Role of amino acid residue 187 of poliovirus polypeptide 2C in determining the guanidine trait.

Although mutations in codons 164 and 179 of poliovirus polypeptide 2C can establish guanidine resistance, the majority of guanidine-resistant and guanidine-dependent viruses contain an M----L change at amino residue 187 along with other critical mutations. The change at residue 187 probably contributes to the guanidine phenotype along with a variety of other modifications in polypeptide 2C, possibly altering conformation of the polypeptide during the initiation step of viral RNA synthesis.

A replication-defective variant of Sabin 3 poliovirus.

A variant of Sabin type 3 poliovirus, 265B, only produces 2-3% of the yields of control virus and has a protracted growth cycle in HeLa cells. Attachment and penetration do not appear to account for defective growth of 265B. Viral protein and RNA syntheses are reduced, and blockage of cell protein synthesis is delayed considerably as compared with the parent virus. Nucleotide sequence analysis failed to detect mutations in protein 2AProt or changes in the 5'-noncoding region that could account for growth properties of the virus. However, a Thr-to-Ala substitution was found in viral peptide 2B of 265B, and this alteration is probably responsible for the replication defect.

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.

Attachment, uncoating and blockage of cell protein synthesis by Sabin derivatives of poliovirus in HeLa and Vero cells.

Virulent Mah type 1 poliovirus and the attenuated Sab types 1, 2 and 3 poliovirus do not efficiently produce plaques or grow as efficiently with Vero cells as with HeLa cells. All viruses attach poorly to simian cells on monolayer and, except for Mah and Sab1, eclipse poorly. Additionally, all three attenuated viruses block initiation of Vero cell protein synthesis less effectively than HeLa cell protein synthesis. The lower rates of attachment and eclipse of these viruses with Vero cells and their limited capacities to block cellular protein synthesis and thereby provide ribosomes for translation of viral RNA probably contribute to their more restricted growth in Vero cells.

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.

Association of poliovirus proteins with the endoplasmic reticulum.

Poliovirus proteins, except P3-7c, are associated with the endoplasmic reticulum after extraction of the cytoplasm and centrifugation of membranes to equilibrium in sucrose gradients. Proteins P3-2, P2-X, and P3-9 are found preferentially among the rough endoplasmic reticulum, whereas P3-7c is located in smooth endoplasmic reticulum fractions. P3-7c is probably not membrane associated, since it can be separated from membranes after centrifugation in buffer. However, P3-4a, P2-5b, P2-X, and P3-9 are avidly bound to membranes and cannot be dislodged with high-ionic-strength buffer containing EDTA or 4 M urea. These proteins are digested by trypsin, indicating peripheral rather than internal localization.

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.

In vivo proteins of defective interfering particles of poliovirus.

DX particles of poliovirus are deletion mutants that do not induce synthesis of capsid proteins or the precursor of capsid proteins (NCVPla) during infection. However, cells infected with DX particles synthesize two proteins, p68 and p25, that are not detected during growth of standard virus, and a protein of 27 000 (p27) which is comparable in molecular weight to VP3. Peptide maps of these proteins were obtained by partial digestion with Staphylococcus aureus V8 protease and elastase. The peptide map of p68 corresponded approximately 70% with the peptide map of NCVPla, and antiserum against virions reacted with p68. These data suggest that p68 is a large fragment of NCVPla. Digestion of purified structural proteins VP1, VP2, and VP3 yielded distinct peptide maps, but p25 was resistant to both V8 protease and elastase and did not react noticeably with anticapsid antibody. Peptide maps obtained for in vivo viral proteins migrating with a molecular weight of 27 000 were complex, indicating the presence of at least two and possibly three proteins. Cells infected with standard gs and gr viruses produced authentic VP3, but cells infected with defective interfering particles did not. However, one gr variant of standard virus contained a mutation in structural protein VP2.

Guanidine-resistant defective interfering particles of poliovirus.

A mixture containing standard poliovirus and D3 particles (mutants with deletions in the capsid locus) was serially passaged in the presence of guanidine. Within five growth cycles, the standard virus was guanidine resistant, but the D3 particles were guanidine sensitive, even after 21 passages with the inhibitor. By passage 40 with guanidine, D3 particles were eliminated, and a new deletion mutant (DX) appeared in the virus population. D3 particles contained a 15% deletion, and DX particles contained a 6% deletion in the capsid locus. Although neither mutant induced the synthesis of NCVP1a or a complete complement of capsid proteins after infection, cells infected with DX particles produced two novel proteins, which had molecular weights of approximately 68,000 and 25,000.

Inhibition of poliovirus polymerase by guanidine in vitro.

Extracts from poliovirus-infected HeLa cells synthesized 35s viral RNA, replicative intermediate, and double-stranded RNA in vitro. Guanidine inhibited the synthesis of all three species of RNA; production of 35s RNA was most sensitive to the inhibitor. Pulse-chase experiments with [3H]UTP indicated that guanidine had no detectable effect on elongation of polynucleotide chains or the release of completed RNA chains from the viral replication complex. Experiments in which short pulses of precursor were used suggest that guanidine blocked the initiation step of RNA synthesis in vitro.

Protein phosphorylations in poliovirus infected cells.

In vivo phosphorylation of proteins that are associated with polysomes of poliovirus-infected VERO (African green monkey kidney) and HeLa (Henrietta Lacks) cells differed from phosphorylations observed with uninfected cells that were fed fresh medium. With both types of cells infection stimulated phosphorylation of proteins with molecular weights of 40 000-41 000, 39 000, 34 000, 32 000, and 24 000. Similarities of phosphorylations in VERO and HeLa cells suggest that they are a specific consequence of infection and might serve a regulatory function during protein synthesis.

Peptide-chain initiation with Lsc poliovirus is intrinsically more resistant to hypertonic environment than is peptide-chain initiation with Mahoney virus and deletion mutants of Mahoney virus.

Peptide-chain initiation with LSc poliovirus was more resistant to hypertonic medium than peptide-chain initiation with Mahoney poliovirus. Protein synthesis of LSc virus retained its relative resistance to high osmolarity created by the addition of excess NaCl to the medium in cells coinfected with Mahoney virus. The data indicate that peptide-chain initiation with LSc virus is intrinsically more resistant to high osmolarity than that of Mahoney virus rather than reflecting different permeability changes in cells after infection. Two Mahoney virus mutants harboring deletions at the 5' end of the viral chromosome exhibited the same sensitivity to excess NaCl as parent virus, suggesting that the original chromosomal region for peptide-chain initiation has not been severely altered by the deletions.

Proteolysis of noncapsid protein 2 of type 3 poliovirus at the restrictive temperature: breakdown of noncapsid protein 2 correlates with loss of RNA synthesis.

Inhibition of RNA synthesis of the LH strain of type 3 poliovirus at the restrictive temperature occurs concurrently with the breakdown of noncapsid virus-specific peptide 2. This finding, along with other reported data (B.D. Korant, In Proteases and Biological Control, p. 621-644, 1975), suggests that noncapsid virus-specific peptide 2 is a component of the viral replicase.

Effect of hypertonic medium on protein synthesis of Mahoney and LSc poliovirus.

The initiation of synthesis of Mahoney virus protein is more sensitive to high osmolarity than initiation of synthesis of LSc virus protein. Production of Mahoney virus protein appears to be only slightly more resistant to high osmolarity than synthesis of HeLa-Rhino cell protein.