Antisera to poly(A)-poly(U)-poly(I) contain antibody subpopulations specific for different aspects of the triple helix.

Rabbit antibodies to the triple-helical polynucleotide poly(A)-poly(U)-poly(I) were fractionated into three major antibody populations, each recognizing a different conformational feature of the triple-helical immunogen. Two distinct populations were purified from precipitates made with poly(A)-poly(U)-poly(U) and poly(A)-poly(I)-poly(I). The former reacted with double-stranded poly(A)-poly(U) or poly(I)-poly(C), and similar populations could be purified with either double-stranded form. The second population recognized the poly(A)-poly(I) region of the triple helix, and the third required all three strands for reactivity. These immunochemical studies suggest that the poly(A) and poly(U) have the same orientation in the triple-helicical poly(A)-poly(U)-poly(I) as in the double-helical poly(A)-poly(U), in which they have Watson-Crick base pairing.     

Action of 3-methylquercetin on poliovirus RNA replication.

3-Methylquercetin is a natural flavone that powerfully blocks poliovirus replication. This compound inhibits selectively poliovirus RNA synthesis both in infected cells and in cell-free systems. Poliovirus double-stranded RNA (replicative forms) is still made in the presence of this inhibitor, whereas the synthesis of single-stranded RNA and the formation of replicative intermediates are drastically blocked.     

Polyadenylic acid on poliovirus RNA. II. poly(A) on intracellular RNAs.

The content, size, and mechanism of synthesis of 3'-terminal poly(A) on the various intracellular species of poliovirus RNA have been examined. All viral RNA species bound to poly(U) filters and contained RNase-resistant stretches of poly(A) which could be analyzed by electrophoresis in polyacrylamide gels. At 3 h after infection, the poly(A) on virion RNA, relicative intermediate RNA, polyribosomal RNA, and total cytoplasmic 35S RNA was heterogeneous in size with an average length of 75 nucleotides. By 6 h after infection many of the intracellular RNA's had poly(A) of over 150 nucleotides in length, but the poly(A) in virion RNA did not increase in size suggesting that the amount of poly(A) which can be encapsidated is limited. At all times, the double-stranded poliovirus RNA molecules had poly(A) of 150 to 200 nucleotides. Investigation of the kinetics of poly(A) appearance in the replicative intermediate and in finished 35S molecules indicated that poly(A) is the last portion of the 35S RNA to be synthesized; no nascent poly(A) could be detected in the replicative intermediate. Although this result indicates that poliovirus RNA is synthesized 5' leads to 3' like other RNA's, it also suggests that much of the poly(A) found in the replicative intermediate is an artifact possibly arising from the binding of finished 35S RNA molecules to the replicative intermediate during extraction. The addition of poly(A) to 35S RNA molecules was not sensitive to guanidene.     

Immunochemical measurement of conformational heterogeneity of poly(inosinic acid).

Several pure poly(I) preparations differed in: (a) their complement fixation reactivity with anti-poly(I) antiserum; (b) their ability to bind to a solid-phase anti-poly(I) antibody-Sepharose column; (c) their ability to inactivate serum complement; and (d) their reactivity with purified antibodies to double-stranded RNA. In particular, poly(I) samples that could induce interferon production differed from non-inducer poly(I)s; the inducers reacted weakly with anti-poly(I) antiserum and were the only ones that reacted with antibodies to double-stranded RNA. One inducer poly(I) did not inactivate complement, and differed from non-inducer poly(I) in quantitative aspects of poly(I) . poly(C) formation with varying amounts of poly(C). An additional type of poly(I) preparation reacted poorly with anti-poly(I) antiserum, did not react with anti-double-stranded-RNA antibodies and failed to induce interferon production. The varying forms of poly(I) were not interconvertible by boiling and rapid chilling. These results indicate that several different stable structural forms of poly(I) may result from a standardized synthetic procedure.     

In Vitro Synthesis of Poliovirus Ribonucleic Acid: Role of the Replicative Intermediate

Poliovirus ribonucleic acid (RNA) polymerase crude extracts could be stored frozen in liquid nitrogen without loss of activity or specificity. The major in vitro product of these extracts was viral single-stranded RNA. However, after short periods of incubation with radioactive nucleoside triphosphates, most of the incorporated label was found in replicative intermediate. When excess unlabeled nucleoside triphosphate was added, the label was displaced from the replicative intermediate and accumulated as viral RNA. It is concluded from this experiment that the replicative intermediate is the precursor to viral RNA. In addition, some of the label was chased into double-stranded RNA. The implications of this finding are discussed.     

Studies on biosynthesis of tobacco mosaic virus. VII. Radioactivity of plus and minus strands in different forms of viral RNA after labelling of infected tobacco leaves

Tobacco leaves were labelled with tritiated undine for 30 or 120 minutes at different times after systemic infection with tobacco mosaic virus. RNA was extracted and separated into three fractions: one enriched in RF (replicative form), one enriched in RI (replicative intermediate), and one containing the bulk of single-stranded RNA. Radioactivity in plus strands (viral RNA) and minus strands (complementary RNA) was determined in each fraction by an isotope dilution assay. The amount of minus strands in the RP and RI fractions and the amount of plus strands in the single-stranded RNA fraction were also determined.Minus-strand synthesis was twice as high a few hours after the outbreak of visible symptoms as during the subsequent large accumulation of plus strands. At the early stage of virus production, the specific radioactivity of the minus strands was three- to fourfold that of the total RNA. Later it was about the same as that of the total RNA. As minus strands constitute a constant part of the total RNA at the later stages, this observation suggests that breakdown of minus strands is small.The specific radioactivity of minus strands was the same in corresponding RF and RI fractions. As the turn-over of minus strands appears to be small, a rapid interconversion of the two RNA types is indicated.In RF and RI the radioactivity in plus strands was between 6 and 50 times greater than that in minus strands. The specific radioactivity of plus strands was greater in RF and RI than in the single-stranded RNA, supporting the concept that both RF and RI have a precursor role for viral RNA.     

Poly(rC) binding proteins mediate poliovirus mRNA stability

The 5'-terminal 88 nt of poliovirus RNA fold into a cloverleaf RNA structure and form ribonucleoprotein complexes with poly(rC) binding proteins (PCBPs; AV Gamarnik, R Andino, RNA, 1997, 3:882-892; TB Parsley, JS Towner, LB Blyn, E Ehrenfeld, BL Semler, RNA, 1997, 3:1124-1134). To determine the functional role of these ribonucleoprotein complexes in poliovirus replication, HeLa S10 translation-replication reactions were used to quantitatively assay poliovirus mRNA stability, poliovirus mRNA translation, and poliovirus negative-strand RNA synthesis. Ribohomopoly(C) RNA competitor rendered wild-type poliovirus mRNA unstable in these reactions. A 5'-terminal 7-methylguanosine cap prevented the degradation of wild-type poliovirus mRNA in the presence of ribohomopoly(C) competitor. Ribohomopoly(A), -(G), and -(U) did not adversely affect poliovirus mRNA stability. Ribohomopoly(C) competitor RNA inhibited the translation of poliovirus mRNA but did not inhibit poliovirus negative-strand RNA synthesis when poliovirus replication proteins were provided in trans using a chimeric helper mRNA possessing the hepatitis C virus IRES. A C24A mutation prevented UV crosslinking of PCBPs to 5' cloverleaf RNA and rendered poliovirus mRNA unstable. A 5'-terminal 7-methylguanosine cap blocked the degradation of C24A mutant poliovirus mRNA. The C24A mutation did not inhibit the translation of poliovirus mRNA nor diminish viral negative-strand RNA synthesis relative to wild-type RNA. These data support the conclusion that poly(rC) binding protein(s) mediate the stability of poliovirus mRNA by binding to the 5'-terminal cloverleaf structure of poliovirus mRNA. Because of the general conservation of 5' cloverleaf RNA sequences among picornaviruses, including C24 in loop b of the cloverleaf, we suggest that viral mRNA stability of polioviruses, coxsackieviruses, echoviruses, and rhinoviruses is mediated by interactions between PCBPs and 5' cloverleaf RNA.     

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.     

Electrophoretic Characterization of Foot-and-Mouth Disease Virus-Specific Ribonucleic Acid

Foot-and-mouth disease virus (FMDV)-specific ribonucleic acid (RNA) was analyzed by electrophoresis on 0.5% agarose gels. Four classes of RNA were resolved as a function of mobility in agarose: two classes of slowly migrating multistranded RNA, the infectious viral RNA with intermediate mobility, and a minor fast-moving class of lower-molecular-weight single-stranded RNA. The major RNA species were infectious viral RNA and the slowest migrating class of multistranded RNA. The latter RNA was polydisperse when analyzed by sucrose gradient centrifugation, it was partially ribonuclease resistant, and it was the predominant RNA species labeled during the initial period of (3)H-uridine triphosphate incorporation in the cell-free system. Heat treatment studies indicated that part of the slowest-moving RNA was degraded at 60 C and almost complete degradation was detected at 100 C. It was concluded that this RNA is the replicative intermediate in viral RNA synthesis. The second class of multistranded RNA contained both a ribonuclease-resistant RNA and a second RNA peak which was detected only after heat treatment at temperatures above 75 C. Fractions of FMDV-specific RNA isolated by sucrose gradient centrifugation were analyzed by agarose-gel electrophoresis. Infectious viral RNA was detected only in the 37S zone and was the major species of RNA in this part of the gradient. The ribonuclease-resistant RNA (the 20S zone) contained about equal amounts of multistranded RNA (both classes) and the low-molecular-weight single-stranded RNA. All sucrose gradient fractions between 20 and 40S were found to contain the replicative intermediate, although the major portion was detected in the 20 to 25S region.     

Virus-Associated Nucleases: Location and Properties of Deoxyribonucleases and Ribonucleases in Purified Frog Virus 3

At least three nuclease activities are associated with purified frog virus 3. These activities are endodeoxyribonuclease (pH 7.5, double-stranded [DS] and single-stranded [SS] deoxyribonucleic acid [DNA]); endodeoxyribonuclease (pH 5.0, DS and SS DNA); endoribonuclease (DS and SS ribonucleic acid [RNA], pH 7.5). These activities are not adsorbed to the surface of the virion but are within the viral capsid and require detergent disruption of virions to unmask enzyme activity. Only one activity, deoxyribonuclease (pH 5.0, SS and DS DNA) appears to be core-associated after detergent disruption of virions. The ribonuclease degrades poliovirus replicative-form RNA, reovirus native RNA, and poly(I) poly(C) to a product with a sedimentation coefficient of about 6S. Qbeta 6S DS RNA and 4S transfer RNA are not degraded. The ribonuclease appears to be a late function of the virus and is elicited in a soluble form as well as a virus-associated form.     

Structure and Infectivity of Picornaviral RNA Encapsidated by Cowpea Chlorotic Mottle Virus Protein

Poliovirus and Mengo virus RNA were shown to associate efficiently with cowpea chlorotic mottle virus protein to form pseudovirions. The sedimentation coefficient for the pseudovirions was similar to that of poliovirus, and electron microscope observations showed the Mengo pseudovirions to be similar in size to Mengo virus. Such pseudovirions were infectious and were more resistant to ribonuclease than viral RNA; however, under our assay conditions, their infectivity was about equal to that of viral RNA.     

Chromatography of Arbovirus Ribonucleic Acid Forms on Columns of Benzoylated-Diethylaminoethyl Cellulose

Benzoylated-diethylaminoethyl cellulose (BD-cellulose) column chromatography was found to be useful in resolving most of the ribonucleic acid (RNA) forms from the replicative cycle of group A arbovirus Semliki Forect virus (SFV). The elution patterns were independent of molecular weight and appeared to be related to the degree of secondary structure in the molecule. Fractions of RNA were taken from a sucrose density gradient of cytoplasmic extracts of SFV-infected chick cells pretreated with actinomycin D. In a linear salt gradient, 16S material cochromatographed with the rapidly eluted ribonuclease resistant core of the double-stranded SFV-RNA and with the homopolymer duplex polyinosinic acid: polycytidylic acid. This fraction, therefore, probably contains an SFV-RNA form similar to the completely double stranded replicative form (RF) of several RNA viruses and bacteriophages. Faster moving (>20S) sucrose gradient fractions eluted more slowly, suggesting a decreasing proportion of secondary structure with increasing sedimentation value. The fractions, therefore, seemed to contain replicative intermediate (RI) structures. The two single stranded forms of SFV-RNA (42S and 26S) could only be eluted from BD-cellulose in the presence of urea or dimethyl sulfoxide, suggesting the presence of minimal secondary structure. Under these conditions, the single-stranded viral RNA forms could not be resolved. Molecular sieve chromatography of the single-stranded RNA forms, performed by passage through an agarose column, also failed to resolve these forms. The viral RNA forms containing a high degree of secondary structure, probably the RF and the RI, could, therefore, be rapidly separated from each other and from the single-stranded forms.     

A premelting conformational transition in poly(dA)-Poly(dT) coupled to daunomycin binding

Circular dichroism and UV absorbance spectroscopy were used to monitor and characterize a premelting conformational transition of poly(dA)-poly(dT) from one helical form to another. The transition was found to be broad, with a midpoint of tm = 29.9 degrees C and delta HVH = +19.9 kcal mol-1. The transition renders poly(dA)-poly(dT) more susceptible to digestion by DNase I and facilitates binding of the intercalator daunomycin. Dimethyl sulfoxide was found to perturb poly(dA)-poly(dT) structure in a manner similar to temperature. These combined results suggest that disruption of bound water might be linked to the observed transition. A thermodynamic analysis of daunomycin binding to poly(dA)-poly(dT) shows that antibiotic binding is coupled to the polynucleotide conformational transition. Daunomycin binding renders poly(dA)-poly(dT) more susceptible to DNase I digestion at low binding ratios, in contrast to the normal behavior of intercalators, indicating that antibiotic binding alters the conformation of the polynucleotide. The unusual thermodynamic profiles previously observed for the binding of many antibiotics to poly(dA)-poly(dT) can be explained by our results as arising from the coupling of ligand binding to the polynucleotide conformational transition. Our data further suggest a physical basis for the temperature dependence of DNA bending.     

Analysis of antigenic profiles of inactivated poliovirus vaccine and vaccine-derived polioviruses by block-ELISA method.

A new block-ELISA test for quantitative evaluation of relative reactivity of antigenic sites was developed and used to reveal the detailed epitope structure of inactivated poliovirus vaccines (IPV) and live poliovirus strains. Poliovirus was captured on ELISA plates coated with rabbit anti-poliovirus IgG and blocked by monoclonal antibodies (Mabs) specific to individual epitopes before the remaining reactive antigenic sites were quantified by polyclonal anti-poliovirus IgG conjugate. The decrease of conjugate binding by the pre-treatment with a Mab reflects its contribution to the overall reactivity of poliovirus antigen. The level of block activity of Mabs for a given antigen can be expressed as a percent of reduction of antigenic reactivity as determined by ELISA test. It can be normalized by expressing this value as a ratio to the block activity of a reference sample. The data on the blocking-activity of a panel of monoclonal antibodies specific to different antigenic sites represents the epitope composition (antigenic profile) of a sample. Quantitative differences in epitope composition were determined for nine samples of inactivated poliovirus vaccine (IPV) and compared with the International Reference Reagent. This method could be used for monitoring consistency of IPV production, comparison of vaccines made by different manufacturers, and for the analysis of antigenically modified strains of attenuated poliovirus. Antigenic structures of two isolates of type 1 vaccine-derived poliovirus (VDPV) were compared with the structures of parental Sabin 1 and wild-type Mahoney strains using 17 monoclonal antibodies and revealed significant differences, suggesting that the method can be used for screening of field isolates and rapid identification of antigenically divergent VDPV strains.     

Studies on the biosynthesis of TMV

Summary The replicative form and the replicative intermediate of TMV-RNA were isolated from synchronously infected tobacco leaves, labeled with H³-uridine for 1 hour. The replicative form is over 90% resistant to RNase and sediments slightly slower than the 16S ribosomal RNA. Sucrose gradient centrifugation of the thermally denatured replicative form revealed that it contains strands of the same size as single-stranded TMV-RNA. The replicative intermediate showed only partial resistance to RNase and heterogeneous sedimentation behavior in sucrose gradients. After mild RNase treatment the replicative intermediate sedimented homogeneously, and with an S value slightly lower than the replicative form.The following abbreviations are used RF replicative form - RI replicative intermediate - STE 0.1 M NaCl-1 mM Tris-HCl-1 mM EDTA, pH 7.4 - SSC 0.15 M NaCl-0.015 M sodium citrate, pH 7.0 - 10xSSC and 0.1xSSC tenfold concentrated and tenfold diluted SSC respectively - MAK methylated albumin coated kieselguhr