Comparative biochemical studies of type 3 poliovirus

A study of the biochemistry of type 3 poliovirus strains which involves the examination of the virus-coded polypeptides in infected cells and the preparation of oligonucleotide maps is reported. The polypeptide patterns were shown to be a relatively stable property of virus strains and distinguished Sabin vaccine strains from wild strains of poliovirus type 3. This approach may be of value in deciding the origin (vaccine or nonvaccine) of field isolates of poliovirus. Oligonucleotide maps were found to be sensitive indicators of differences among strains and appear to form a basis for determining genetic relationships among strains. The nucleotide maps of two viruses isolated from human cases of paralytic poliomyelitis temporally associated with the administration of attenuated vaccine suggested a vaccine origin for the strain. In one case the nucleotide map was indistinguishable from that of the vaccine strain.     

Further studies of specificity of antibodies contained in antiserum against poliovirus.

Antigenic components of Mahoney strain (poliovirus type 1) involved in virus neutralization reaction were analyzed with mutant Mahoney strains resistant to inhibitors in equine serum (inhibitor-resistant mutants) by means of the kinetic neutralization test. It was shown that absorption of anti-Mahoney serum with five inhibitor-resistant mutants yielded sera with different antibodies, of which three had distinct specificities and two specificities possibly partly related to one of those three sera. Further, it was found that step wise selection of Mahoney variants resistant to one, two, three and four different inhibitors resulted in gradual deviation of its antigenic composition from that of the original strain. From these results, the possible presence of three or more distinct antigenic determinant sites on the surface of Mahoney strain was indicated.     

Preliminary studies of crystals of poliovirus type I

Poliovirus contains 60 copies each of four coat protein subunits arranged on a T = 1 icosahedral surface. Crystals of the Mahoney and the Sabin strains of the type I serotype of polio have been obtained. Crystals of the Mahoney strain belong to the orthorhombic space group P2₁2₁2 with $\text{a = 324}\text{A}\text{?}\text{, b = 359}\text{A}\text{?}\text{, c = 381}\text{A}\text{?}$, contain $\text{1}\text{2}$ virus particle in the asymmetric unit, and diffract to at least 2.5 Å resolution. The crystals are apparently identical to those characterized by Finch & Klug (1959). Collection of three-dimensional X-ray diffraction data to 2.9 Å resolution is in progress.     

Degradation of cellular proteins during poliovirus infection: studies by two-dimensional gel electrophoresis.

Picornaviruses encode for their own proteinases, which are responsible for the proteolytic processing of the polyprotein encoded in the viral genome to produce the mature viral polypeptides. The two poliovirus proteinases, known as proteins 2A and 3C, use the poliovirus-encoded polyprotein as a substrate. The possibility that these poliovirus proteinases also degrade cellular proteins remains largely unexplored. High-resolution two-dimensional gel electrophoresis indicates that a few cellular proteins disappear after poliovirus infection. Thus, at least nine acidic and five basic cellular proteins, ranging in Mr from 120 to 30 kilodaltons, are clearly degraded during poliovirus infection of HeLa cells. The degradation of these cellular polypeptides is very specific because it does not occur upon infection of HeLa cells with encephalomyocarditis virus or Semliki Forest virus. Moreover, inhibitors of poliovirus replication, such as cycloheximide or 3-methylquercetin, block the disappearance of these polypeptides. These results suggest that the input virions are not responsible for this degradation and that active poliovirus replication is required for the proteolysis to occur. Analysis of the time course of the disappearance of these polypeptides indicates that it does not occur during the first 2 h of infection, clearly suggesting that this phenomenon is not linked to the poliovirus-induced shutoff of host protein synthesis. This conclusion is strengthened by the finding that 3-methylquercetin blocks proteolysis without preventing shutoff of host translation.     

Postabsorption neutralization of poliovirus.

Nineteen neutralizing murine monoclonal antibodies against poliovirus type 1, including representatives reacting with each of the antigenic sites on the virion, were tested for their abilities to neutralize the virus either before or after attachment to susceptible cells. All antibodies neutralized unattached virus; six had reasonable titers of postabsorption neutralization (PAN). Experiments with antibodies lacking PAN activity showed that Fc-specific rabbit anti-mouse antibodies could confer PAN activity. PAN was shown to involve the prevention of the cell-mediated conversion of virus to 135S and 80S particles. Evidence that one of the PAN-positive antibodies probably bound bivalently to preabsorbed virions, whereas a PAN-negative antibody bound monovalently, is presented. Two PAN-positive antibodies were added to an excess of virus in suspension, and only one antibody caused the virus to aggregate.     

Inactivation of poliovirus by chloramine-T.

Since concern has recently been expressed about the presence of genotoxic substances due to chlorination of water and wastewater, chloramine-T (CAT) is proposed as an alternative disinfectant to chlorine. The viricidal properties of chlorine and CAT were compared. Kinetics of inactivation of poliovirus type 2 by chlorine and CAT in chlorine demand-free water were investigated by using a kinetic apparatus. Inactivation of the virus by chlorine and CAT occurred in two steps. The initial linear part of the inactivation curve followed a pseudo-first-order reaction with the virus. An obvious dose-response relationship was demonstrated with CAT. The rate of inactivation of the virus by CAT was faster in acid medium than in alkaline medium. Inactivation kinetic studies were performed at different temperatures, and the kinetic, Arrhenius, and thermodynamic parameters were evaluated. The rate of inactivation of poliovirus type 2 by chlorine was faster than that by CAT under identical conditions. A mechanism for the viral inactivation in acid conditions was proposed which led to a rate equation consistent with the experimental results. The results indicate that CAT may be an effective viricide against poliovirus type 2 in an acid medium.     

Reconcentration of poliovirus from sewage.

Virus can be adsorbed from effluents of sewage treatment plants on large-surface membranes. Subsequent elution of virus requires large volumes, which in turn requires reconcentration of virus for assay. However, reconcentration of such viral eluates on small adsorbent surfaces is difficult because certain soluble sewage components are adsorbed along with the virus on the initial virus adsorbent and are removed along with the virus by the eluent. Upon acidification of the initial eluate to reconcentrate the virus on smaller membrane surfaces, flocs are formed that interfere with the reconcentration process. To circumvent this problem, the interfering sewage components can be removed by activated carbon and ion-exchange resins. The virus is then readily reconcentrated on small membranes.     

Protein processing map of poliovirus.

Five previously unmapped proteins (5a, 7d, 8, 9b, and 10) were located on the proteolytic processing map of the polyprotein. One of the proteins, 9b, appears to be the sister fragment of a cleavage reaction (P3-9 leads to P3-9b + VPg). Two of the other newly mapped proteins, 8 and 10, have been identified as sister fragments of X-related proteins 3b and 5b; thus, P2-3b leads to P2-8 + P2-5b and P2-5b leads to P2-10 + P2-X. The remaining proteins, 5a and 7d, mapped in the 1b protein and appear to result from the cleavages P3-1b leads to P3-5a + P3-6b and P3-4b leads to P3-7d + P3-6b. These assignments account for over 95% of the total polioviral proteins and complete the mapping of the major processing pathways.     

Reconcentration of poliovirus from sewage.

Virus can be adsorbed from effluents of sewage treatment plants on large-surface membranes. Subsequent elution of virus requires large volumes, which in turn requires reconcentration of virus for assay. However, reconcentration of such viral eluates on small adsorbent surfaces is difficult because certain soluble sewage components are adsorbed along with the virus on the initial virus adsorbent and are removed along with the virus by the eluent. Upon acidification of the initial eluate to reconcentrate the virus on smaller membrane surfaces, flocs are formed that interfere with the reconcentration process. To circumvent this problem, the interfering sewage components can be removed by activated carbon and ion-exchange resins. The virus is then readily reconcentrated on small membranes.     

Carboxy-terminal analysis of poliovirus proteins: termination of poliovirus RNA translation and location of unique poliovirus polyprotein cleavage sites.

The carboxy-terminal amino acids of a number of poliovirus proteins were determined by carboxypeptidase A analysis. The nonstructural proteins P3-2, P3-4b and their precursor. P3-1b, were found to be coterminal with a sequence of -Ser-Phe-COOH. As these proteins are coded for at the extreme 3' end of the viral RNA, it is possible to establish the termination site of translation at nucleotide 7,361, 73 nucleotides before the start of the polyadenylic acid tract of the RNA. Two additional nonstructural proteins, P2-X and its precursor, P2-3b, were also found to be coterminal with a sequence of -Phe-Gln-COOH. This result confirms the existence of at least one Gln-Gly proteolytic cleavage site. These Gln-Gly cleavage sites are predicted from the nucleotide sequence to be ubiquitous throughout the poliovirus genome. The only exceptions are the cleavage sites at the carboxy termini of the structural protein VP4 and VP1. Carboxypeptidase A analysis of VP1 establishes a terminal sequence of -Thr-Tyr-COOH, and similar analysis of VP4 shows Asn to be the terminal amino acid residue, observations that prove the existence of the exceptional C-terminal amino acids. In none of the analyzed cases has C-terminal trimming after cleavage been observed.     

Polyadenylic acid on poliovirus RNA. III. In vitro addition of polyadenylic acid to poliovirus RNAs.

A crude RNA polymerase preparation was made from HeLa cells infected for 3 h with poliovirus. All virus-specific RNA species labeled in vitro (35S RNA, replicative intermediate RNA [RI], and double-stranded RNA [dsRNA]) would bind to poly(U) filters and contained RNase-resistant stretches of poly(A) which could be analyzed by electrophoresis in polyacrylamide gels. After incubation for 45 min with [3-H]ATP in the presence of the other three nucleoside triphosphates, the labeled poly(A) on the RI and dsRNA migrated on gels as relatively homogenous peaks approximately 200 nucleotides in length. In contrast, the poly(A) from the 35S RNA had a heterogeneous size distribution ranging from 50 to 250 nucleotides. In the absence of UTP, CTP, and GTP, the size of the newly labeled poly(A) on the dsRNA and RI RNA was the same as it was in the presence of all four nucleoside triphosphates. However the poly(A) on the 35S RNA lacked the larger sequences seen when the other three nucleoside triphosphates were present. When [3-H]ATP was used as the label in infected and uninfected extracts, heterogeneous single-stranded RNA sedimenting at less than 28S was also labeled. This heterogeneous RNA probably represents HeLa cytoplasmic RNA to which small lengths of poly(A) (approximately 15 nucleotides) had been added. These results indicate that in the in vitro system poly(A) can be added to both newly synthesized and preexisting RNA molecules. Furthermore, an enzyme capable of terminal addition of poly(A) exists in both infected and uninfected extracts.     

The genome-linked protein of picornaviruses. VII. Genetic mapping of poliovirus VPg by protein and RNA sequence studies

The poliovirus genome-linked protein (VPg) has been subjected to radiochemical microsequence analysis. Sequence studies of virion RNA by a modification of Sanger's dideoxy method have revealed a base sequence corresponding to the amino acid analysis. This result proves that VPg is virus-encoded. The RNA sequence has allowed us to predict the total amino acid sequence of VPg and part of its precursor. VPg is, at most, 27 amino acids long. It maps within the 3' terminal segment of the viral genome that encodes the precursor polypeptide NCVP1b for the virus-specific RNA polymerase NCVP4.