George F. Vande Woude

Studies on foot-and-mouth disease virus ribonucleic acid synthesis

Abstract Ribonucleic acid (RNA) was extracted from baby hamster kidney cells infected with type A119 foot-and-mouth disease virus (FMDV) in the presence of actinomycin-D. Virus-specific RNA components, resolved by ultracentrifugation in sucrose gradients, were first detected between 90 and 180 minutes after infection; the maximum rate of synthesis was found 5 hours after infection. The virus-specific RNA components which have been identified are viral RNA and a complex having a sedimentation coefficient of about 20 S which is partially ribonuclease (RNase) resistant. The RNase-resistant RNA is synthesized in amounts similar to viral RNA, and “pulse-labeling” experiments indicate that it is formed before virus RNA.

Isolation of the structural polypeptides of foot-and-mouth disease virus and analysis of their C-terminal sequences

Abstract The three polypeptides—VP 1 , VP 2 and VP 3 —which comprise approximately 91% of the protein of foot-and-mouth disease virus, type A 12 -119, were separated by polyacrylamide gel electrophoresis (PAGE) in sufficient purity and quantity for determination of their amino acid compositions as well as for analysis of their C-terminal sequences and molecular weights by reaction with carboxypeptidase-A. At least seven distinct differences in amino acid compositions were observed among the three polypeptides, but their average composition was nearly identical to that of the total protein of the virus, VP 0–4 . C-Terminal amino acid sequences for VP 1 and VP 3 appear to to be (—serine-glutamine) and (—glutamine-alanine-leucine), respectively, and the C-terminal amino acid of VP 2 is probably glutamic acid. Molecular weights for VP 1 and VP 3 calculated from C-terminal data were in the 27,500–31,000 dalton range compared with values of 28,500 determined previously by PAGE.

Chemical and physical properties of foot-and-mouth disease virus: A comparison with maus elberfeld virus

The chemical and physical properties of foot-and-mouth disease (FMD) and Maus Elberfeld (ME) viruses have been compared, and it is concluded that these viruses are structurally similar. Thus, the total FMD virion mass is ca. 8.3 × 106 daltons based on an RNA mass of 2.6 × 106 daltons. The FMD virus capsid protein, ca. 5.7 × 106 daltons, is predominantly comprised of 60 identical 85,500 dalton protein subunits, each unit containing three non-identical polypeptides, VP1–3. Two additional components are present at levels of 33/virion (VP4; 13,700) and 2/virion (VP0; 39,500). The reported molecular weight for the FMD-12 S virion subparticle (2.5 – 3.0 × 105 daltons) may indicate that it is a trimer of the 85,500 dalton protein subunit, and is centered about a three-fold axis of the icosahedral virus.

The inactivation of foot-and-mouth disease virus at ionic-strength dependent isoelectric points

Abstract Foot-and-mouth disease virus (FMDV) isoelectric point (pI) values, determined by cellulose acetate electrophoresis, were linear with respect to the square root of the ionic strength. This effect for proteins is attributed to the preferential binding of anions. The Longsworth and Jacobsen equation, pI=pI0−(AΓ/2)/(1+BΓ/2), was evaluated for FMDV; Γ/2 is the ionic strength, pI0 is the isoionic point 7.6, and constants A and B are 21.5 and 4.2, respectively. With pI values of 3.25−7.5 estimated from this equation, it was found that FMDV was always inactivated at its pl, while at 2 pH units above its pI there was no loss of infectivity. Storing virus at conditions greater than 2 pH units above isoelectric points at alkaline pH also resulted in inactivation. Thus, virus inactivation or stability can be predetermined over a range of hydrogen ion concentrations and ionic strengths. Inactivation of FMDV below pH 7.0 in isotonic solution or degradation at pH 7.5 in hypotonic solution (Γ/2 0.002) which previously appeared to occur by two independent mechanisms are both the result of isoelectric inactivation.

Ribosomal factors effecting the stimulation of cell-free protein synthesis in the presence of foot-and-mouth disease virus ribonucleic acid☆

Abstract Ribosomal associated factors dissociated with high salt mediate the binding of N-acetyl-aminoacyl-tRNA to salt washed (s-) ribosomes in the presence of exogenous messenger RNA (e.g., foot-and-mouth disease virus (FMDV)-RNA). Only ribosomal factors isolated from FMDV-infected cells could show a stimulation of N-acetyl-aminoacyl-tRNA binding in the presence of FMDV-RNA. Amino acid incorporation studies also corroborated the need for ribosomal associated factors from FMDV-infected cells for stimulation. Polyacrylamide gel analysis of the cell-free product synthesized in the presence of FMDV-RNA indicated that amino acid labeled protein appeared to be a large molecular weight and rather heterogeneous protein.

Amino acid composition and C-terminal sequence of foot-and-mouth disease virus protein.

Abstract The amino acid composition of foot-and-mouth disease virus (FMDV) protein from type A virus, strain 119, and sequence of amino acids at the C-terminus were determined. Previous evidence from disc gel electrophoresis and gel filtration experiments has indicated that the protein of FMDV is built up from a homogeneous polypeptide. The amino acid composition of the protein agreed closely with that determined earlier on whole virus, where a large correction factor for glycine was necessitated. The amino acids with basic, hydroxy, acidic, and hydrocarbon (including tyrosine) side chains comprised about 12, 17, 19, and 42% of the protein, respectively. Methionine, tryptophan, and half-cystine were present in the least amounts, approximately 1.5, 1.0, and 0.8%, respectively. Analysis showed the half-cystine to exist in the protein coat in situ entirely as cysteine. The rate of release of amino acids from the protein by carboxypeptidase A together with hydrazinolysis of the protein indicated as C-terminal sequence of —(Val, Asp, Thr, Asn, Phe, Glu, Gly)-Ser-Ala-Leu-Gln, the sequence within the parentheses being uncertain. An extinction coefficient, E 1% 276m μ = 11.1, calculated for the protein from quantitative amino acid analysis, was identical to that determined earlier by quantitation of the protein mass.

Inhibition of ribonucleic acid methylation in the foot-and-mouth disease virus-infected host cell.

Abstract Inhibition of baby hamster kidney (BHK) host cell protein and RNA synthesis following infection with foot-and-mouth disease virus (FMDV) has been described (Brown et al. , 1966; Polatnick et al. , in press). Foot-and-mouth disease virus (type A, strain 119) infection causes a more drastic inhibition of host cell protein synthesis than of RNA synthesis throughout the infectious cycle. For example, early in infection (90 min postinfection [PI]), and at peak virus production (300 min PI), protein synthesis is inhibited approximately 50 and 80%, respectively, while RNA synthesis is inhibited only 20 and 50% (Polatnick et al. , in press). In this report, the effect of FMDV infection on host cell RNA methylation is examined at 300 min PI. Methylation of total host cell RNA, fractionated 1 M NaCl insoluble RNA and sRNA is shown to be inhibited to the same degree as host cell protein synthesis in the FMDV infected cell.

The influence of the host cell, ionic strength and chymotrypsin on foot-and-mouth disease virus electrophoretic mobility

Cellulose acetate zone electrophoretic analyses of foot-and-mouth disease virus (FMDV), type A, strain 119, with different passage histories revealed differences in homogeneity and response to chymotrypsin induced mobility changes. Foot-and-mouth disease virus with a high passage history in calf kidney cells migrated as a single electrophoretic component at the pH and ionic strengths examined. This virus, purified after a single additional passage in baby hamster kidney cells, clone 13, also gave a single zone. In contrast, virus purified after a single passage in uncloned baby hamster kidney cells revealed two electrophoretic zones, 1 and 2, as determined by staining and infectivity. Zone 1 migrated the same distance as the single virus zone prepared from the high passage calf kidney cells. All FMDV with low passage histories had only a single electrophoretic zone. After incubation with chymotrypsin, differences between FMDV with low and high passage histories were indicated by the increase in mobility of only high passage virus. It was further demonstrated that only the mobility of zone 1 of the heterogeneous virus was changed by the enzyme. Trypsin, ribonuclease, and diisopropylfluorophosphate inactivated chymotrypsin had no effect.