determined by flow microfluorimetry, and DNA chain elongation was retarded as measured by fiber autoradiography. Infection with polyomavirus did not bypass the defect in cell DNA synthesis, and the mutant did not support virus DNA replication at the restrictive temperature. After shift down to the permissive temperature, cell DNA synthesis was restored whereas virus DNA synthesis was not. Analysis of virus DNA synthesized at the restrictive temperature showed that the synthesis of form I and replicative intermediate DNA decreased concurrently and that the rate of completion of virus DNA molecules remained constant with increasing time at the restrictive temperature. These studies indicated that the mutation inhibited ongoing DNA synthesis at a step early in elongation of nascent chains. The defect in virus and cell DNA synthesis was expressed in vitro. [3H]dTTP incorporation was reduced, consistent with the in vivo data. The addition of a high-salt extract prepared from wild-type 3T3 cells preferentially stimulated the incorporation of [3H]dTTP into the DNA of mutant cells at the restrictive temperature. A similar extract prepared from mutant cells was less effective and was more heat labile as incubation of it at the restrictive temperature for 1 h destroyed its ability to stimulate DNA synthesis in vitro, whereas wild-type extract was not inactivated until incubated at that temperature for 3 h.     

Characterization of influenza virus NS1 protein by using a novel helper-virus-free reverse genetic system

We have developed a novel helper-virus-free reverse genetic system to genetically manipulate influenza A viruses. The RNPs, which were purified from the influenza A/WSN/33 (WSN) virus, were treated with RNase H in the presence of NS (nonstructural) cDNA fragments. This specifically digested the NS RNP. The NS-digested RNPs thus obtained were transfected into cells together with the in vitro-reconstituted NS RNP. The NS-digested RNPs alone did not rescue viruses; however, cotransfection with the NS RNP did. This protocol was also used to rescue the NP transfectant. We obtained two NS1 mutants, dl12 and N110, using this protocol. The dl12 NS gene contains a deletion of 12 amino acids at positions 66 to 77 near the N terminus. This virus was temperature sensitive in Madin-Darby bovine kidney (MDBK) cells as well as in Vero cells. The translation of all viral proteins as well as cellular proteins was significantly disrupted during a later time of infection at the nonpermissive temperature of 39 degrees C. The N110 mutant consists of 110 amino acids which are the N-terminal 48% of the WSN virus NS1 protein. Growth of this virus was significantly reduced at any temperature. In the virus-infected cells, translation of the M1 protein was reduced to 10 to 20% of that of the wild-type virus; however, the translation of neither the nucleoprotein nor NS1 was significantly interfered with, indicating the important role of NS1 in translational stimulation of the M1 protein.     

Regulation of the production of infectious genotype 1a hepatitis C virus by NS5A domain III

Although hepatitis C virus (HCV) assembly remains incompletely understood, recent studies with the genotype 2a JFH-1 strain suggest that it is dependent upon the phosphorylation of Ser residues near the C terminus of NS5A, a multifunctional nonstructural protein. Since genotype 1 viruses account for most HCV disease yet differ substantially in sequence from that of JFH-1, we studied the role of NS5A in the production of the H77S virus. While less efficient than JFH-1, genotype 1a H77S RNA produces infectious virus when transfected into permissive Huh-7 cells. The exchange of complete NS5A sequences between these viruses was highly detrimental to replication, while exchanges of the C-terminal domain III sequence (46% amino acid sequence identity) were well tolerated, with little effect on RNA synthesis. Surprisingly, the placement of the H77S domain III sequence into JFH-1 resulted in increased virus yields; conversely, H77S yields were reduced by the introduction of domain III from JFH-1. These changes in infectious virus yield correlated well with changes in the abundance of NS5A in RNA-transfected cells but not with RNA replication or core protein expression levels. Alanine replacement mutagenesis of selected Ser and Thr residues in the C-terminal domain III sequence revealed no single residue to be essential for infectious H77S virus production. However, virus production was eliminated by Ala substitutions at multiple residues and could be restored by phosphomimetic Asp substitutions at these sites. Thus, despite low overall sequence homology, the production of infectious virus is regulated similarly in JFH-1 and H77S viruses by a conserved function associated with a C-terminal Ser/Thr cluster in domain III of NS5A.     

Regulation of type VI collagen synthesis in transformed mesenchymal cells.

We have analysed the effects of oncogenic transformation on the expression of type VI collagen in mesenchymal cells. Synthesis of type VI collagen was almost completely inhibited in fibroblasts transformed by DNA or RNA tumour viruses or in cells derived from spontaneous mesenchymal tumours. Inhibition of type VI collagen synthesis appears, therefore, to be a common phenomenon of transformed mesenchymal cells. When introduced into normal cells by viral vectors, the 'nuclear' oncogene v-myc had an inhibitory effect similar to that of the 'cytoplasmic' oncogene v-src. Fibroblasts infected with a temperature-sensitive strain of Rous sarcoma virus (NY68) produced type VI collagen at the restrictive, but not at the permissive temperature. If such cells were shifted from the permissive to the restrictive temperature, synthesis of the individual subunits of type VI collagen was co-ordinately induced. These results demonstrate that the activity of a single oncogene product is sufficient to inhibit type VI collagen expression.     

Characterization of ts 16, a temperature-sensitive mutant of vaccinia virus.

We have characterized a temperature-sensitive mutant of vaccinia virus, ts16, originally isolated by Condit et al. (Virology 128:429-443, 1983), at the permissive and nonpermissive temperatures. In a previous study by Kane and Shuman (J. Virol 67:2689-2698, 1993), the mutation of ts16 was mapped to the I7 gene, encoding a 47-kDa protein that shows partial homology to the type II topoisomerase of Saccharomyces cerevisiae. The present study extends previous electron microscopy analysis, showing that in BSC40 cells infected with ts16 at the restrictive temperature (40 degrees C), the assembly was arrested at a stage between the spherical immature virus and the intracellular mature virus (IMV). In thawed cryosections, a number of the major proteins normally found in the IMV were subsequently localized to these mutant particles. By using sucrose density gradients, the ts16 particles were purified from cells infected at the permissive and nonpermissive temperatures. These were analyzed by immunogold labelling and negative-staining electron microscopy, and their protein composition was determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. While the ts16 virus particles made at the permissive temperature appeared to have a protein pattern identical to that of wild-type IMV, in the mutant particles the three core proteins, p4a, p4b, and 28K, were not proteolytically processed. Consistent with previous data the sucrose-purified particles could be labelled with [3H]thymidine. In addition, anti-DNA labelling on thawed cryosections suggested that most of the mutant particles had taken up DNA. On thawed cryosections of cells infected at the permissive temperature, antibodies to I7 labelled the virus factories, the immature viruses, and the IMVs, while under restrictive conditions these structures were labelled much less, if at all. Surprisingly, however, by Western blotting (immunoblotting) the I7 protein was present in similar amounts in the defective particles and in the IMVs isolated at the permissive temperature. Finally, our data suggest that at the nonpermissive temperature the assembly of ts16 is irreversibly arrested in a stage at which the DNA is in the process of entering but before the particle has completely sealed, as monitored by protease experiments.     

Regulation of coliphage f1 single-stranded DNA synthesis by a DNA-binding protein

Control of single-strand DNA synthesis in coliphage f1 was studied with the use of mutants which are temperature sensitive in gene 2, a gene essential for phage DNA replication. Cells were infected at a restrictive temperature with such a mutant, and the DNA synthesized after a shift to permissive temperature was examined. When cells were held at 42 °C for ten or more minutes after infection, only single-stranded DNA was synthesized immediately after the shift to permissive temperature. This indicated that the accumulation of a pool of double-stranded, replicative form DNA molecules is not an absolute requirement for the synthesis of single-stranded DNA, although replicative form DNA accumulation precedes single-strand synthesis in cells infected with wild-type phage. Cells infected at restrictive temperature with the mutant phage do not replicate the infecting DNA, but do accumulate a substantial amount of gene 5 protein, a DNA-binding protein essential for single-strand synthesis. It is proposed that this accumulated gene 5 protein, by binding to the limited number of replicating DNA molecules formed following the shift to the permissive temperature, acts to prevent the synthesis of double-stranded replicative form DNA, thus causing the predominant appearance of single strands. This explanation implies an intermediate common to both single and double-stranded DNA synthesis. The kinetics of gene 5 protein synthesis indicates that it is the ratio of the gene 5 protein to replicating DNA molecules which determines whether an intermediate will synthesize double or single-stranded DNA.     

Effects of Influenza A Virus NS1 Protein on Protein Expression: the NS1 Protein Enhances Translation and Is Not Required for Shutoff of Host Protein Synthesis

The influenza A virus NS1 protein, a virus-encoded alpha/beta interferon (IFN-alpha/beta) antagonist, appears to be a key regulator of protein expression in infected cells. We now show that NS1 protein expression results in enhancement of reporter gene activity from transfected plasmids. This effect appears to be mediated at the translational level, and it is reminiscent of the activity of the adenoviral virus-associated I (VAI) RNA, a known inhibitor of the antiviral, IFN-induced, PKR protein. To study the effects of the NS1 protein on viral and cellular protein synthesis during influenza A virus infection, we used recombinant influenza viruses lacking the NS1 gene (delNS1) or expressing truncated NS1 proteins. Our results demonstrate that the NS1 protein is required for efficient viral protein synthesis in COS-7 cells. This activity maps to the amino-terminal domain of the NS1 protein, since cells infected with wild-type virus or with a mutant virus expressing a truncated NS1 protein-lacking approximately half of its carboxy-terminal end-showed similar kinetics of viral and cellular protein expression. Interestingly, no major differences in host cell protein synthesis shutoff or in viral protein expression were found among NS1 mutant viruses in Vero cells. Thus, another viral component(s) different from the NS1 protein is responsible for the inhibition of host protein synthesis during viral infection. In contrast to the earlier proposal suggesting that the NS1 protein regulates the levels of spliced M2 mRNA, no effects on M2 protein accumulation were seen in Vero cells infected with delNS1 virus.     

Permanent cell lines established from ts-COS cells that regulate by temperature the amplification and expression of cloned genes.

Temperature-sensitive COS cells have been transformed at restrictive temperature with SV40 replicons containing the neo or pac markers. Puromycin-resistant cell clones maintained at the restrictive temperature contain the pac gene integrated into the cell DNA. However, when the cells are shifted to the permissive temperature the pac gene is amplified in episomal forms up to 2-4 X 10(4) copies per cell. Concomitant with this, an induction of 35-300 fold in the levels of puromycin acetyl transferase activity is observed, leading to the accumulation of the enzyme up to 10-60 mU/mg of total cell protein. A band of apparent molecular weight 26,500 daltons is observed by polyacrylamide gel electrophoresis of induced culture extracts, that accounts for approximately 3% of the newly synthesized protein. The expression of non-selectable genes can also be regulated, as shown by the induction of influenza virus nucleoprotein synthesis in transformed cells. These results indicate that the ts-COS cells can be used as a highly efficient, regulable mammalian expression system.     

Role of the serine-threonine kinase PAK-1 in myxoma virus replication

Subversion or appropriation of cellular signal transduction pathways is a common strategy employed by viruses to promote an environment within infected cells that supports the viral replicative cycle. Using subsets of 3T3 murine fibroblasts previously shown to differ in their ability to support myxoma virus (MV) replication, we investigated the role of host serine-threonine kinases (STKs) as potential mediators of the permissive phenotype. Both permissive and nonpermissive 3T3 cells supported equivalent levels of virion binding, entry, and early virus gene expression, indicating that MV tropism in 3T3 cells was not determined by receptor-mediated entry. In contrast, late virus gene expression and viral DNA replication were selectively compromised in restrictive 3T3 cells. Addition of specific protein kinase inhibitors, many of which shared the ability to influence the activity of the STKs p21-activated kinase 1 (PAK-1) and Raf-1 attenuated MV replication in permissive 3T3 cells. Western blot detection of the phosphorylated forms of PAK-1 (Thr423) and Raf-1 (Ser338) confirmed activation of these kinases in permissive cells after MV infection or gamma interferon treatment, but the activated forms of both kinases were greatly reduced or absent in restrictive 3T3 cells. The biological significance of these activations was demonstrated by using the autoinhibitory domain of PAK-1 (amino acids 83 to 149), expression of which reduced the efficiency of MV infection in permissive 3T3 cells concurrent with a decrease in PAK-1 activation. In comparison, overexpression of a constitutively active PAK-1 (T423E) mutant increased MV replication in restrictive 3T3 cells. These observations suggest that induced signaling via cellular STKs may play important roles in determining the permissiveness of host cells to poxvirus infection.     

Inherited resistance to N- and B-tropic murine leukemia viruses in vitro: titration patterns in strains SIM and SIM.R congenic at the Fv-1 locus.

We have investigated the titration patterns of murine leukemia viruses on mouse embryo cultures derived from a pair of congenic strains differing at the Fv-1 locus. XC plaque and infectious center assays carried out with N- and B-tropic viruses on both SIM (Fv-1nn) and SIM.R(Fv-1bb) host cells yielded results that were best approximated by Poisson one-hit curves. Titration curves of N-tropic virus by direct XC plaque assay were linear and parallel on the different hosts, with titers 1.8 to 2.7 log10 lower on SIM.R and on (SIM X SIM.R)F1 than on SIM cells; similar linear and parallel curves were found for B-tropic virus, with titers 1.4 to 2.0 log10 lower on SIM and (SIM XSIM-R)F1 than on SIM-R cells. In the infectious center assays, the proportion of infected cells was linearly related to multiplicity of infection on both permissive (N- on SIM and B- on SIM.R) restrictive (B- on SIM and N- on SIM.R) genotypes at multiplicities of infection below 0.5; the line relating the variables was about 1 log10 lower in the restrictive than in the permissive situations. At multiplicities of infection where the proportion of infected cells reached a plateau, differences between the results on permissive and restrictive genotypes were considerably reduced. This appeared to be due to the action of non-Fv-1 factors in permissive host. We conclude that the major action of the restrictive allele at the Fv-1 locus in this system is to reduce the probability of successful murine leukemia virus infection without a change in hitness.     

NS2 is required for efficient translation of viral mRNA in minute virus of mice-infected murine cells.

Detailed analysis of five NS2 mutants of the autonomous parvovirus minute virus of mice (MVMp) has revealed the following. At low multiplicities of infection, NS2 mutants killed NB324K cells as well as wild-type (wt) MVM did and grew to high titers, while in contrast they grew poorly and did not readily kill murine A9 cells. Following CaPO4 transfection of murine fibroblasts, NS2 mutant infectious clones generated approximately 10-fold less monomer replicative-form DNA than wt and no detectable progeny single-stranded DNA. On nonmurine semipermissive NB324K cells, however, these mutant plasmid clones generated near wt levels of all replicative DNA forms. After infection of highly synchronized murine fibroblasts by NS2 mutant virus at inputs equivalent to those of the wt, mutant monomer replicative-form DNA was decreased 5- to 10-fold compared with that of the wt, and progeny single-stranded DNA accumulation was decreased to an even greater extent. Both total and cytoplasmic NS2 mutant RNA was decreased, but the amount of total viral mRNA generated, relative to accumulated viral DNA in the same experiments, was similar to that seen in wt infection. The accumulation of virus-generated proteins was also decreased in NS2 mutant infection; however, the magnitude of this decrease, compared with that of wt infections, was significantly greater than the concomitant decrease in mutant-generated levels of accumulated cytoplasmic RNA, and this effect was most dramatic for VP2. There was no such disparity between the relative accumulation of mutant-generated RNA and protein in cells permissive for the growth of these mutants. These results suggest that translation of MVM viral RNA is specifically reduced in NS2 mutant infection of restrictive cells. Because the affected viral proteins are required for the efficient production of viral replicative DNA forms, these results reveal a fundamental, although perhaps not the only, role for NS2 in parvovirus infection.     

Three additional genes required for deoxyribonucleic acid synthesis in Saccharomyces cerevisiae

Deoxyribonucleic acid (DNA) synthesis was examined in asynchronous and synchronous cultures of a number of cdc (cell division cycle) temperature-sensitive mutant strains. The kinetics of DNA synthesis after a shift to the restrictive temperature was compared with that obtained after inhibition of protein synthesis at the permissive temperature, a condition that specifically blocks the initiation of new rounds of DNA replication, but does not block those in progress. Mutations in three genes (cdc 4, 7, and 28) appear to block a precondition for DNA synthesis since cells carrying these lesions cannot start new rounds of DNA replication after a shift from permissive to restrictive temperature, but can finish rounds that were in progress. These three genes are classified as having roles in the "initiation" of DNA synthesis. Mutations in two genes (cdc 8 and 21) block DNA synthesis, itself, since cells harboring these lesions that had started DNA synthesis at the permissive temperature arrest synthesis abruptly upon a shift to the restrictive temperature. Mutations in 13 other cdc genes do not impair DNA synthesis in the first cell cycle at the restrictive temperature.     

Liquid holding recovery in a DEB-Inactivated rad3 strain of Saccharomyces cerevisiae carrying a thermosensitive prt mutation

Summary The mutation prt1-1 with a thermosensitive block in initiation of protein synthesis was introduced into a rad3 strain to study the effect of inhibition of protein synthesis on liquid holding recovery (LHR) from the lethal effects of diepoxybutane (DEB). Liquid holding of the prt1-1rad3 strain under restrictive conditions did not decrease the level of recovery as compared with the permissive temperature. Post-incubation of cells in growth medium under permissive conditions prior to LH resulted in the loss of capacity for LHR, while cells post-incubated under restrictive conditions were fully capable of LHR. The results are interpreted as indicating that protein synthesis during LH is not required for the increase in survival and that the occurrence of protein synthesis prior to liquid holding abolishes the capacity for LHR.     

Use of specific single stranded DNA probes cloned in M13 to study the RNA synthesis of four temperature-sensitive mutants of HK/68 influenza virus.

Specific single stranded DNA probes have been obtained for both influenza virion RNA (vRNA) and complementary RNA (cRNA) by cloning a hemagglutinin gene fragment in the single stranded DNA phase M13. These probes were used for hybridization with the total labeled RNA from cytoplasmic extracts of infected cells. MDCK cells were infected with temperature-sensitive mutants of influenza HK/68 and the production of the virus specific RNA species was analysed at both permissive and restrictive temperatures. Results show that two NP mutants which undergo intracistronic complementation exhibit two different phenotypes at the non permissive temperature: ts2C is poly A cRNA and vRNA negative whereas ts463 is RNA positive. Two mutants of P genes were also analysed and we discuss the relationship existing between the synthesis of the three RNA species especially between poly A and non poly A cRNA.     

Genetic analysis of influenza virus NS1 gene: a temperature-sensitive mutant shows defective formation of virus particles

To perform a genetic analysis of the influenza A virus NS1 gene, a library of NS1 mutants was generated by PCR-mediated mutagenesis. A collection of mutant ribonucleic proteins containing the nonstructural genes was generated from the library that were rescued for an infectious virus mutant library by a novel RNP competition virus rescue procedure. Several temperature-sensitive (ts) mutant viruses were obtained by screening of the mutant library, and the sequences of their NS1 genes were determined. Most of the mutations identified led to amino acid exchanges and concentrated in the N-terminal region of the protein, but some of them occurred in the C-terminal region. Mutant 11C contained three mutations that led to amino acid exchanges, V18A, R44K, and S195P, all of which were required for the ts phenotype, and was characterized further. Several steps in the infection were slightly altered: (i) M1, M2, NS1, and neuraminidase (NA) accumulations were reduced and (ii) NS1 protein was retained in the nucleus in a temperature-independent manner, but these modifications could not justify the strong virus titer reduction at restrictive temperature. The most dramatic phenotype was the almost complete absence of virus particles in the culture medium, in spite of normal accumulation and nucleocytoplasmic export of virus RNPs. The function affected in the 11C mutant was required late in the infection, as documented by shift-up and shift-down experiments. The defect in virion production was not due to reduced NA expression, as virus yield could not be rescued by exogenous neuraminidase treatment. All together, the analysis of 11C mutant phenotype may indicate a role for NS1 protein in a late event in virus morphogenesis.     

Influenza virus NS1 protein enhances the rate of translation initiation of viral mRNAs.

The effect of NS1 protein on the efficiency of influenza virus mRNA translation was evaluated by determining the accumulation of nucleoprotein (NP) or M1 mRNAs in the cytoplasm of cells expressing either of these genes alone or in combination with the NS1 gene, as well as the total cell accumulation of NP or M1 protein. Coexpression of NS1, but not of NS2 protein, led to increases in the translation of these mRNAs in the range of 5- to 100-fold. This translation enhancement was specific for viral mRNAs, since the translation of neither cat nor lacZ mRNAs was affected by the coexpression of NS1 protein. The use of chimeric cat genes containing the 5'-extracistronic sequences of the influenza virus mRNAs corresponding to segment 2, 7, or 8 indicated that these sequences can in part account for the observed effect. The enhancement of viral mRNA translation mediated by NS1 protein was due to an increase in the translation initiation rate, since the sizes of NP-specific polysomes, but not those of lacZ-specific polysomes, was significantly higher in cells coexpressing NS1 protein than in those expressing only the NP gene.     

Characterization of T antigen in cells infected with a temperature-sensitive mutant of simian virus 40.

T antigen induced in African green monkey kidney cells by a temperature-sensitive mutant of simian virus 40, defective in a function required for cell transformation, was characterized. The number of T antigen-positive cells estimated by an immunofluorescent techniques was almost equal at permissive (32.5 C) and restrictive (38.5 C) temperatures, but was slightly reduced when the infected cells were incubated at a higher temperature (40.5 C). However, a complement fixation test indicated that the amount of T antigen induced by the mutant is not significantly different from that induced by wild-type virus at 40.5 C. These results suggest that the T antigen-inducing ability of the mutant is not defective. Two distinct molecular species of T antigen were induced by the mutant at the permissive temperature, whereas only one form was observed at the restrictive temperature. The larger molecular form (14 to 15S) induced by the mutant at the permissive temperature was more heat labile than that induced by wild-type virus, suggesting that the mutated gene product is a component of the larger molecular form.