Guanidine-resistant Mutants Research Articles

Selection and characterisation of guanidine-resistant mutants of human enterovirus 71

The replication of human enterovirus 71 (HEV71) in cell culture is inhibited by concentrations of guanidine that do not have an observable adverse effect on host cell metabolism. Although the HEV71 non-structural protein 2C is known to play an important role in viral RNA replication, its precise biochemical activities and structure have not been fully determined. Here we describe amino acid substitutions in HEV71 protein 2C that confer resistance to guanidine. Three guanidine-resistant virus populations were independently isolated and found to contain five mutations in protein 2C, one of which, A4657T (2C-M193L), was present in two of the independently selected populations. This mutation was introduced into a HEV71 infectious cDNA clone and was sufficient to confer complete resistance to growth inhibition in the presence of 4mM guanidine. In the first guanidine-resistant population selected, the 2C-M193L mutation occurred in association with an additional mutation, A4459G (2C-I127V), located in the putative cis-acting replication element (cre) of coding region 2C. This mutation conferred only partial guanidine resistance when introduced into the HEV71-26M infectious clone. When the 2C-I127V and 2C-M193L mutations were introduced into HEV71-26M together, the 2C-I127V mutation did not increase the level of guanidine resistance due to the 2C-M193L mutation alone. This study confirms that guanidine resistance can be readily selected in HEV71 and is attributable to mutations within protein 2C.

Dynamics of picornavirus RNA replication within infected cells

Replication of many picornaviruses is inhibited by low concentrations of guanidine. Guanidine-resistant mutants are readily isolated and the mutations map to the coding region for the 2C protein. Using in vitro replication assays it has been determined previously that guanidine blocks the initiation of negative-strand synthesis. We have now examined the dynamics of RNA replication, measured by quantitative RT-PCR, within cells infected with either swine vesicular disease virus (an enterovirus) or foot-and-mouth disease virus as regulated by the presence or absence of guanidine. Following the removal of guanidine from the infected cells, RNA replication occurs after a significant lag phase. This restoration of RNA synthesis requires de novo protein synthesis. Viral RNA can be maintained for at least 72 h within cells in the absence of apparent replication but guanidine-resistant virus can become predominant. Amino acid substitutions within the 2C protein that confer guanidine resistance to swine vesicular disease virus and foot-and-mouth disease virus have been identified. Even when RNA synthesis is well established, the addition of guanidine has a major impact on the level of RNA replication. Thus, the guanidine-sensitive step in RNA synthesis is important throughout the virus life cycle in cells.

Mutations in the 2C region of poliovirus responsible for altered sensitivity to benzimidazole derivatives.

MRL-1237, [1-(4-fluorophenyl)-2-(4-imino-1,4-dihydropyridin-1-yl) methylbenzimidazole hydrochloride], is a potent and selective inhibitor of the replication of enteroviruses. To reveal the target molecule of MRL-1237 in viral replication, we selected spontaneous MRL-1237-resistant poliovirus mutants. Of 15 MRL-1237-resistant mutants obtained, 14 were cross-resistant to guanidine hydrochloride (mrgr), while 1 was susceptible (mrgs). Sequence analysis of the 2C region revealed that the 14 mrgr mutants contained a single nucleotide substitution that altered an amino acid residue from Phe-164 to Tyr. The mrgs mutant, on the other hand, contained a substitution of Ile-120 to Val. Through the construction of a cDNA-derived mutant, we confirmed that the single mutation at Phe-164 was really responsible for the reduced susceptibility to MRL-1237. MRL-1237 inhibited poliovirus-specific RNA synthesis in HeLa cells infected with a wild strain but not with an F164Y mutant. We furthermore examined the effect of mutations of the 2C region on the drug sensitivity of cDNA-derived guanidine-resistant and -dependent mutants. Two guanidine-resistant mutants were cross-resistant to MRL-1237 but remained susceptible to another benzimidazole, enviroxime. Either MRL-1237 or guanidine stimulated the viral replication of two guanidine-dependent mutants, but enviroxime did not. These results indicate that MRL-1237, like guanidine, targets the 2C protein of poliovirus for its antiviral effect.

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.

Effect of temperature on growth of guanidine-resistant mutants of poliovirus.

Guanidine-resistant (gr) mutants of poliovirus were previously categorized into four groups by electrophoretic properties and peptide maps of nonstructural virus protein 2C. Growth of mutants in the presence of guanidine depends upon temperature of incubation. The four groups of gr variants respond differently to temperature when guanidine is included in the culture medium. The data suggest clustering of gr mutations at several sites in the guanidine locus.

Guanidine-resistant poliovirus mutants produce modified 37-kilodalton proteins.

Eighteen spontaneous, guanidine-resistant mutants of poliovirus were obtained by plaque selection. Isoelectric focusing demonstrated charge changes in a 37-kilodalton protein, pX, among three of the mutants. The precursor of pX, NCVP5b , also exhibited charge changes among the three mutants. pX of 12 mutants was also examined by peptide mapping with Staphylococcus aureus V8 protease. Nine of the mutants presented modified maps, and seven of these maps were identical. The demonstration of mutational changes in pX in 12 of 18 mutants suggests a role for this protein in determining the guanidine trait of poliovirus and corroborates studies with foot-and-mouth disease virus.

Guanidine-resistant mutants of aphthovirus induce the synthesis of an altered nonstructural polypeptide, P34.

Extracts of cells infected with guanidine-resistant mutants of aphthovirus were examined for differences in virus-induced polypeptides by using electrofocusing. Four of 1 independent spontaneous mutants induced the synthesis of an altered nonstructural polypeptide, P34. The precursor of P34, P52, and a previously unmapped polypeptide, P20c, also carried these charge-change mutations. No mutations in other regions of the genome were detected, and the remaining six guanidine-resistant mutants appeared entirely normal by electrofocusing. However, when the P34 of one of the latter mutants was examined by tryptic peptide fingerprinting, it too differed from that of the guanidine-sensitive parent. The frequency of P34 alterations among guanidine-resistant mutants suggests that P34 is functionally involved in the antiviral action of guanidine.

Adsorption of Enteroviruses to Soil Cores and Their Subsequent Elution by Artificial Rainwater

The adsorption and elution of a variety of human enteroviruses in a highly permeable, sandy soil was studied by using cores (43 by 125 mm) collected from an operating recharge basin on Long Island. Viruses studied included field and reference strains of polioviruses types 1 and 3 and reference strains of coxsackie virus B3 and echovirus types 1 and 6. Viruses suspended in treated sewage effluent were allowed to percolate through soil cores, and the filtrate was assayed for unadsorbed viruses. To determine the likelihood of desorption and mobilization, soil-bound viruses were subjected to a rinse with either treated sewage effluent or simulated rainwater which reflected the anion, cation, and pH characteristics of a typical northeastern United States rainfall. The results demonstrated that all polioviruses tested, including both reference and field strains, adsorbed extremely well to cores. Adsorption was somewhat reduced when clean, unconditioned soils were used. Soil-bound poliovirus strain LSc was not significantly mobilized by flooding columns with either a sewage effluent or rainwater rinse. One virus was mobilized by both types of rinses. The amount of viruses mobilized by rainwater rinses ranged from 24 to 66%. Variable adsorption-elution results were observed with other enteroviruses. Two guanidine-resistant mutants of poliovirus LSc demonstrated a soil adsorption-elution profile different from that of the parent strain. The data support the conclusion that soil adsorption-elution behavior is strain dependent and that poliovirus, particularly strain LSc, represents an inappropriate model.

CHARACTERS OF GUANIDINE RESISTANT MUTANTS OF TYPE 1 POLIOVIRUS.

Following exposure of type 1 poliovirus (Mahoney and LSc 2ab strains) to guanidine hydrochloride, the presence of mutants resisting the action of the inhibitor could be demonstrated by means of the plaque method. Triple exposure to guanidine in increasing concentrations (70–150–250 μg/ml.) resulted in the isolation of highly resistant mutants, as compared to the parental strains, while a single exposure to 20–40 μg/ml. led to the demonstration of strains displaying an intermediate degree of resistance. Quantitative investigations of the resistance to 35 μg/ml. guanidine hydrochloride (i. e. to a concentration which still permits comparison with the parental strains) have shown the difference of susceptibility between the clonal strains obtained by the first method and the respective parental strains to be of the order of 106 in mutants of Mahoney virus and of the order of 105 in mutants of the LSc 2ab strain. The high degree of resistance of these mutants was further demonstrated by the fact that the maximum guanidine concentrations tolerated by the host cells (175–225 μg/ml.) still allowed the above mentioned clonal strains to display a plaque forming capacity of 10−1.2–10−4 as compared to that of the initial population, whereas similar resistance rates were displayed by the parental population only at concentrations as low as 10–25 μg/ml. Theg − character (guanidine resistance) proved to be stable in the course of 10 passages in the absence of the inhibitor, and the virus reconstituted from the infectious RNA extracted fromg − mutants maintained this character. A study of the correlation between guanidine resistance on the one hand, and the markersrct 40°, MS, E, as well as neurovirulence in monkeys, on the other, has shown that the presence of theg − character is not associated with any change in these markers, as compared to their pattern in theg + parental strains. This shows that guanidine resistance is controlled by a distinct portion of the genetic material and does not display the phenomenon of covariation with neurovirulence.

Genetic recombination with poliovirus type 1: Studies of crosses between a normal horse serum-resistant mutant and several guanidine-resistant mutants of the same strain

Mixed infection of HeLa cells with two mutants of type 1 poliovirus yielded progeny which were resistant to both inhibitors. One mutant ( ho) was resistant to the plaque-forming inhibitor in certain horse sera and the other was resistant to the virus inhibitory effects of guanidine. Mutants of poliovirus were isolated which had different degrees of resistance to the effects of guanidine. Some of these mutants were stable during repeated cultivation in HeLa cells in the absence of the drug. Mutants were also isolated which were dependent on guanidine for growth. Mixed infection with an ho mutant (resistant to the plaque inhibitory effects of horse serum) and a g 100 mutant (resistant to the inhibitory effects of 100 μg of guanidine per milliliter in agar overlay) under conditions of high and approximately equal multiplicity of infection yielded progeny containing 0.42% of the recombinant mutant ho g 100 . All double mutants tested possessed the full resistance to guanidine. Mixed infection with mutants ho and g 20 or ho and g 27 yielded approximately 0.4% of the recombinants ho g 20 or ho g 27 . While the spontaneous rate of g mutants to ho g was fairly high, the proportion of double mutants found was 15–20 times that expected as a result of mixed infection and, by crossing g 100 with a strain ho Δ r, the occurrence of heat-resistant clones in the recombinant progeny ruled out the possibility that the spontaneous mutation was the sole source of the double mutants. The frequency of ho g recombinants increased with time after mixed infection. The first new virus contained about 0.2% recombinants, and this proportion increased to about 0.4% at the time of complete virus maturation. This result suggests that RNA produced in the cell as the result of infection may act as a template for further RNA production and may recombine so that the RNA produced may be recombinant.