Sindbis virus glycoprotein E1 is divided into two discrete domains at amino acid 129 by disulfide bridge connections

The E1 membrane glycoprotein of Sindbis virus contains structural and functional domains, which are conformationally dependent on the presence of intramolecular disulfide bridges (B. A. Abell and D. T. Brown, J. Virol. 67:5496-5501, 1993; R. P. Anthony, A. M. Paredes, and D. T. Brown, Virology 190:330-336, 1992). We have examined the disulfide bonds in E1 and have determined that the E1 membrane glycoprotein contains two separate sets of interconnecting disulfide linkages, which divide the protein into two domains at amino acid 129. These separate sets of disulfides may stabilize and define the structural and functional regions of the E1 protein.     

Sindbis virus membrane fusion is mediated by reduction of glycoprotein disulfide bridges at the cell surface.

We have examined the role of thiol-disulfide exchange reactions during the penetration of cells by Sindbis virus. The protein-protein association that form the rigid icosahedral lattice of the Sindbis virus envelope have been shown to be stabilized by disulfide bridges, and reduction of these critical disulfide bridges during cell penetration may be the mechanism by which the rigid protein lattice is disrupted prior to fusion (R. Anthony and D. T. Brown, J. Virol. 65:1187-1194, 1991; R. Anthony, A. Paredes, and D. T. Brown, Virology 190:330-336, 1992). Reduction of disulfide bridges occurs at near neutral pHs via thiol-disulfide exchange reactions, and these reactions can be blocked by covalent modification of the thiol involved. In this study, the effects of the reducing agent 2-mercaptoethanol on Sindbis virus-mediated cell-cell fusion from without and the effects of the membrane-impermeable thiol-alkylating reagent 5,5'-dithiobis(2-nitrobenzoic acid) on Sindbis virus penetration were determined. The presence of exogenous reducing agent was found to induce fusion from without under conditions unfavorable to both typical Sindbis virus-mediated fusion from without and cysteine-mediated thiol-disulfide exchange reactions. In addition, the thiol-alkylating reagent was found to inhibit Sindbis virus entry when present during infection. These results are consistent with a model for Sindbis virus entry in which reduction of critical disulfide bridges at the cell surface disrupts the rigid protein-protein associations of the envelope, allowing membrane fusion and release of the viral genome into the cell.     

Disulfide bridge-mediated folding of Sindbis virus glycoproteins.

The Sindbis virus envelope is composed of 80 E1-E2 (envelope glycoprotein) heterotrimers organized into an icosahedral protein lattice with T=4 symmetry. The structural integrity of the envelope protein lattice is maintained by E1-E1 interactions which are stabilized by intramolecular disulfide bonds. Structural domains of the envelope proteins sustain the envelope's icosahedral lattice, while functional domains are responsible for virus attachment and membrane fusion. We have previously shown that within the mature Sindbis virus particle, the structural domains of the envelope proteins are significantly more resistant to the membrane-permeative, sulfhydryl-reducing agent dithiothreitol (DTT) than are the functional domains (R. P. Anthony, A. M. Paredes, and D. T. Brown, Virology 190:330-336, 1992). We have used DTT to probe the accessibility of intramolecular disulfides within PE2 (the precursor to E2) and E1, as these proteins fold and are assembled into the spike heterotrimer. We have determined through pulse-chase analysis that intramolecular disulfide bonds within PE2 are always sensitive to DTT when the glycoproteins are in the endoplasmic reticulum. The reduction of these disulfides results in the disruption of PE2-E1 associations. E1 acquires increased resistance to DTT as it folds through a series of disulfide intermediates (E1alpha, -beta, and -gamma) prior to assuming its native and most compact conformation (E1epsilon). The transition from a DTT-sensitive form into a form which exhibits increased resistance to DTT occurs after E1 has folded into its E1beta conformation and correlates temporally with the dissociation of BiP-E1 complexes and the formation of PE2-E1 heterotrimers. We propose that the disulfide bonds within E1 which stabilize the protein domains required for maintaining the structural integrity of the envelope protein lattice form early within the folding pathway of E1 and become inaccessible to DTT once the heterotrimer has formed.     

Location and role of free cysteinyl residues in the Sindbis virus E1 and E2 glycoproteins

Sindbis virus is a single-stranded positive-sense RNA virus. It is composed of 240 copies of three structural proteins: E1, E2, and capsid. These proteins form a mature virus particle composed of two nested T=4 icosahedral shells. A complex network of disulfide bonds in the E1 and E2 glycoproteins is developed through a series of structural intermediates as virus maturation occurs (M. Mulvey and D. T. Brown, J. Virol. 68:805-812, 1994; M. Carleton et al., J. Virol. 71:1558-1566, 1997). To better understand the nature of this disulfide network, E1 and E2 cysteinyl residues were labeled with iodoacetamide in the native virus particle and analyzed by liquid chromatography-tandem mass spectrometry. This analysis identified cysteinyl residues of E1 and E2, which were found to be label accessible in the native virus particle, as well as those that were either label inaccessible or blocked by their involvement in disulfide bonds. Native virus particles alkylated with iodoacetamide demonstrated a 4-log decrease in viral infectivity. This suggests that the modification of free cysteinyl residues results in the loss of infectivity by destabilizing the virus particle or that a rearrangement of disulfide bonds, which is required for infectivity, is blocked by the modification. Although modification of these residues prevented infectivity, it did not alter the ability of virus to fuse cells after exposure to acidic pH; thus, modification of free cysteinyl residues biochemically separated the process of infection from the process of membrane fusion.     

Proteolytic processing of the Sindbis virus membrane protein precursor PE2 is nonessential for growth in vertebrate cells but is required for efficient growth in invertebrate cells.

We have shown previously that processing of the Sindbis virus envelope protein precursor PE2 to envelope protein E2 is not required for virus maturation in cultured vertebrate fibroblast cells and that unprocessed PE2 can be incorporated into infectious virus in place of E2 (J. F. Presley and D. T. Brown, J. Virol. 63:1975-1980, 1989; D. L. Russell, J. M. Dalrymple, and R. E. Johnston, J. Virol. 63:1619-1629, 1989). To better understand the role of this processing event in the invertebrate vector portion of the alphavirus life cycle, we have examined the maturation of Sindbis virus mutants defective in PE2 processing in cultured mosquito cells. We found that although substantial amounts of structural proteins PE2, E1, and C were produced in infected mosquito (aedine) cell lines, very little infectious virus was released. When the period of infection was extended, plaque size variants appeared, some of which exhibited a restored ability to grow in mosquito cells. The nucleotide sequences of two such variants were determined. These variants contained point mutations that restored PE2 cleavage, indicating a genetic linkage between failure to cleave PE2 and failure to grow in mosquito cells.     

Identification of a uniquely immunodominant, cross-reacting site in the human immunodeficiency virus endonuclease protein.

One of the features of the life cycle of retroviruses is insertion of the proviral DNA into host chromosomes. A protein encoded by the 3' end of the pol gene of the virus genome has been shown to possess endonuclease activity (D. P. Grandgenett, A. C. Vora, and R. D. Schiff, Virology 89:119-132, 1978), which is necessary for DNA integration. Sera from the majority of human immunodeficiency virus (HIV)-infected individuals react with endonuclease protein p31 in serological tests (J. S. Allan, J. E. Coligan, T.-H. Lee, F. Barin, P. J. Kanki, S. M'Boup, M. F. McLane, J. E. Groopman, and M. Essex, Blood 69:331-333, 1987; E. F. Lillehoj, F. H. R. Salazar, R. J. Mervis, M. G. Raum, H. W. Chan, N. Ahmad, and S. Venkatesan, J. Virol. 62:3053-3058, 1988; K. S. Steimer, K. W. Higgins, M. A. Powers, J. C. Stephans, A. Gyenes, G. George-Nascimento, P. A. Liciw, P. J. Barr, R. A. Hallewell, and R. Sanchez-Pescador, J. Virol. 58:9-16, 1986). It is not known, however, which part of the protein represents the target(s) for antibody response. To study this, we synthesized peptides and used them in an enzyme-linked immunosorbent assay system to map the reactivity of human immunodeficiency virus type 1 (HIV-1) antibody-positive sera to the different regions of the HIV endonuclease. A uniquely antigenic, HIV-1- and HIV-2-cross-reacting site was identified in the central part of this protein from Phe-663 to Trp-670.     

Transformation by the oncogenic latent membrane protein correlates with its rapid turnover, membrane localization, and cytoskeletal association.

The latent membrane protein (LMP) of Epstein-Barr virus (EBV) has a short half-life (V. R. Baichwal and B. Sugden, J. Virol, 61:866-875, 1987; K.P. Mann and D. Thorley-Lawson, J. Virol, 61:2100-2108, 1987), is localized in patches in the membrane (D. Liebowitz, D. Wang, and E, Kieff, J. Virol, 58:233-237, 1986), and associates with the cytoskeleton in EBV-immortalized B lymphocytes (D. Liebowitz, R. Kopan, E. Fuchs, J. Sample, and E. Kieff, Mol. Cell. Biol. 7:2299-2308, 1987; K. P. Mann and D. Thorley-Lawson, J. Virol. 61:2100-2108, 1987). Deletion mutants of LMP that are either positive or negative in the induction both of anchorage-independent growth of BALB/c 3T3 cells (V. R. Baichwal and B. Sugden, Oncogene 4:67-74, 1989) and of cytotoxicity in a variety of cells (W. Hammerschmidt, B. Sugden, and V. R. Baichwal, J. Virol. 63:2469-2475, 1989) have been studied to identify the biochemical properties of this protein that correlate with its effects on cell growth. Mutant LMP proteins that are metabolically stable, do not associate with the cytoskeleton, and exhibit a diffuse plasma membrane localization also do not induce anchorage-independent growth in rodent cells or cytotoxicity in B lymphoblastoid cells. In contrast, a mutant of LMP that is functionally identical to the wild-type protein has a half-life, membrane localization, and cytoskeletal association similar or identical to those of LMP. These results are consistent with the hypothesis that LMP's rapid turnover, association with the cytoskeleton, and patching in the membrane are required for it to affect cell growth.     

Unique amino acid substitutions in the capsid proteins of foot-and-mouth disease virus from a persistent infection in cell culture.

Maintenance of a persistent foot-and-mouth disease virus (FMDV) infection in BHK-21 cells involves a coevolution of cells and virus (J. C. de la Torre, E. Martínez-Salas, J. Díez, A. Villaverde, F. Gebauer, E. Rocha, M. Dávila, and E. Domingo, J. Virol. 62:2050-2058, 1988). The resident FMDV undergoes a number of phenotypic changes, including a gradual decrease in virion stability. Here we report the nucleotide sequence of the P1 genomic segment of the virus rescued after 100 passages of the carrier cells (R100). Only 5 of 15 mutations in P1 of R100 were silent. Nine amino acid substitutions were fixed on the viral capsid during persistence, and three of the variant amino acids are not represented in the corresponding position of any picornavirus sequenced to date. Cysteine at position 7 of VP3, that provides disulfide bridges at the FMDV fivefold axis, was substituted by valine, as determined by RNA, cDNA, and protein sequencing. The modified virus shows high buoyant density in cesium chloride and depicts the same sensitivity to photoinactivation by intercalating dyes as the parental FMDV C-S8c1. Amino acid substitutions fixed in VP1 resulted in altered antigenicity, as revealed by reactivity with monoclonal antibodies. In addition to defining at the molecular level the alterations the FMDV capsid underwent during persistence, the results show that positions which are highly invariant in an RNA genome may change when viral replication occurs in a modified environment.     

Disulfide bond-coupled folding of bovine pancreatic trypsin inhibitor derivatives missing one or two disulfide bonds.

The disulfide bond-coupled folding and unfolding mechanism (at pH 8.7, 25 degrees C in the presence of oxidized and reduced dithiothreitol) was determined for a bovine pancreatic trypsin inhibitor mutant in which cysteines 30 and 51 were replaced with alanines so that only two disulfides, between cysteines 14 and 38 and cysteines 5 and 55, remain. Similar studies were made on a chemically-modified derivative of the mutant retaining only the 5-55 disulfide. The preferred unfolding mechanism for the Ala30/Ala51 mutant begins with reduction of the 14-38 disulfide. An intramolecular rearrangement via thiol-disulfide exchange, involving the 5-55 disulfide and cysteines 14 and/or 38, then occurs. At least five of six possible one-disulfide bond species accumulate during unfolding. Finally, the disulfide of one or more of the one-disulfide bond intermediates (excluding that with the 5-55 disulfide) is reduced giving unfolded protein. The folding mechanism seems to be the reverse of the unfolding mechanism; the observed folding and unfolding reactions are consistent with a single kinetic scheme. The rate constant for the rate-limiting intramolecular folding step--rearrangements of other one-disulfide bond species to the 5-55 disulfide intermediate--seems to depend primarily on the number of amino acids separating cysteines 5 and 55 in the unfolded chain. The energetics and kinetics of the mutant's folding mechanism are compared to those of wild-type protein [Creighton, T. E., & Goldenberg, D. P. (1984) J. Mol. Biol. 179, 497] and a mutant missing the 14-38 disulfide [Goldenberg, D. P. (1988) Biochemistry 27, 2481]. The most striking effects are destabilization of the native structure and a large increase in the rate of unfolding.     

Retention of secretory proteins in an intermediate compartment and disappearance of the Golgi complex in an END4 mutant of Chinese hamster ovary cells

Mutant V.24.1, a member of the End4 complementation group of temperature-sensitive CHO cells, is defective in secretion at the restrictive temperature (Wang, R.-H., P. A. Colbaugh, C.-Y. Kao, E. A. Rutledge, and R. K. Draper. 1990. J. Biol. Chem. 265:20179-20187; Presley, J. F., R. K. Draper, and D. T. Brown. 1991. J. Virol. 65:1332-1339). We have further investigated the secretory lesion and report three main findings. First, the block in secretion is not due to aberrant folding or oligomerization of secretory proteins in the endoplasmic reticulum because the hemagglutinin of influenza virus folded and oligomerized at the same rate in mutant and parental cells at the restrictive temperature. Second, secretory proteins accumulated in a compartment intermediate between the ER and the Golgi. Several lines of evidence support this conclusion, the most direct being the colocalization by immunofluorescence microscopy of influenza virus hemagglutinin with a 58-kD protein that is known to reside in an intermediate compartment. Third, at the resolution of fluorescence microscopy, the Golgi complex in the mutant cells vanished at the restrictive temperature.     

DNA of Epstein-Barr virus. III. Identification of restriction enzyme fragments that contain DNA sequences which differ among strains of Epstein-Barr virus.

Previous kinetic and absorption hybridization experiments had demonstrated that the DNA of the B95-8 strain of Epstein-Barr virus was missing approximately 10% of the DNA sequences present in the DNA of the HR-1 strain (R.F. Pritchett, S.D. Hayward, and E. Kieff, J. Virol. 15:556-569, 1975; B. Sugder, W.C. Summers, and G. Klein, J. Virol. 18:765-775, 1976). The HR-1 strain differs from other laboratory strains, including the B95-8 and W91 strains, and from virus present in throat washings from patients with infectious mononucleosis in its inability to transform lymphocytes into lymphoblasts capable of long-term growth in culture (P. Gerber, Lancet i:1001, 1973; J. Menezes, W. Leibold, and G. Klein, Exp. Cell. Res. 92:478-484, 1975; G. Miller, D. Coope, J. Niederman, and J. Pagano, J. Virol. 18:1071-1080, 1976; G. Miller, J. Robinson, L. Heston, and M. Lipman, Proc. Natl. Acad. Sci. U.S.A. 71:4006-4010, 1974). In the experiments reported here, the restriction enzyme fragments of Epstein-Barr virus DNA which contain sequences which differ among the HR-1, B95-8, and W91 strains have been identified. The DNA of the HR-1, B95-8, and W91 strains each differed in complexity. The sequences previously shown to be missing in the B95-8 strain were contained in the EcoRI-C and -D and Hsu I-E and -N fragments of the HR-1 strain and in the EcoRI-C and Hsu I-D and -E fragments of the W91 strain. The HR-1 strain was missing DNA contained in EcoRI fragments A and J through K and Hsu I fragment B of the B95-8 strain and in the EcoRI-A and Hsu I-B fragments of the W91 strain. The relationship of these data to the linkage map of restriction enzyme fragments of the DNA of the B95-8 and W91 strains (E. Kieff, N. Raab-Traub, D. Given, W. King, A.T. Powell, R. Pritchett, and T. Dambaugh, In F. Rapp and G. de-The, ed., Oncogenesis and Herpesviruses III, in press; D. Given and E. Kieff, submitted for publication) and the possible significance of the data are discussed.     

Kinetic analysis of the folding and unfolding of a mutant form of bovine pancreatic trypsin inhibitor lacking the cysteine-14 and -38 thiols

The kinetics of the disulfide-coupled unfolding-refolding transition of a mutant form of bovine pancreatic trypsin inhibitor (BPTI) lacking Cys-14 and -38 were measured and compared to previous results for the wild-type protein and other modified forms. The altered cysteines, which were changed to serine in the mutant protein, are normally paired in a disulfide in the native protein but from disulfides with Cys-5 in two-disulfide kinetic intermediates during folding. Although the mutant protein could fold efficiently, the kinetics of both folding and unfolding were altered, reflecting the roles of these cysteines in the two-disulfide intermediates with "wrong" disulfides. The intramolecular rate constant for the formation of the second disulfide of the native mutant protein was more than 10(3)-fold lower than that for the formation of a second disulfide during the refolding of the wild-type protein. The observed rate of unfolding of the mutant protein was also lower than that of the wild-type protein, demonstrating that the altered cysteines are involved in the intramolecular rearrangements that are the rate-determining step in the unfolding of the wild-type protein. These results confirm the previous conclusion [Creighton, T.E. (1977) J. Mol. Biol. 113, 275-293] that the energetically preferred pathway for folding and unfolding of BPTI includes intramolecular rearrangements of intermediates in which Cys-14 and -38 are paired in disulfides not present in the native protein. The present results are also consistent with other, less detailed, studies with similar mutants lacking Cys-14 and -38 [Marks, C.B., Naderi, H., Kosen, P.A., Kuntz, I.D., & Anderson, S. (1987) Science (Washington, D.C.) 235, 1370-1371].     

Phenotypes of Epstein-Barr virus LMP1 deletion mutants indicate transmembrane and amino-terminal cytoplasmic domains necessary for effects in B-lymphoma cells.

The Epstein-Barr virus (EBV) latent infection membrane protein 1 (LMP1) has previously been shown to cause EBV-negative B-lymphoma cells to grow in large clumps and to alter expression of surface activation and adhesion molecules (D. Wang, D. Liebowitz, F. Wang, C. Gregory, A. Rickinson, R. Larson, T. Springer, and E. Kieff, J. Virol. 62:1473-4184, 1988; F. Wang, C. Gregory, C. Sample, M. Rowe, D. Liebowitz, R. Murray, A. Rickinson, and E. Kieff, J. Virol. 64:2309-2318, 1990). In order to identify functional elements in the amino-terminal cytoplasmic domain and the first four transmembrane domains which were previously shown to be essential for LMP1 activity, three smaller deletion mutants were constructed and tested for their activity in B-lymphoma cells. The results of the present study indicate that the amino-terminal cytoplasmic domain, the first transmembrane domain, and the third and fourth transmembrane domains each contribute to LMP1's effects on B lymphocytes.     

The formation of intramolecular disulfide bridges is required for induction of the Sindbis virus mutant ts23 phenotype.

The Sindbis virus envelope protein spike is a hetero-oligomeric complex composed of a trimer of glycoprotein E1-E2 heterodimers. Spike assembly is a multistep process which occurs in the endoplasmic reticulum (ER) and is required for the export of E1 from the ER. PE2 (precursor to E2), however, can transit through the secretory pathway and be expressed at the cell surface in the absence of E1. Although oligomer formation does not appear to be required for the export of PE2, there is evidence that defects in E1 folding can affect PE2 transit from the ER. Temperature-sensitive mutant ts23 of Sindbis virus contains two amino acid substitutions in E1, while PE2 and capsid protein have the wild-type sequence; however, at the nonpermissive temperature, both E1 and PE2 are retained within the ER and can be isolated in protein aggregates with the molecular chaperone GRP78-BiP. We previously demonstrated that the temperature sensitivity for ts23 was lost as oligomer formation took place at the permissive temperature, suggesting that temperature sensitivity is initiated early in the process of viral spike assembly (M. Carleton and D. T. Brown, J. Virol. 70:952-959, 1996). Experiments described herein investigated the defects in envelope protein maturation that occur in ts23-infected cells and which result in retention of both envelope proteins in the ER. The data demonstrate that in ts23-infected cells incubated at the nonpermissive temperature, E1 folding is disrupted early after synthesis, resulting in the rapid incorporation of both E1 and PE2 into disulfide-stabilized aggregates. Furthermore, the aberrant E1 conformation which is responsible for induction of the ts phenotype requires the formation of intramolecular disulfide bridges formed prior to E1 association with PE2 and the completion of E1 folding.     

Delay of vaccinia virus-induced apoptosis in nonpermissive Chinese hamster ovary cells by the cowpox virus CHOhr and adenovirus E1B 19K genes.

The infection of vaccinia virus in Chinese hamster ovary (CHO) cells produces a rapid shutdown in protein synthesis, and the infection is abortive (R.R. Drillien, D. Spehner, and A. Kirn, Virology 111:488-499, 1978; D.E. Hruby, D.L. Lynn, R. Condit, and J.R. Kates, J. Gen. Virol. 47:485-488, 1980). Cowpox virus, which can productively infect CHO cells, had previously been shown to contain a host range gene, CHOhr, which confers on vaccinia virus the ability to replicate in CHO cells (D. Spehner, S. Gillard, R. Drillien, and A. Kirn, J. Virol. 62:1297-1304, 1988). We found that CHO cells underwent apoptosis when infected with vaccinia virus. The expression of the CHOhr gene in vaccinia virus allowed for the expression of late virus genes. CHOhr also delayed or prevented vaccinia virus-induced apoptosis in CHO cells such that there was sufficient time for replication of the virus before the cell died. The E1B 19K gene from adenovirus also delayed vaccinia virus-induced apoptosis; however, there was no detectable expression of late virus genes. Furthermore, E1B 19K also delayed cell death in CHO cells which had been productively infected with vaccinia virus. This study identifies a new antiapoptotic gene from cowpox virus, CHOhr, for which the protein contains an ankyrin-like repeat and shows no significant homology to other proteins. This work also indicates that an antiapoptotic gene from one virus family can delay cell death in an infection of a virus from a different family.     

Genome complexities of the three mRNA species of snowshoe hare bunyavirus and in vitro translation of S mRNA to viral N polypeptide.

The genome complexities of the principal intracellular viral complementary RNA species of the snowshoe hare bunyavirus have been analyzed by duplex analyses involving hybridization of complementary RNA to individual 32P-labeled viral RNA species (large, L; medium, M; and small, S), recovery of nuclease-resistant duplexes, and determination of the oligonucleotide fingerprints of the protected 32P-labeled viral sequences. The result for the M RNA (which codes for the glycoproteins G1 and G2; J. R. Gentsch and D. H. L. Bishop, J. Virol. 30:767-770, 1979) indicates that there is a single polycistronic M mRNA. Similar results were obtained for the L and S RNA species. In vitro translation studies with the S complementary RNA species of snowshoe hare virus as well as melted purified S duplexes substantiate earlier genetic and molecular studies (J. R. Gentsch and D. H. L. Bishop, J. Virol. 28:417-419, 1978; J. Gentsch, D. H. L. Bishop, and J. F. Obijeski, J. Gen. Virol. 34-257-268, 1977), which indicate that S mRNA codes for the virion nucleocapsid protein N.     

A single amino acid change in the E2 glycoprotein of Sindbis virus confers neurovirulence by altering an early step of virus replication.

Amino acid changes in the envelope glycoproteins of Sindbis virus have been linked to neurovirulence; however, the molecular mechanisms by which these amino acid changes alter neurovirulence are not known. Recombinant-virus studies have mapped an important determinant of neurovirulence in adult mice to a single amino acid change, glutamine to histidine, at position 55 of the E2 glycoprotein (P. C. Tucker, E. G. Strauss, R. J. Kuhn, J. H. Strauss, and D. E. Griffin, J. Virol. 67:4605-4610, 1993). To investigate how histidine confers neurovirulence, we examined the various stages of the virus life cycle in neural (N18) and nonneural (BHK) cells. In BHK cells, recombinant viruses 633 (E255Q) and TE (E255H) replicated similarly. In contrast, in N18 neuroblastoma cells, TE established infection more efficiently, replicated faster, and achieved higher rates of virus release than did 633. Viral structural protein synthesis was similar in 633- and TE-infected BHK cells, while in N18 cells, structural protein synthesis was detected only in TE-infected cells at 6 h and remained higher for at least 16 h postinfection. Viral RNA synthesis was initiated more rapidly and was up to fivefold greater in TE- versus 633-infected N18 cells. Taken together with other data demonstrating minimal effects on virus binding and entry (P. C. Tucker, S. H. Lee, N. Bui, D. Martinie, and D. E. Griffin, J. Virol. 71:6106-6112, 1997), these data suggest that E2 position 55 plays an important role at early stages of infection of neural cells, thereby facilitating neurovirulence.     

Guanidine-resistant mutants of poliovirus have distinct mutations in peptide 2C.

In previous work in our laboratory, 12 guanidine-resistant (gr) mutants of poliovirus were selected from 12 separate stocks of plaque-purified guanidine-sensitive (gs) viruses (K. Anderson-Sillman, S. Bartal, and D. R. Tershak, J. Virol. 50:922-928, 1984). Peptide mapping of protein 2C and evaluation of virus growth at different temperatures enabled us to subdivide these mutants into several distinct groups (D. R. Tershak, Can. J. Microbiol. 31:1166-1168, 1985; Anderson-Sillman et al., J. Virol.). Studies by Pincus et al. indicate that two nucleotide changes in codon 179 of protein 2C leads to an Asn-to-Gly or Asn-to-Ala change and that these missense modifications account for guanidine resistance (S. E. Pincus, H. Rohl, and E. Wimmer, Virology 157:83-88, 1987; S. E. Pincus and E. Wimmer, J. Virol. 60:793-796, 1986). In the present report, we confirm their findings but also show that a single amino acid change of Phe to Tyr in residue 164 of protein 2C or a Met-to-Leu replacement in amino acid 187 coupled with mutations in codons 233 or 295 and 309 could confer guanidine resistance.