Molecular characterization of mouse-virulent poliovirus type 1 Mahoney mutants: involvement of residues of polypeptides VP1 and VP2 located on the inner surface of the capsid protein shell

Most poliovirus (PV) strains, including PV PV-1/Mahoney, are unable to cause paralysis in mice. Determinants for restriction of PV-1/Mahoney in mice have been identified by manipulating PV-1 cDNA and located on the viral capsid protein VP1. These determinants consist of a highly exposed amino acid sequence on the capsid surface corresponding to the B-C loop (M. Murray, J. Bradley, X. Yang, E. Wimmer, E. Moss, and V. Racaniello, Science 241:213-215, 1988; A. Martin, C. Wychowski, T. Couderc, R. Crainic, J. Hogle, and M. Girard, EMBO J. 7:2839-2847, 1988) and of residues belonging to the N-terminal sequence located on the inner surface of the protein shell (E. Moss and V. Racaniello, EMBO J. 10:1067-1074, 1991). Using an in vivo approach, we isolated two mouse-neurovirulent PV-1 mutants in the mouse central nervous system after a single passage of PV-1/Mahoney inoculated by the intracerebral route. Both mutants were subjected to two additional passages in mice, plaque purified, and subsequently characterized. The two cloned mutants, Mah-NK13 and Mah-NL32, retained phenotypic characteristics of the parental PV-1/Mahoney, including epitope map, heat lability, and temperature sensitivity. Mah-NK13 exhibited slightly smaller plaques than did the parental virus. The nucleotide sequences of the mutant genomes were determined, and mutations were identified. Mutations were independently introduced into the parental PV-1/Mahoney genome by single-site mutagenesis. Mutated PV-1/Mahoney viruses were then tested for their neurovirulence in mice. A single amino acid substitution in the capsid proteins VP1 (Thr-22-->Ile) and VP2 (Ser-31-->Thr) identified in the Mah-NK13 and Mah-NL32 genomes, respectively, conferred the mouse-virulent phenotype to the mouse-avirulent PV-1/Mahoney. Ile-22 in VP1 was responsible for the small-plaque phenotype of Mah-NK13. Both mutations arose during the first passage in the mouse central nervous system. We thus identified a new mouse adaptation determinant on capsid protein VP1, and we showed that at least one other capsid protein, VP2, could also express a mouse adaptation determinant. Both determinants are located in the inside of the three-dimensional structure of the viral capsid. They may be involved in the early steps of mouse nerve cell infection subsequent to receptor attachment.     

Evaluation of the genetic stability of the temperature-sensitive PB2 gene mutation of the influenza A/Ann Arbor/6/60 cold-adapted vaccine virus

A single-gene reassortant bearing the PB2 gene of the A/Ann Arbor/6/60 cold-adapted virus in the background of the A/Korea/82 (H3N2) wild-type virus is a temperature-sensitive (ts) virus with an in vitro shutoff temperature of 38 degrees C. A single mutation at amino acid (aa) at 265 (Asp-Ser) of the PB2 protein is responsible for the ts phenotype. This ts single-gene PB2 reassortant virus was serially passaged at elevated temperatures in Madin-Darby canine kidney cells to generate ts+ phenotypic revertant viruses. Four ts+ phenotypically revertant viruses were derived independently, and each possessed a shutoff temperature for replication in vitro of > 40 degrees C. Each of the four phenotypically revertant viruses replicated efficiently in the upper and lower respiratory tracts of mice and hamsters, unlike the PB2 single-gene reassortant virus, confirming that the ts phenotype was responsible for the attenuation of this virus in rodents. Mating the ts+ revertants with wild-type virus yielded ts progeny in high frequency, indicating that the loss of ts phenotype was due to a suppressor mutation which was mapped to the PA gene in each of the four independently derived ts phenotypic revertants. Nucleotide sequence analysis confirmed the absence of new mutations on the PB2 gene and the presence of predicted amino acid changes in the PA proteins of the revertant viruses. These studies suggest that single amino acid changes at aa 245 (Glu-Lys) or 347 (Asp-Asn) of the PA protein can completely suppress the ts and attenuation phenotypes specified by the Asp-Ser mutation at aa 265 of the PB2 protein of the A/Ann Arbor/6/60 cold-adapted virus.     

Sequences within the poliovirus internal ribosome entry segment control viral RNA synthesis

The 5' untranslated region of poliovirus RNA has been reported to possess two functional elements: (i) the 5' proximal 88 nucleotides form a cloverleaf structure implicated in positive-strand RNA synthesis during viral replication, and (ii) nucleotides 134 to at least 556 function as a highly structured internal ribosome entry segment (IRES) during cap-independent, internal initiation of translation. We show here that the IRES itself is bifunctional and contains sequences necessary for viral RNA synthesis per se. For this purpose, we used a dicistronic poliovirus RNA in which the translation of the viral non-structural (replication) proteins is uncoupled from the poliovirus IRES. In this system, RNA synthesis is readily detectable in transfected cells, even when the poliovirus IRES is inactivated by point mutation. However, deletion of the major part of the poliovirus IRES renders viral-specific RNA synthesis undetectable. Using the same system, we show that a three nucleotide deletion at position 500 in the 5' untranslated region drastically affects both translation efficiency and RNA synthesis. Furthermore, disruption of the secondary structure of the IRES around nucleotide 343 has minimal effects on IRES function, but dramatically reduces viral RNA replication. Taken together, these results provide direct evidence that sequences essential for viral RNA synthesis are located in the 3' region of the poliovirus IRES.     

Second-site mutation of Ala-220 to Glu or Asp suppresses the mutation of Asp-285 to Asn in the transposon Tn10-encoded metal-tetracycline/H+ antiporter of Escherichia coli

A carboxyl group of Asp-285 is essential for tetracycline/H+ antiport mediated by the transposon Tn10-encoded metal-tetracycline/H+ antiporter (TetA) of Escherichia coli (Yamaguchi, A., Akasaka, T., Ono, N., Someya, Y., Nakatani, M., and Sawai, T. (1992) J. Biol. Chem. 267, 7490-7498). Spontaneous tetracycline resistance revertants were isolated from E. coli cells carrying the Asn-285 mutant tetA gene. All of the revertants were due to the second-site mutation at codon 220 of GCG (Ala) to GAG (Glu). The Km value of the tetracycline transport mediated by the revertant TetA protein was about 4-fold higher than that of the wild-type, indicating that the revertant is a low affinity mutant. A Glu-220 and Asn-285 double mutant constructed by site-directed mutagenesis showed the same properties as the revertants, confirming that the mutation of Ala-220 is solely responsible for the suppression. The Asp-220 mutation of the Asn-285 mutant resulted in a lower level of restoration of the tetracycline resistance and the transport activity than in the case of the Glu-220 mutation. A single mutation replacing Ala-220 with Glu or Asp caused about a 2-4-fold decrease in the tetracycline resistance, but no crucial change in the transport activity. It is not likely that Glu-220 is required for a charge-neutralizing salt bridge because an unpaired negative charge in a Glu-220 or Asp-220 single mutant did not cause a serious change in the activity. An alternative explanation is reasonable; Asp-285 directly contributes to the binding of a cationic substrate, metal-tetracycline chelation complex, or proton, and an acidic residue at position 220 can take over the role of Asp-285.     

Left ventricular apex visualization: the value of magnetic resonance imaging

We have previously shown that prolonged interferon-beta (IFN-beta) treatment of RS485 cells (NIH3T3 cells transformed with multiple copies of an LTR-cHa-ras oncogene) resulted in the phenotypic reversion of 1%-5% of the culture, depending on the conditions used. This reversion persisted after IFN-beta was discontinued, although the revertants retained the LTR-cHa-ras and continued to express ras mRNA and p21. Clones were prepared of such persistent revertant cell lines (PRs). Expression of lysyl oxidase (LOX), which appears to act as a suppressor of ras transformation, was downregulated in RS485 and upregulated in the PRs. When retinoic acid (RA) was combined with IFN-beta treatment of the RS485 cultures, a different mechanism of reversion predominated. Following 60 days of treatment with 20 IU/ml of IFN-beta and 10 microM RA, all of the multiple (3-5) copies of the transforming LTR-c-Ha-ras originally present in RS485 cells were deleted from the genome in 72% of 54 revertant cell lines isolated. As in the case of revertants observed after treatment with IFN-beta alone, LOX mRNA expression was upregulated in all of the revertants that resulted from the treatment with IFN plus RA. The level of LOX mRNA expression acts, therefore, as an indicator of transformation in this system.     

Intratympanic gentamicin in Meniere''s disease

In Chlamydomonas reinhardtii, mutants defective in the cytochrome pathway of respiration lack the capacity to grow under heterotrophic conditions (in darkness on acetate). In the dark- strain duM18, a + 1 T addition in a run of four Ts, located at codon 145 of the mitochondrial cox1 gene encoding subunit I of cytochrome c oxidase, is responsible for the mutant phenotype. A leaky revertant (su11) that grows heterotrophically at a lower rate than wild-type cells was isolated from dum18. Its respiration sensitivity to cyanide was low and its cytochrome c oxidase activity was only 4% of that of the wild-type enzyme. Meiotic progeny obtained from crosses between revertant and wild-type cells inherited the phenotype of the mt- parent, showing that the suppressor mutation, like dum18 itself, is located in the mitochondrial genome. In order to map the su11 mutation relative to dum18, a recombinational analysis was performed on the diploid progeny. It demonstrated that su11 was very closely linked to the dum18 mutation less than 20-30 bp away. The cox1 gene of the su11 revertant was then sequenced. In addition to the + 1 T frameshift mutation still present at codon 145, an A-->C substitution was found at codon 146, leading to the replacement of a glutamic acid by an alanine in the polypeptide chain. No other mutations were detected in the cox1 coding sequence. As the new GCG codon (Ala) created at position 146 is very seldom used in the mitochondrial genome of C. reinhardtii, we suggest that the partial frameshift suppression by the nearby substitution is due to an occasional abnormal translocation of the ribosome (+ 1 base shift) facilitated both by the run of Ts and the low level of weak interaction of alanyl-tRNA.     

RNA structure adjacent to the attenuation determinant in the 5'-non-coding region influences poliovirus viability

In attenuated Sabin strains, point mutations within stem-loop V of the 5'-non-coding region (NCR) reduce neurovirulence and cell-specific cap-independent translation. The stem-loop V attenuation determinants lie within the highly structured internal ribosome entry site. Although stem-loop V Sabin mutations have been proposed to alter RNA secondary structure, efforts to identify such conformational changes have been unsuccessful. A previously described linker-scanning mutation (X472) modified five nucleotides adjacent to the attenuation determinant at nt 480 [for poliovirus (PV) type 1]. Transfection of X472 RNA generated only pseudo-revertants in HeLa (cervical carcinoma) or SK-N-SH (neuroblastoma) cells. Pseudo-revertants from both cell types contained nucleotide changes within the X472 linker. In addition, some neuroblastoma-isolated revertants revealed second site mutations within the pyrimidine-rich region located approximately 100 nt distal to the original lesion. Enzymatic RNA structure probing determined that the X472 linker substitution did not disrupt the overall conformation of stem-loop V but abolished base pairing adjacent to the attenuation determinant. Our analyses correlated increased base pairing proximal to the stem-loop V attenuation determinant with growth of X472 revertant RNAs (measured by northern blot analysis). Potential roles of second site mutations in the pyrimidine-rich region are discussed. In addition, our enzymatic structure probing results are shown on a consensus secondary structure model for stem-loop V of the PV 5'-NCR.     

Alterations to both the primary and predicted secondary structure of stem-loop IIIc of the hepatitis C virus 1b 5' untranslated region (5'UTR) lead to mutants severely defective in translation which cannot be complemented in trans by the wild-type 5'UTR sequence

Cap-independent translation of the hepatitis C virus (HCV) genomic RNA is mediated by an internal ribosome entry site (IRES) within the 5' untranslated region (5'UTR) of the virus RNA. To investigate the effects of alterations to the primary sequence of the 5'UTR on IRES activity, a series of HCV genotype 1b (HCV-1b) variant IRES elements was generated and cloned into a bicistronic reporter construct. Changes from the prototypic HCV-1b 5'UTR sequence were identified at various locations throughout the 5'UTR. The translation efficiencies of these IRES elements were examined by an in vivo transient expression assay in transfected BHK-21 cells and were found to range from 0.4 to 95.8% of the activity of the prototype HCV-1b IRES. Further mutational analysis of the three single-point mutants most severely defective in activity, whose mutations were all located in or near stem-loop IIIc, demonstrated that both the primary sequence and the maintenance of base pairing within this stem structure were critical for HCV IRES function. Complementation studies indicated that defective mutants containing either point mutations or major deletions within the IRES elements could not be complemented in trans by a wild-type IRES.     

Isolation of flat revertants from human papillomavirus type 18 E6E7 transformed 3Y1 cells by transfection with a rat embryo fibroblast cDNA expression library

A rat embryo fibroblast (REF) cDNA expression library was transfected into 3Y1 cells transformed by human papillomavirus type 18 E6 and E7 genes and 10 flat revertants were isolated. These revertants expressed the same levels of E6 and E7 mRNA as the parent cells, but had greatly reduced ability to form colonies in soft agar. Suppression of transformation was dominant in cell hybrids generated by fusing each revertant with the parental transformed cells. Furthermore, loss of transfected cDNA was observed in re-transformed cell hybrids derived from one flat revertant. Overexpression of the cDNA suppresses the colony-forming efficiency of the cells transformed by E6 and E7 genes.     

Expression of the poliovirus receptor in intestinal epithelial cells is not sufficient to permit poliovirus replication in the mouse gut

Although the initial site of poliovirus replication in humans is the intestine, previously isolated transgenic mice which carry the human poliovirus receptor (PVR) gene (TgPVR mice), which develop poliomyelitis after intracerebral inoculation, are not susceptible to infection by the oral route. The low levels of PVR expressed in the TgPVR mouse intestine might explain the absence of poliovirus replication at that site. To ascertain whether PVR is the sole determinant of poliovirus susceptibility of the mouse intestine, we have generated transgenic mice by using the promoter for rat intestine fatty acid binding protein to direct PVR expression in mouse gut. Pvr was detected by immunohistochemistry in the enterocytes and M cells of transgenic mouse (TgFABP-PVR) small intestine. Upon oral inoculation with poliovirus, no increase in virus titer was detected in the feces of TgFABP-PVR mice, and no virus replication was observed in the small intestine, although poliovirus replicated in the brain after intracerebral inoculation. The failure of poliovirus to replicate in the TgFABP-PVR mouse small intestine was not due to lack of virus binding sites, because poliovirus could attach to fragments of small intestine from these mice. These results indicate that the inability of poliovirus to replicate in the mouse alimentary tract is not solely due to the absence of virus receptor, and other factors are involved in determining the ability of poliovirus to replicate in the mouse gut.     

A single mutation that increases maize seed weight

The maize endosperm-specific gene shrunken2 (Sh2) encodes the large subunit of the heterotetrameric starch synthetic enzyme adenosine diphosphoglucose pyrophosphorylase (AGP; EC 2.7.7.27). Here we exploit an in vivo, site-specific mutagenesis system to create short insertion mutations in a region of the gene known to be involved in the allosteric regulation of AGP. The site-specific mutagen is the transposable element dissociation (Ds). Approximately one-third (8 of 23) of the germinal revertants sequenced restored the wild-type sequence, whereas the remaining revertants contained insertions of 3 or 6 bp. All revertants retained the original reading frame 3' to the insertion site and involved the addition of tyrosine and/or serine. Each insertion revertant reduced total AGP activity and the amount of the SH2 protein. The revertant containing additional tyrosine and serine residues increased seed weight 11-18% without increasing or decreasing the percentage of starch. Other insertion revertants lacking an additional serine reduced seed weight. Reduced sensitivity to phosphate, a long-known inhibitor of AGP, was found in the high seed-weight revertant. This alteration is likely universally important since insertion of tyrosine and serine in the potato large subunit of AGP at the comparable position and expression in Escherichia coli also led to a phosphate-insensitive enzyme. These results show that single gene mutations giving rise to increased seed weight, and therefore perhaps yield, are clearly possible in a plant with a long history of intensive and successful breeding efforts.     

Mutations of G158 and their second-site revertants in the plasma membrane H(+)-ATPase gene (pma1) in Saccharomyces cerevisiae

A G158D mutation residing near the cytoplasmic end of transmembrane segment 2 of the H(+)-ATPase from Saccharomyces cerevisiae appears to alter electrogenic proton transport by the proton pump (Perlin et al. (1988) J. Biol. Chem. 263, 18118-18122.) The mutation confers upon whole cells a pronounced growth sensitivity to low pH and a resistance to the antibiotic hygromycin B. The isolated enzyme retains high activity (70% of wild type) but is inefficient at pumping protons in a reconstituted vesicle system, suggesting that this enzyme may be partially uncoupled (Perlin et al. (1989) J. Biol. Chem. 264, 21857-21864). In this study, the acid-sensitive growth phenotype of the pma1-D158 mutant was utilized to isolate second site suppressor mutations in an attempt to probe structural interactions involving amino acid 158. Site-directed mutagenesis of the G158 locus was also performed to explore its local environment. Nineteen independent revertants of pma1-G158D were selected as low pH-resistant colonies. Four were full phenotypic revertants showing both low pH resistance and hygromycin B sensitivity. Of three full revertants analyzed further, one restored the original glycine residue at position 158 while the other two carried compensatory mutations V336A or F830S, in transmembrane segments 4 and 7, respectively. Partial revertants, which could grow on low pH medium but still retained hygromycin B resistance, were identified in transmembrane segments 1 (V127A) and 2 (C148T, G156C), as well as in the cytoplasmic N-terminal domain (E110K) and in the cytoplasmic loop between transmembrane segments 2 and 3 (D170N, L275S). Relative to the G158D mutant, all revertants showed enhanced net proton transport in whole-cell medium acidification assays and/or improved ATP hydrolysis activity. Small polar amino acids (Asp and Ser) could be substituted for glycine at the 158 position to produce active, albeit somewhat defective, enzymes; larger hydrophobic residues (Leu and Val) produced more severe phenotypes. These results suggest that G158 is likely to reside in a tightly packed polar environment which interacts, either directly or indirectly, with transmembrane segments 1, 4 and 7. The revertant data are consistent with transmembrane segments 1 and 2 forming a conformationally sensitive helical hairpin structure.     

One of two NTP binding sites in poliovirus RNA polymerase required for RNA replication

The poliovirus RNA-dependent RNA polymerase (3Dpol) has been shown to contain two NTP binding sites by chemical cross-linking of oxidized nucleotide to the intact protein. Only one site (Lys-61) was shown to be essential for RNA chain elongation activity by purified enzyme; however, a full-length viral RNA, coding for an altered lysine residue (K276L) in the second site, generated virus with a minute plaque phenotype that rapidly reverted to a wild-type phenotype with Arg-276 replacing Leu-276 in 3D. Viruses with lysine to leucine substitutions in other positions of the second binding site of their polymerase proteins grew with wild-type phenotype. To test the significance of the second binding site, poliovirus 3Dpol was generated with lysine (wild-type), leucine, or arginine at residue 276 and tested for NTP cross-linking using 32P-oxidized GTP. Analysis of cyanogen bromide peptides of each 3D preparation showed that the second NTP binding site had severely reduced NTP binding in mu276(Leu) but not in the revertant mu276(Arg), despite the reported requirement for lysine in the cross-linking reaction. To eliminate the possibility that 32P-oxidized GTP cross-linked to Arg at residue 276, a model system was designed with unmodified amino acid or acetylated (alpha-amino) amino acid and 32P-oxidized GTP. Cross-linking to lysine, but not leucine or arginine, was observed thus eliminating the possibility that NTP could be cross-linked to residue 276 in 3D. We conclude that NTP binding at the second site in poliovirus 3D is at lysine residues at positions other than 276 (278 or 283), and nucleotide binding at these sites has no bearing on elongation activity or replication of the virus. Nucleotide binding only at the site including Lys-61 is essential for RNA replication.     

Sequences within a small yeast RNA required for inhibition of internal initiation of translation: interaction with La and other cellular proteins influences its inhibitory activity

We recently reported purification, determination of the nucleotide sequence, and cloning of a 60-nucleotide RNA (I-RNA) from the yeast Saccharomyces cerevisiae which preferentially blocked cap-independent, internal ribosome entry site (IRES)-mediated translation programmed by the poliovirus (PV) 5' untranslated region (UTR). The I-RNA appeared to inhibit IRES-mediated translation by virtue of its ability to bind a 52-kDa polypeptide which interacts with the 5' UTR of viral RNA. We demonstrate here that the HeLa 52-kDa I-RNA-binding protein is immunologically identical to human La autoantigen. Moreover, I-RNA-mediated purified La protein. By using I-RNAs with defined deletions, we have identified sequences of I-RNA required for inhibition of internal initiation of translation. Two smaller fragments of I-RNA (16 and 25 nucleotides) inhibited PV UTR-mediated translation from both monocistronic and bicistronic RNAs. When transfected into HeLa cells, these derivatives of I-RNA inhibited translation of PV RNA. A comparison of protein binding by active and inactive I-RNA mutants demonstrates that in addition to the La protein, three other polypeptides with apparent molecular masses of 80, 70, and 37 kDa may influence the translation-inhibitory activity of I-RNA.     

Selective growth of transformed cell lines by rat liver perfusate

Perfused rat liver releases growth-promoting activity for viral, spontaneous, and chemically transformed cells. After 5 days of incubation with perfusate, cell lines 3T12-NY (a spontaneous fibroblast transformant), NQ-T1 (a chemically transformed fibroblast line), W-8 (a chemically transformed epithelial rat liver cell line increase in cell growth above controls. Their respective normal counterparts: 3T3 Cl 42, A31-714, K-16, and HEF are not so stimulated. Within another set, the virally transformed mouse fibroblast cell line, SV3T3, exhibits a 27-fold increase in growth; however, 3T3 (mouse, fibroblasts), Py3T3 (polyomatransformed 3T3 cells), SV-Fl2-101 (a flat revertant line), and SV-Py-3T3 (a doubly transformed line) are nonresponsive. Perfused rat liver also release survival activity for SV-3T3 cells. The growth-stimulating activity in liver perfusate is selective for transformed cells. It is suggested that the liver may play a role in suporting neoplasia in vivo.     

Life-cycle, longevity and fecundity of Blomia tropicalis (Acari: Glycyphagidae) in a tropical laboratory

HSV-1716 is a replication-restricted, neuroattenuated ICP 34.5 gene mutant of herpes simplex virus type 1 (HSV-1). Because of the attenuated phenotype of ICP 34.5 mutants in rodent models of HSV disease, they have been promoted as potential vaccine strains and gene therapy vectors and have been used by us and others as therapeutic agents for the treatment of experimental malignant tumors. However, all data on the phenotype of HSV-1716 and other ICP 34.5 mutants are from animal model systems, while humans are the natural hosts of HSV-1. To achieve an initial characterization of the phenotype of 1716 in human tissue, we have studied its replication in mature human skin xenografts on SCID mice. We find that replication of 1716 is severely restricted in such human skin grafts relative to both parental wild-type HSV-1 strain 17+ and the HSV-1716 revertant virus 1716R, in which the 759-bp ICP 34.5 gene deletions have been repaired. Moreover, the replication of both 1716 and 17+ is significantly better in the human skin grafts than it is in mouse skin. The implications of these findings are discussed.     

Vegetative tissue lectins of peanut (A. hypogaea)

Lectin activities in roots, nodules, stems and leaves of 1-6 week old peanut plant (A. hypogaea) were checked by erythrocyte (human and rabbit) agglutination and sugar inhibition assays. Human and rabbit erythrocyte agglutinating activities were specifically inhibited by lactose/cellobiose (SLII) and methyl alpha-mannoside (SLI) respectively. Seeds, embryos and cotyledons agglutinated neuraminidase treated human erythrocytes and that activity was inhibited by T-disaccharide. In the roots of field grown plants SLI was the major activity, while nodules showed both activities (SLI and SLII). Specific activities of SLI and SLII were maximal in stem tissue and hypocotyl exhibited minimal levels. Actively growing tissues like newly emerging young leaves and elongating stem contained more SLII activity in comparison to the mature tissues. Immunological test indicated that all the vegetative tissue lectins are serologically related.