Targeted Recombination within the Spike Gene of Murine Coronavirus Mouse Hepatitis Virus-A59: Q159 Is a Determinant of Hepatotropism

Previous studies of a group of mutants of the murine coronavirus mouse hepatitis virus (MHV)-A59, isolated from persistently infected glial cells, have shown a strong correlation between a Q159L amino acid substitution in the S1 subunit of the spike gene and a loss in the ability to induce hepatitis and demyelination. To determine if Q159L alone is sufficient to cause these altered pathogenic properties, targeted RNA recombination was used to introduce a Q159L amino acid substitution into the spike gene of MHV-A59. Recombination was carried out between the genome of a temperature-sensitive mutant of MHV-A59 (Alb4) and RNA transcribed from a plasmid (pFV1) containing the spike gene as well as downstream regions, through the 3′ end, of the MHV-A59 genome. We have selected and characterized two recombinant viruses containing Q159L. These recombinant viruses (159R36 and 159R40) replicate in the brains of C57BL/6 mice and induce encephalitis to a similar extent as wild-type MHV-A59. However, they exhibit a markedly reduced ability to replicate in the liver or produce hepatitis compared to wild-type MHV-A59. These viruses also exhibit reduced virulence and reduced demyelination. A recombinant virus containing the wild-type MHV-A59 spike gene, wtR10, behaved essentially like wild-type MHV-A59. This is the first report of the isolation of recombinant viruses containing a site-directed mutation, encoding an amino acid substitution, within the spike gene of any coronavirus. This technology will allow us to begin to map the molecular determinants of pathogenesis within the spike glycoprotein.     

Importance of individual amino acids in the Switch I region in eEF2 studied by functional complementation in S. cerevisiae

Elongation factor 2 (eEF2) is a member of the G-protein super family. G-proteins undergo conformational changes associated with binding of the guanosine nucleotide and hydrolysis of the bound GTP. These structural rearrangements affects the Switch I region (also known as the Effector loop). We have studied the role of individual amino acids in the Switch I region (amino acids 25-73) of S. cerevisiae eEF2 using functional complementation in yeast. 21 point mutations in the Switch I region were created by site-directed mutagenesis. Mutants K49R, E52Q, A53G, F55Y, K60R, Q63A, T68S, I69M and A73G were functional while mutants R54H, F55N, D57A, D57E, D57S, R59K, R59M, Q63E, R65A, R65N, T68A and T68M were inactive. Expression of mutants K49R, A53G, Q63A, I69M and A73G was associated with markedly decreased growth rates and yeast cells expressing mutants A53G and I69M became temperature sensitive. The functional capacity of eEF2 in which the major part Switch I (amino acids T56 to I69) was converted into the homologous sequence found in EF-G from E. coli was also studied. This protein chimera could functionally replace yeast eEF2 in vivo. Yeast cells expressing this mutant grew extremely slowly, showed increased cell death and became temperature sensitive. The ability of the mutant to replace authentic eEF2 in vivo indicates that the structural rearrangement of Switch I necessary for eEF2 function is similar in eukaryotes and bacteria. The effect of two point mutations in the P-loop was also studied. Mutant A25G but not A25V could functionally replace yeast eEF2 even if cells expressing the mutant grew slowly. The A25G mutation converted the consensus sequences AXXXXGK[T/S] in eEF2 to the corresponding motif GXXXXGK[T/S] found in all other G-proteins, suggesting that the alanine found in the P-loop of peptidyltranslocases are not essential for function.     

Immunogenic peptide comprising a mouse hepatitis virus A59 B-cell epitope and an influenza virus T-cell epitope protects against lethal infection.

The coronavirus spike protein S is responsible for important biological activities including virus neutralization by antibody, cell attachment, and cell fusion. Recently, we have elucidated the amino acid sequence of an S determinant common in murine coronaviruses (W. Luytjes, D. Geerts, W. Posthumus, R. Meloen, and W. Spaan, J. Virol. 63:1408-1412, 1989). A monoclonal antibody directed to this determinant (MAb 5B19.2) protected mice against acute fatal infection. In this study, BALB/c mice were immunized with a synthetic peptide of 13 amino acids corresponding to the binding site of MAb 5B19.2, which was either extended with an amino acid sequence of influenza virus hemagglutinin or conjugated to keyhole limpet hemocyanin. Both immunogens induced S-specific antibodies in mice, but only the hemagglutinin-peptide construct protected them against lethal challenge. In contrast to mouse hepatitis virus type 4 (MHV-4), MHV-A59 was not neutralized in vitro by MAb 5B19.2. Neither MHV-A59 nor MHV-4 was neutralized in vitro by antibodies comprising by the synthetic peptides. Our results demonstrated that antibodies elicited with a synthetic peptide comprising a B-cell epitope and a T-helper cell determinant can protect mice against an acute fetal mouse hepatitis virus infection.     

Structure/function mapping of amino acids in the N-terminal zinc finger of the human immunodeficiency virus type 1 nucleocapsid protein: residues responsible for nucleic acid helix destabilizing activity

The nucleocapsid protein (NC) of HIV-1 is 55 amino acids in length and possesses two CCHC-type zinc fingers. Finger one (N-terminal) contributes significantly more to helix destabilizing activity than finger two (C-terminal). Five amino acids differ between the two zinc fingers. To determine at the amino acid level the reason for the apparent distinction between the fingers, each different residue in finger one was incrementally replaced by the one at the corresponding location in finger two. Mutants were analyzed in annealing assays with unstructured and structured substrates. Three groupings emerged: (1) those similar to wild-type levels (N17K, A25M), (2) those with diminished activity (I24Q, N27D), and (3) mutant F16W, which had substantially greater helix destabilizing activity than that of the wild type. Unlike I24Q and the other mutants, N27D was defective in DNA binding. Only I24Q and N27D showed reduced strand transfer in in vitro assays. Double and triple mutants F16W/I24Q, F16W/N27D, and F16W/I24Q/N27D all showed defects in DNA binding, strand transfer, and helix destabilization, suggesting that the I24Q and N27D mutations have a dominant negative effect and abolish the positive influence of F16W. Results show that amino acid differences at positions 24 and 27 contribute significantly to finger one's helix destabilizing activity.     

Conformational changes in the spike glycoprotein of murine coronavirus are induced at 37 degrees C either by soluble murine CEACAM1 receptors or by pH 8

The spike glycoprotein (S) of the murine coronavirus mouse hepatitis virus (MHV) binds to viral murine CEACAM receptor glycoproteins and causes membrane fusion. On virions, the 180-kDa S glycoprotein of the MHV-A59 strain can be cleaved by trypsin to form the 90-kDa N-terminal receptor-binding subunit (S1) and the 90-kDa membrane-anchored fusion subunit (S2). Incubation of virions with purified, soluble CEACAM1a receptor proteins at 37 degrees C and pH 6.5 neutralizes virus infectivity (B. D. Zelus, D. R. Wessner, R. K. Williams, M. N. Pensiero, F. T. Phibbs, M. deSouza, G. S. Dveksler, and K. V. Holmes, J. Virol. 72:7237-7244, 1998). We used liposome flotation and protease sensitivity assays to investigate the mechanism of receptor-induced, temperature-dependent virus neutralization. After incubation with soluble receptor at 37 degrees C and pH 6.5, virions became hydrophobic and bound to liposomes. Receptor binding induced a profound, apparently irreversible conformational change in S on the viral envelope that allowed S2, but not S1, to be degraded by trypsin at 4 degrees C. Various murine CEACAM proteins triggered conformational changes in S on recombinant MHV strains expressing S glycoproteins of MHV-A59 or MHV-4 (MHV-JHM) with the same specificities as seen for virus neutralization and virus-receptor activities. Increased hydrophobicity of virions and conformational change in S2 of MHV-A59 could also be induced by incubating virions at pH 8 and 37 degrees C, without soluble receptor. Surprisingly, the S protein of recombinant MHV-A59 virions with a mutation, H716D, that precluded cleavage between S1 and S2 could also be triggered to undergo a conformational change at 37 degrees C by soluble receptor at neutral pH or by pH 8 alone. A novel 120-kDa subunit was formed following incubation of the receptor-triggered S(A59)H716D virions with trypsin at 4 degrees C. The data show that unlike class 1 fusion glycoproteins of other enveloped viruses, the murine coronavirus S protein can be triggered to a membrane-binding conformation at 37 degrees C either by soluble receptor at neutral pH or by alkaline pH alone, without requiring previous activation by cleavage between S1 and S2.     

Murine coronavirus spike protein determines the ability of the virus to replicate in the liver and cause hepatitis

Recombinant mouse hepatitis viruses (MHV) differing only in the spike gene, containing A59, MHV-4, and MHV-2 spike genes in the background of the A59 genome, were compared for their ability to replicate in the liver and induce hepatitis in weanling C57BL/6 mice infected with 500 PFU of each virus by intrahepatic injection. Penn98-1, expressing the MHV-2 spike gene, replicated to high titer in the liver, similar to MHV-2, and induced severe hepatitis with extensive hepatocellular necrosis. S(A59)R13, expressing the A59 spike gene, replicated to a somewhat lower titer and induced moderate to severe hepatitis with zonal necrosis, similar to MHV-A59. S4R21, expressing the MHV-4 spike gene, replicated to a minimal extent and induced few if any pathological changes, similar to MHV-4. Thus, the extent of replication and the degree of hepatitis in the liver induced by these recombinant viruses were determined largely by the spike protein.     

Contributions of the viral genetic background and a single amino acid substitution in an immunodominant CD8+ T-cell epitope to murine coronavirus neurovirulence

The immunodominant CD8+ T-cell epitope of a highly neurovirulent strain of mouse hepatitis virus (MHV), JHM, is thought to be essential for protection against virus persistence within the central nervous system. To test whether abrogation of this H-2Db-restricted epitope, located within the spike glycoprotein at residues S510 to 518 (S510), resulted in delayed virus clearance and/or virus persistence we selected isogenic recombinants which express either the wild-type JHM spike protein (RJHM) or spike containing the N514S mutation (RJHM(N514S)), which abrogates the response to S510. In contrast to observations in suckling mice in which viruses encoding inactivating mutations within the S510 epitope (epitope escape mutants) were associated with persistent virus and increased neurovirulence (Pewe et al., J Virol. 72:5912-5918, 1998), RJHM(N514S) was not more virulent than the parental, RJHM, in 4-week-old C57BL/6 (H-2b) mice after intracranial injection. Recombinant viruses expressing the JHM spike, wild type or encoding the N514S substitution, were also selected in which background genes were derived from the neuroattenuated A59 strain of MHV. Whereas recombinants expressing the wild-type JHM spike (SJHM/RA59) were highly neurovirulent, A59 recombinants containing the N514S mutation (SJHM(N514S)/RA59) were attenuated, replicated less efficiently, and exhibited reduced virus spread in the brain at 5 days postinfection (peak of infectious virus titers in the central nervous system) compared to parental virus encoding wild-type spike. Virulence assays in BALB/c mice (H-2d), which do not recognize the S510 epitope, revealed that attenuation of the epitope escape mutants was not due to the loss of a pathogenic immune response directed against the S510 epitope. Thus, an intact immunodominant S510 epitope is not essential for virus clearance from the CNS, the S510 inactivating mutation results in decreased virulence in weanling mice but not in suckling mice, suggesting that specific host conditions are required for epitope escape mutants to display increased virulence, and the N514S mutation causes increased attenuation in the context of A59 background genes, demonstrating that genes other than that for the spike are also important in determining neurovirulence.     

Characterization of active-site mutants of Schizosaccharomyces pombe phosphoglycerate mutase. Elucidation of the roles of amino acids involved in substrate binding and catalysis.

The roles of a number of amino acids present at the active site of the monomeric phosphoglycerate mutase from the fission yeast Schizosaccharomyces pombe have been explored by site-directed mutagenesis. The amino acids examined could be divided broadly into those presumed from previous related structural studies to be important in the catalytic process (R14, S62 and E93) and those thought to be important in substrate binding (R94, R120 and R121). Most of these residues have not previously been studied by site-directed mutagenesis. All the mutants except R14 were expressed in an engineered null strain of Saccharomyces cerevisiae (S150-gpm:HIS) in good yield. The R14Q mutant was expressed in good yield in the transformed AH22 strain of S. cerevisiae. The S62A mutant was markedly unstable, preventing purification. The various mutants were purified to homogeneity and characterized in terms of kinetic parameters, CD and fluorescence spectra, stability towards denaturation by guanidinium chloride, and stability of phosphorylated enzyme intermediate. In addition, the binding of substrate (3-phosphoglycerate) to wild-type, E93D and R120,121Q enzymes was measured by isothermal titration calorimetry. The results provide evidence for the proposed roles of each of these amino acids in the catalytic cycle and in substrate binding, and will support the current investigation of the structure and dynamics of the enzyme using multidimensional NMR techniques.     

Fusion-defective mutants of mouse hepatitis virus A59 contain a mutation in the spike protein cleavage signal.

Infection of primary mouse glial cell cultures with mouse hepatitis virus strain A59 results in a productive, persistent infection, but without any obvious cytopathic effect. Mutant viruses isolated from infected glial cultures 16 to 18 weeks postinfection replicate with kinetics similar to those of wild-type virus but produce small plaques on fibroblasts and cause only minimal levels of cell-to-cell fusion under conditions in which wild type causes nearly complete cell fusion. However, since extensive fusion is present in mutant-infected cells at late times postinfection, the defect is actually a delay in kinetics rather than an absolute block in activity. Addition of trypsin to mutant-infected fibroblast cultures enhanced cell fusion a small (two- to fivefold) but significant degree, indicating that the defect could be due to a lack of cleavage of the viral spike (fusion) protein. Sequencing of portions of the spike genes of six fusion-defective mutants revealed that all contained the same single nucleotide mutation resulting in a substitution of aspartic acid for histidine in the spike cleavage signal. Mutant virions contained only the 180-kDa form of spike protein, suggesting that this mutation prevented the normal proteolytic cleavage of the 180-kDa protein into the 90-kDa subunits. Examination of revertants of the mutants supports this hypothesis. Acquisition of fusion competence correlates with the replacement of the negatively charged aspartic acid with either the wild-type histidine or a nonpolar amino acid and the restoration of spike protein cleavage. These data confirm and extend previous reports concluding cleavage of S is required for efficient cell-cell fusion by mouse hepatitis virus but not for virus-cell fusion (infectivity).     

Do rates of intercellular adhesion measure the cell affinities reflected in cell-sorting and tissue-spreading configurations?

These experiments constitute the first experimental test of the hypothesis that the rates of adhesion between cells measure the intensities of adhesion or tissue affinities that could explain cell sorting and tissue spreading. For any set of relative adhesive intensities between cells in a heterogeneous population, a corresponding minimal free energy configuration can be calculated. This is the cell distribution toward which both cell sorting and tissue spreading should lead. Equilibrium configurations were determined for combinations of 7-day embryonic retina (R) with liver (L) and heart (H), both of which became completely enveloped by R. To produce these results, the adhesive intensities would have to fall in the sequences: L-L > L-R > R-R; and H-H > H-R > R-R. To determine whether the rates of adhesion fall into these same sequences, we have devised a new technique which measures the rates of adhesion between pairs of already-formed cell aggregates of like and unlike kinds. These fall in the sequence L-L > or = H-H > L-H > R-R > H-R > L-R. If these rates paralleled the corresponding intensities of adhesion at configurational equilibrium, both L and H should have become only partially enveloped by R. Thus the rates at which adhesions are initiated do not predict the relative adhesive intensities that could explain the observed tissue configurations.     

N-terminal region of FKBP12 is essential for binding to the skeletal ryanodine receptor

It is known that the two types of FK506-binding proteins FKBP12 and FKBP12.6 are tightly associated with the skeletal (RyR1) and cardiac ryanodine receptors (RyR2), respectively, and their interactions are important for channel functions of the RyR. In the case of cardiac muscle, three amino acid residues (Gln-31, Asn-32, and Phe-59) of FKBP12.6 could be essential for the selective binding to RyR2 (Xin, H. B., Rogers, K., Qi, Y., Kanematsu, T., and Fleischer, S. (1999) J. Biol. Chem. 274, 15315-15319). In this study to identify amino acid residues of FKBP12 that are important for the selective binding to RyR1, we mutated 9 amino acid residues of FKBP12 that differ from the counterparts of FKBP12.6 (Q3E, R18A, E31Q, D32N, M49R, R57A, W59F, H94A, and K105A), and we examined binding properties of these mutants to RyR1 by in vitro binding assay by using glutathione S-transferase-fused proteins of the mutants and Triton X-100-solubilized, FKBP12-depleted rabbit skeletal sarcoplasmic reticulum vesicles. Among the nine mutants tested, only Q3E and R18A lost their selective binding ability to RyR1. Furthermore, co-immunoprecipitation of RyR1 with 33 various mutants for the 9 positions produced by introducing different size, charge, and hydrophobicity revealed that an integration of the hydrogen bonds by the irreplaceable Gln-3 and the hydrophobic interactions by the residues Arg-18 and Met-49 could be a possible mechanism for the binding of FKBP12 to RyR1. Therefore, these results suggest that the N-terminal regions of FKBP12 (Gln-3 and Arg-18) and Met-49 are essential and unique for binding of FKBP12 to RyR1 in skeletal muscle.     

Analysis of the receptor-binding site of murine coronavirus spike protein.

It has been found that a domain composed of 330 amino acids of the N terminus of murine coronavirus spike protein [S1N(330)] is involved in receptor-binding activity (H. Kubo, Y.K. Yamada, and F. Taguchi, J. Virol. 68:5403-5410, 1994). To delineate the amino acid sequences involved in receptor-binding activity, we have compared the S1N(330) proteins of seven different mouse hepatitis virus MHV strains that are able to utilize the MHV receptor protein. Three conserved regions (sites I, II, and III) were found to consist of more than 10 identical amino acids, and they were analyzed for receptor-binding activity by site-directed mutagenesis. S1N(330) with a substitution at position 62 from the N terminus of S1 in region I and that with substitutions at positions 212, 214, and 216 in region II showed no receptor-binding activity. The S1N(330) mutants without receptor-binding activity were not able to prevent virus binding to the receptor. These results suggest that the receptor-binding site on S1N(330) is composed of regions located apart from each other in the protein's primary structure, in which Thr at position 62 as well as amino acids located at positions 212, 214, and 216 are particularly important.     

Three amino acid residues determine selective binding of FK506-binding protein 12.6 to the cardiac ryanodine receptor.

FK506-binding protein (FKBP12) has been found to be associated with the skeletal muscle ryanodine receptor (RyR1) (calcium release channel), whereas FKBP12.6, a novel isoform of FKBP, is selectively associated with the cardiac ryanodine receptor (RyR2). For both RyRs, the stoichiometry is 4 FKBP/RyR. Although FKBP12.6 differs from FKBP12 by only 18 of 108 amino acids, FKBP12.6 selectively binds to RyR2 and exchanges with bound FKBP12.6 of RyR2, whereas both FKBP isoforms bind to RyR1 and exchange with bound FKBP12 of RyR1. To assess the amino acid residues of FKBP12.6 that are critical for selective binding to RyR2, the residues of FKBP12.6 that differ with FKBP12 were mutated to the respective residues of FKBP12. RyR2 of cardiac sarcoplasmic reticulum, prelabeled by exchange with [35S]FKBP12.6, was used as assay system for binding/exchange with the mutants. The triple mutant (Q31E/N32D/F59W) of FKBP12.6 was found to lack selective binding to the cardiac RyR2, comparable with that of FKBP12.0. In complementary studies, mutations of FKBP12 to the three critical amino acids of FKBP12.6, conferred selective binding to RyR2. Each of the FKBP12.6 and FKBP12 mutants retained binding to the skeletal muscle RyR1. We conclude that three amino acid residues (Gln31, Asn32, and Phe59) of human FKBP12.6 account for the selective binding to cardiac RyR2.     

The N-terminal domain of the murine coronavirus spike glycoprotein determines the CEACAM1 receptor specificity of the virus strain

Using isogenic recombinant murine coronaviruses expressing wild-type murine hepatitis virus strain 4 (MHV-4) or MHV-A59 spike glycoproteins or chimeric MHV-4/MHV-A59 spike glycoproteins, we have demonstrated the biological functionality of the N-terminus of the spike, encompassing the receptor binding domain (RBD). We have used two assays, one an in vitro liposome binding assay and the other a tissue culture replication assay. The liposome binding assay shows that interaction of the receptor with spikes on virions at 37 degrees C causes a conformational change that makes the virions hydrophobic so that they bind to liposomes (B. D. Zelus, J. H. Schickli, D. M. Blau, S. R. Weiss, and K. V. Holmes, J. Virol. 77: 830-840, 2003). Recombinant viruses with spikes containing the RBD of either MHV-A59 or MHV-4 readily associated with liposomes at 37 degrees C in the presence of soluble mCEACAM1(a), except for S(4)R, which expresses the entire wild-type MHV-4 spike and associated only inefficiently with liposomes following incubation with soluble mCEACAM1(a). In contrast, soluble mCEACAM1(b) allowed viruses with the MHV-A59 RBD to associate with liposomes more efficiently than did viruses with the MHV-4 RBD. In the second assay, which requires virus entry and replication, all recombinant viruses replicated efficiently in BHK cells expressing mCEACAM1(a). In BHK cells expressing mCEACAM1(b), only viruses expressing chimeric spikes with the MHV-A59 RBD could replicate, while replication of viruses expressing chimeric spikes with the MHV-4 RBD was undetectable. Despite having the MHV-4 RBD, S(4)R replicated in BHK cells expressing mCEACAM1(b); this is most probably due to spread via CEACAM1 receptor-independent cell-to-cell fusion, an activity displayed only by S(4)R among the recombinant viruses studied here. These data suggest that the RBD domain and the rest of the spike must coevolve to optimize function in viral entry and spread.     

The conserved sequence NXX[S/T]HX[S/T]QDXXXT of the lactate/pyruvate:H(+) symporter subfamily defines the function of the substrate translocation pathway

In Saccharomyces cerevisiae Jen1p is a lactate/proton symporter belonging to the lactate/pyruvate:H(+) symporter subfamily (TC#2.A.1.12.2) of the Major Facilitator Superfamily. We investigated structure-function relationships of Jen1p using a rational mutational analysis based on the identification of conserved amino acid residues. In particular, we studied the conserved sequence (379)NXX[S/T]HX[S/T]QDXXXT(391). Substitution of amino acid residues N379, H383 or D387, even with very similar amino acids, resulted in a dramatic reduction of lactate and pyruvate uptake, but conserved measurable acetate transport. Acetate transport inhibition assays showed that these mutants conserve the ability to bind, but do not transport, lactate and pyruvate. More interestingly, the double mutation H383D/D387H, while behaving as a total loss-of-function allele for lactate and pyruvate uptake, can fully restore the kinetic parameters of Jen1p for acetate transport. Thus, residues N379, H383 or D387 affect both the transport capacity and the specificity of Jen1p. Substitutions of Q386 and T391 resulted in no or moderate changes in Jen1p transport capacities for lactate, pyruvate and acetate. On the other hand, Q386N reduces the binding affinities for all Jen1p substrates, while Q386A increases the affinity specifically for pyruvate. We also tested Jen1p specificity for a range of monocarboxylates. Several of the mutants studied showed altered inhibition constants for these acids. These results and 3D in silico modelling by homology threading suggest that the conserved motif analyzed is part of the substrate translocation pathway in the lactate/pyruvate:H(+) symporter subfamily.     

In vitro random mutagenesis of the D1 protein of the photosystem II reaction center confers phototolerance on the cyanobacterium Synechocystis sp. PCC 6803.

The D1 protein of the photosystem II reaction center is thought to be the most light-sensitive component of the photosynthetic machinery. To understand the mechanisms underlying the light sensitivity of D1, we performed in vitro random mutagenesis of the psbA gene that codes for D1, transformed the unicellular cyanobacterium Synechocystis sp. PCC 6803 with mutated psbA, and selected phototolerant transformants that did not bleach in high intensity light. A region of psbA2 coding for 178 amino acids of the carboxyl-terminal portion of the peptide was subjected to random mutagenesis by low fidelity polymerase chain reaction amplification or by hydroxylamine treatment. This region contains the binding sites for Q(B), D2 (through Fe), and P680. Eighteen phototolerant mutants with single and multiple amino acid substitutions were selected from a half million transformants exposed to white light at 320 micromol m(-2) s(-1). A strain transformed with non-mutagenized psbA2 became bleached under the same conditions. Site-directed mutagenesis has confirmed that one or more substitutions of amino acids at residues 234, 254, 260, 267, 322, 326, and 328 confers phototolerance. The rate of degradation of D1 protein was not appreciably affected by the mutations. Reduced bleaching of mutant cyanobacterial cells may result from continued buildup of photosynthetic pigment systems caused by changes in redox signals originating from D1.     

The N-terminal region of the murine coronavirus spike glycoprotein is associated with the extended host range of viruses from persistently infected murine cells

Although murine coronaviruses naturally infect only mice, several virus variants derived from persistently infected murine cell cultures have an extended host range. The mouse hepatitis virus (MHV) variant MHV/BHK can infect hamster, rat, cat, dog, monkey, and human cell lines but not the swine testis (ST) porcine cell line (J. H. Schickli, B. D. Zelus, D. E. Wentworth, S. G. Sawicki, and K. V. Holmes, J. Virol. 71:9499-9507, 1997). The spike (S) gene of MHV/BHK had 63 point mutations and a 21-bp insert that encoded 56 amino acid substitutions and a 7-amino-acid insert compared to the parental MHV strain A59. Recombinant viruses between MHV-A59 and MHV/BHK were selected in hamster cells. All of the recombinants retained 21 amino acid substitutions and a 7-amino-acid insert found in the N-terminal region of S of MHV/BHK, suggesting that these residues were responsible for the extended host range of MHV/BHK. Flow cytometry showed that MHV-A59 bound only to cells that expressed the murine glycoprotein receptor CEACAM1a. In contrast, MHV/BHK and a recombinant virus, k6c, with the 21 amino acid substitutions and 7-amino-acid insert in S bound to hamster (BHK) and ST cells as well as murine cells. Thus, 21 amino acid substitutions and a 7-amino-acid insert in the N-terminal region of the S glycoprotein of MHV/BHK confer the ability to bind and in some cases infect cells of nonmurine species.     

The importance of the Q motif in the ATPase activity of a viral helicase

NS3 proteins of flaviviruses contain motifs which indicate that they possess protease and helicase activities. The helicases are members of the DExD/H box helicase superfamily and NS3 proteins from some flaviviruses have been shown to possess ATPase and helicase activities in vitro. The Q motif is a recently recognised cluster of nine amino acids common to most DExD/H box helicases which is proposed to regulate ATP binding and hydrolysis. In addition a conserved residue occurs 17 amino acids upstream of the Q motif ('+17'). We have analysed full-length and truncated NS3 proteins from Powassan virus (a tick-borne flavivirus) to investigate the role that the Q motif plays in the hydrolysis of ATP by a viral helicase. The Q motif appears to be essential for the activity of Powassan virus NS3 ATPase, however NS3 deletion mutants that contain the Q motif but lack the '+17' amino acid have ATPase activity albeit at a reduced level.     

Identification of spike protein residues of murine coronavirus responsible for receptor-binding activity by use of soluble receptor-resistant mutants.

We previously demonstrated by site-directed mutagenesis analysis that the amino acid residues at positions 62 and 214 to 216 in the N-terminal region of mouse hepatitis virus (MHV) spike (S) protein are important for receptor-binding activity (H. Suzuki and F. Taguchi, J. Virol. 70:2632-2636, 1996). To further identify the residues responsible for the activity, we isolated the mutant viruses that were not neutralized with the soluble form of MHV receptor proteins, since such mutants were expected to have mutations in amino acids responsible for receptor-binding activity. Five soluble-receptor-resistant (srr) mutants isolated had mutations in a single amino acid at three different positions: one was at position 65 (Leu to His) (srr11) in the S1 subunit and three were at position 1114 (Leu to Phe) (srr3, srr4, and srr7) and one was at position 1163 (Cys to Phe) (srr18) in the S2 subunit. The receptor-binding activity examined by a virus overlay protein blot assay and by a coimmunoprecipitation assay showed that srr11 S protein had extremely reduced binding activity, while the srr7 and srr18 proteins had binding activity similar to that of wild-type cl-2 protein. However, when cell surface receptors were used for the binding assay, all srr mutants showed activity similar to that of the wild type or only slightly reduced activity. These results, together with our previous observations, suggest that amino acids located at positions 62 to 65 of S1, a region conserved among the MHV strains examined, are important for receptor-binding activity. We also discuss the mechanism by which srr mutants with a mutation in S2 showed high resistance to neutralization by a soluble receptor, despite their sufficient level of binding to soluble receptors.     

Identification of the catalytic residues involved in the carboxyl transfer of pyruvate carboxylase

To clarify the mechanism of carboxyl transfer from carboxylbiotin to pyruvate, the following conserved amino acid residues present in the carboxyl transferase domain of Bacillus thermodenitrificans pyruvate carboxylase were converted to homologous amino acids: Asp543, Glu576, Glu592, Asp649, Lys712, Asp713, and Asp762. The carboxylase activity of the resulting mutants, D543E, E576D, E576Q, E592Q, D649N, K712R, K712Q, D713E, D713N, D762E, and D762N, was generally less than that of the wild type from mutation, but it decreased the most to 5% or even less than that of the wild type with D543E, D576Q, D649N, K712R, and K712Q. The decrease in activity observed for Asp543, Asp649, and Lys712 mutants was not for structural reasons because their structures seemed to remain intact as assessed by gel filtration and circular dichroism. On the basis of these data, a mechanism is proposed where Lys712 and Asp543 serve as the key acid and base catalyst, respectively.