Comparative sequence analysis of the inverted terminal repetition in the genomes of animal and avian adenoviruses

The nucleotide sequences at the inverted terminal repetitions from two animal adenoviruses, infectious canine hepatitis virus and equine adenovirus, and from one avian adenovirus, CELO, were analyzed. DNAs from infectious canine hepatitis virus and equine adenovirus contain a homologous region which is 23 nucleotides long from the terminus. The first 17 nucleotides of this region are identical to the ones in human adenovirus type 2 DNA. The striking homologous sequence of 14 nucleotides, conserved in the inverted terminal repetitions of several human adenovirus strains and in simian adenovirus type 7, is only partially conserved in the two animal and one avian adenoviruses reported here.     

Nucleotide sequence of the coat protein cistron and the 3' noncoding region of cucumber green mottle mosaic virus (watermelon strain) RNA

Double-stranded cDNA copies of cucumber green mottle mosaic virus (watermelon strain, CGMMV-W) RNA polyadenylated in vitro were cloned into the pBR322 at the PstI site. The sequence of 1071 nucleotides from the Tend of the genomic RNA was determined using two recombinant plasmids and the genomic RNA. The coat protein cistron was located in residues 176-661 from the 3' end. The coat protein was composed of 160 amino acid residues with the molecular weight of 17,261. The 3' noncoding region of the CGMMVW genome was 175 nucleotides long and highly homologous to that of the common strain of TMV. The assembly origin of reconstitution is positioned within the coat protein cistron as predicted previously. In the 5' flanking region of the coat protein cistron a long open frame, probably of 30K protein, was found. The predicted 30K and the coat protein cistron would overlap each other as is the case of the cowpea strain of TMV.     

Natural variants of the Sabin type 1 vaccine strain of poliovirus and correlation with a poliovirus neutralization site

Independent substitution mutations have been detected in capsid polypeptide VP1 of the type 1 oral poliovirus vaccine isolated from normal infant vaccine recipients. These mutations map at amino acid residues 142 and 147 of VP1, a region only minimally hydrophilic. A synthetic peptide, corresponding to residues 141 to 147 of VP1 was synthesized, conjugated to a carrier polypeptide of bovine serum albumin. The conjugate was found to elicit a weak poliovirus neutralizing antibody response. It was also capable of priming the immune system for the production of IgG-type antibodies able to neutralize greater than 99.999% of infectious type 1 virus. It is suggested that region 141 to 147 of VP1 may be involved in neutralization of the virus and that the mutants may have accumulated by antibody selection.     

Isoelectric focusing and 2D-analysis of poliovirus proteins.

The polypeptides of poliovirus were isoelectrically focused in urea containing polyacrylamide gels after dissociation of virus particles in urea. The bands obtained were correlated to the known four structural polypeptides of poliovirus by the two-dimensional technique with sodium dodecyl sulfate-polyacrylamide gel electrophoresis in the second dimension. The measured isoelectric points are: VP1, 8.1; VP2, 6.4; VP3, 6.0; VP4, 7.3. Additional bands caused by charge modification of the polypeptides during storage of the virus can be found.     

Quantitation of the relatedness of reovirus serotypes 1, 2, and 3 at the gene level

As judged by the ability of their genomes to hybridize, reovirus serotypes 1 and 3 are related to the extent of about 70% to each other and about 10% to serotype 2. Two techniques were developed to measure the extent to which the individual cognate genes of these serotypes are related. Both involve comparison of heterologous hybrid genes that contain plus and minus strands of genes of different serotypes with homologous hybrid genes (molecules formed by reannealing the separated plus and minus strands of the same gene). In the first technique, the amounts in such hybrids of material sensitive to ribonuclease under standard conditions were compared; in the second, their relative electrophoretic mobilities. The results obtained with the two techniques agreed well. They showed that for the three reovirus isolates examined (the Lang strain of serotype 1, the D5 Jones strain of serotype 2, and the Dearing strain of serotype 3), all 10 genes of serotypes 1 and 3 are much more closely related to each other than to the genes of serotype 2. Although this result relates to only three isolates of mammalian reovirus, it suggests that the gene sets of reovirus serotypes 1, 2, and 3 evolved independently of each other. Apparently double infection of hosts with strains of two reovirus serotypes, which would very likely yield recombinants, occurs infrequently and/or such recombinants have a lower survival advantage than strains containing “pure” gene sets. The results also show that the gene that has diverged most markedly during evolution is the S1 gene, the gene that encodes the minor outer shell capsid protein σ1 which is the reovirus cell attachment protein and hemagglutinin and possesses the most type-specific antigenic determinants. The serotype 1 and 3 S1 genes are about 10% homologous, serotype 2 and serotype 1 or 3 S1 genes about 3%. The genes that have diverged least are the three L genes (85–90% homology for the serotype 1 and 3 L genes). In all cases, the serotype 2 and serotype 1 or 3 genes exhibit no more than 20%, and often less than 10% homology. In spite of this high degree of divergence, the antigenic determinants on proteins encoded by genes of serotype 2 on the one hand and serotypes 1 and 3 on the other hand are, with the exception of those on proteins σ1, highly conserved, and the 60 to 80 nucleotides at the 5′- and 3′-termini of at least three sets of cognate genes (L3, M3, and S2) of all three serotypes, serotype 1 and 3 as well as serotype 2, are highly homologous and in some instances almost identical. Thus, while some regions of reovirus genes have diverged greatly during evolution, others have been highly conserved.     

Comparative examination of the polypeptides of herpes simplex virus: types 1 and 2

Polypeptides synthesized during productive infection of HSV-1 and HSV-2 were found to possess distinct characteristics in regard to localization within the cell, DNA-binding properties, and phosphorylation after synthesis. Continuous labeling for 14 hr or pulse-labeling at successive periods during the replicative cycle with radioactive precursors revealed two types of polypeptide localization: (a) selective accumulation or enhancement within the cytoplasm or nucleus with barely detectable concentrations elsewhere and (b) accumulation in significant concentrations within both cytoplasm and nucleus showing little selective enhancement. Of the polypeptides made during HSV-1 infection 22 were phosphorylated as compared with 16 phosphoproteins specified by HSV-2. Phosphorylation was also implicated in the generation of the four molecular forms comprising the ICP 5–8 complex. Twenty-three polypeptides with affinity for DNA were detected after either type of infection. Sufficient comparisons were made to provide a basis for the tentative listing of 20 polypeptides of HSV-1 with corresponding polypeptides of HSV-2.     

Cleavage sites in the polypeptide precursors of poliovirus protein P2-X

Partial amino-terminal sequence analysis has been performed on the three major polypeptide products (P2-3b, P2-5b, and P2-X) from the central region (P2) of the poliovirus polyprotein, and this analysis precisely locates the amino termini of these products with respect to the nucleotide sequence of the poliovirus RNA genome. Like most of the products of the replicase region (P3), the amino termini of P2-5b and P2-X are generated by cleavage between glutamine and glycine residues. Thus, P2-5b and P2-X are probably both produced by the action of a single (virus-encoded?) proteinase. The amino terminus of P2-3b, on the other hand, is produced by a cleavage between the carboxy-terminal tyrosine of VP1 and the glycine encoded by nucleotides 3381–3383. This result may suggest that more than one proteolytic activity is required for the complete processing of the poliovirus polyprotein.     

Isolation and characterization of defective-interfering particles of poliovirus Sabin 1 strain

Defective-interfering (DI) particles of the Sabin strain of type 1 poliovirus were generated on serial high m.o.i. passaging. The deletions, measured by agarose gel electrophoresis, appeared to comprise approximately 10% of the total genome. Analysis of the RNAs, after digestion with RNase T1, by two-dimensional polyacrylamide gel electrophoresis revealed that the locations of the deleted genome regions were similar to those of the DI particles of the Mahoney strain of type 1 poliovirus (A. Nomoto, A. Jacobson, Y. F. Lee, J. Dunn, and E. Wimmer, (1979), J. Mol. Biol. 128, 179-196). Taking the known nucleotide sequences of the total genome and large RNase T1-resistant oligonucleotides into account, the deletions of almost all DI RNAs were found to exist between nucleotide positions 1307 and 2630, a genome region encoding capsid polypeptides VP2, VP3, and VP1. In cells coinfected with the purified DI particles and the Sabin strain of type 2 or type 3 poliovirus, particles containing the DI genomes were effectively produced. These results suggest that encapsidation signals are conserved in all three serotypes of polioviruses. However, only a very small amount of similar DI particles appeared to be produced in cells coinfected with coxsackie virus B1, although the genomes of polioviruses and coxsackie viruses have common sequences and therefore these viruses are considered to have arisen from a common ancestor. These data may suggest differences in encapsidation signals between polioviruses and coxsackie viruses.     

Amino-terminal sequence of adenovirus type 2 proteins: Hexon,fiber, component IX,and early protein 1B-15K

The partial amino-terminal amino acid sequence has been determined for four adenovirus 2 proteins: hexon, fiber, component IX, and early protein E1B-15K. A comparison of these sequences with the nucleotide sequence of the region of the genome encoding each of these proteins has identified the initiation sites for protein synthesis. Each protein is initiated at the AUG codon nearest the 5′ end of its mRNA. The initiating methionine is retained by fiber and component IX while it is removed from hexon and protein E1B-15K.     

Comparative sequence and structure of different isolates of citrus exocortis viroid

The nucleotide sequences of two new Australian isolates of citrus exocortis viroid (CEV), CEV-DE25 and CEV-DE26, have been determined and compared with the previously sequenced Australian isolate, CEV-A (J. E. Visvader, A. R. Gould, G. E. Breuning, and R. H. Symons. FEBS Lett.137, 288-292 (1982).) and a Californian isolate, CEV-C (H. J. Gross, G. Krupp, H. Domdey, M. Raba, P. Jank, C. Lossow, H. Alberty, K. Ramm, and H. L. Sanger. Eur. J. Biochem.121, 249-257 (1982).). Sequencing of recombinant DNA clones prepared from purified CEV-A showed sequence heterogeneity at four nucleotide residues which was not observed by direct RNA sequencing. The data indicated that purified CEV-A contained minor amounts of at least one other CEV species designated CEV-AM. All isolates contain 371 residues. In comparison with the sequence of CEV-A, four residues are different in CEV-C and CEV-DE25 while CEV-DE26 has 27 nucleotide differences which include exchanges, insertions, and deletions. Most of the nucleotide differences in CEV-C, CEV-DE25, and CEV-DE26 alter the proposed secondary structure relative to CEV-A. No nucleotide differences were found to occur in the highly conserved central region previously shown to be present in the predicted native structures of CEV, potato spindle tuber viroid, chrysanthemum stunt viroid, and coconut cadang-cadang viroid.     

Restriction enzyme analysis of the genomes of Plodia interpunctella and Pieris rapae granulosis viruses

The DNAs from the granulosis viruses of Plodia interpunctella and Pieris rapae were clearly distinguishable based on restriction endonuclease fragment patterns. By sizing single- and double-digestion products approximate molecular weights of 72 x 10(2) and 75 x 10(6), respectively, were estimated for the viral genomes.     

Comparative analysis of the enveloped virions of two granulosis viruses of the armyworm, Pseudaletia unipuncta

The enveloped virions of two strains of a granulosis virus, a synergistic Hawaiian (GVH) and a nonsynergistic Oregonian (GVO) strain which infect the armyworm, Pseudaletia unipuncta, were liberated from their capsules with 0.02 N NaOH and purified by filtration through membrane filters. The envelopes were solubilized with 0.1% Triton X-100. The enveloped virions, analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), had 9 to 12 polypeptides, 3 of which were associated with the naked virion. The two strains differed in their electrophoretic patterns mainly in the presence of two heavy polypeptides (89,000 and 97,000 daltons) and a 52,000-dalton polypeptide in GVH and in the absence of the heavy polypeptides and the presence of a 57,000-dalton polypeptide in GVO. The two heavy polypeptides were sensitive to proteinase. Two two-dimensional electrophoreses (first dimension with agarose gel electrophoresis and second dimension with SDS-PAGE and immunoelectrophoresis) of the envelope of GVH resolved two polypeptides, 37,000 and 48,000 daltons, which were serologically related to the synergistic factor present in the capsule of GVH; however their molecular weights differed from that of the GVH synergistic factor. In GVO, no polypeptide serologically related to the synergistic factor was detected in the envelope. The enveloped virions purified by differential filtration were infectious when fed to armyworm larvae, but the naked virions, free of envelopes, were not infectious.     

Use of temperature-sensitive mutants to study the morphogenesis of poliovirus

Three temperature-sensitive (ts) mutants of poliovirus (type 1 Mahoney) were isolated after nitrous acid treatment and characterized as phenotypically RNA⁺. When cells were infected at 37° with two of the three RNA⁺ts mutants (ts109 and ts739), reduced levels of 14 S particles were synthesized. One RNA⁺ mutant (ts520) synthesized significant amount of viral 14 S particle subunits. All of the mutants synthesized reduced amounts of procapsids and virions at 37°. At 39.5°, with all three ts mutants, the production of all virus-related particles in infected cells was markedly suppressed. Isoelectric focusing of the viral-related particles produced at 37° by the ts mutants and electrophoretic analysis of their structural polypeptides revealed the following: (i) ts739 synthesized an altered VP0 polypeptide and produced 14 S particles with an altered isoelectric point; (ii) ts109 produced 14 S particles with a normal pI but containing what appeared to be an altered VP1; (iii) ts520 produced normal 14 S particles as demonstrated by their pI, the electrophoretic behavior of their constituent structural polypeptides in SDS-PAGE, their ability to self-assemble, and their ability to form procapsid-like structures when incubated in extracts from wild-type (wt) virus-infected cells. However, ts520-infected cells contained few, if any, procapsids and extracts made therefrom were unable to assemble ts520 or wt 14 S particles into detectable amounts of pI 6.8 empty capsids. These and other findings are consistent with ts739 (and probably ts109) possessing an altered structural protein and ts520 being mutant in its morphopoietic factor.     

Evidence for common, intertypic antigenic determinants on poliovirus capsid polypeptides

Polyclonal antibodies prepared against individualized type 1 poliovirus structural polypeptides VP1, VP2, and VP3 were used to analyze the presence of common antigenic determinants among the three poliovirus serotypes. Each anti-VP antiserum immunoprecipitated specifically the polypeptide against which it had been prepared, as well as the corresponding polypeptide of the heterotypic viruses. Anti-VP1 antisera also reacted with type 1, type 2, and type 3 heat-denaturated poliovirions (C particles), whereas anti-VP2 and anti-VP3 sera formed immune complexes with type 1 and type 3, but not with type 2, C particles. It was concluded that capsid polypeptides VP1, VP2, and VP3 of the three serotypes share common antigenic determinants which are masked in mature virions (D particles), but can be unmasked, at least partially, upon heat inactivation of the virus.     

Alterations in the genomes of avian sarcoma viruses

We have identified polypeptides specific to region Elb (map position [mp] 4.6–112) of adenovirus 2 (Ad2) that are synthesized in six lines of Ad-transformed rat or human cells (F17, F4, T2C4, 8617, 5RK clone I, 293), and in Ad2 early infected KB cells. [³⁵S]Methionine-labeled polypeptides were immunoprecipitated using antisera against F17 cells, an Ad2-transformed rat cell line that retains only El. To determine whether they are viral coded, these polypeptides were compared by tryptic peptide mapping with polypeptides translated in vitro from Ela-specific mRNA (mp 1.3–4.5) and Elb-specific mRNA. Polypeptides of 19,000 daltons early infected KB cells. The 19K, 20K, and 53K could be translated from Elb-specific mRNA and thus are coded by Elb. The 19K was precipitated from all transformed cell lines, the 20K was immunoprecipitated from F4, 8617, and T2C4 cells, and the 53K was immunoprecipitated from F4, 8617, T2C4, and 293 cells. These results suggest that the 19K, and perhaps the 20K and 53K, may be important in adenovirus-induced cell transformation. The 20K and 53K share methionine-containing tryptic peptides with each other, but not with the 19K. These results, together with the Ad2 Elb DNA sequence (T. Gingeras and R. Roberts, personal communication), suggest that 19K is translated in a different reading frame from 53K and 20K.     

Inhibition of uncoating of poliovirus by arildone, a new antiviral drug.

The antiviral effects of a new drug, arildone (4-[6-(2-chloro-4-methoxyphenoxy)hexyl]-3,5-heptanedione, on poliovirus type 2 replication and host cell functions are described. Arildone inhibits poliovirus replication at a minimal inhibitory concentration (MIC) of 0.2 μM, while transport of radioactively labeled precursors and synthesis of DNA, RNA, and protein in uninfected HeLa cells are not inhibited. This drug is not virucidal and does not interfere with adsorption or penetration. Arildone inhibits uncoating of poliovirus and thereby prevents virus-induced shutoff of host cell protein synthesis. The possible mechanisms by which arildone interacts with the poliovirus icosahedral capsid to prevent uncoating are discussed.     

Comparative studies of wild-type and cold-mutant (temperature-sensitive) influenza viruses: nonrandom reassortment of genes during preparation of live virus vaccine candidates by recombination at 25 degrees between recent H3N2 and H1N1 epidemic strains and cold-adapted A/An Arbor/6/60.

Genetic compositions of 35 recombinant cold-adapted influenza A(H3N2 and H1N1) candidate live attenuated vaccine strains have been determined. The viruses, which had been obtained by recombination (reassortment) at 25° between contemporary epidemic wild-type strains and cold-adapted A/Ann Arbor/6/60(H2N2), followed by selection for growth at 25° of virus with wild-type HA and NA, have a highly restricted genetic composition. Eighteen of the thirty-five recombinants had RNAs coding for the three polymerase (P) proteins, NP, M, and NS, from the cold-adapted mutant A/Ann Arbor/6/60 had only the HA and NA of the wild-type strains. Only 4 out of 64 theoretically possible combinations of genes coding for nonglycoprotein viral products were detected. The restricted genetic composition of cold-adapted recombinants produced at 25° supports the evaluation of this method of preparing live vaccine strains to determine whether recombinants with constant gene composition have predictable levels of attenuation for man.     

The RNAs of cucumoviruses: 3'-terminal sequence analysis of two strains of tomato aspermy virus

The sequences of 189 residues from the 3' terminus of three RNAs of one strain and two RNAs of another strain of tomato aspermy virus have been determined; there was almost complete sequence homology between the RNAs. A base-paired transfer RNA-like structure is proposed for tomato aspermy virus RNAs which is similar in many aspects to the structure proposed for the 3'-terminal 172 residues of RNA 4 of the Q-strain of cucumber mosaic virus (R. H. Symons, Nucleic Acids Res.7, 825-837, 1979). These 172 residues of cucumber mosaic virus RNA 4 can be aligned to show 73% sequence homology with the 3'-terminal 189 residues of the tomato aspermy virus RNAs.     

Identification of poliovirus precursor proteins by immunoprecipitation

Precursors of poliovirus capsid polypeptides were detected using immunoprecipitation followed by SDS-PAGE. Two common VPO-3-1 precursors were found, and the occurrence of alternative cleavage pathways was confirmed.     

Homologies among the nucleotide sequences of the genomes of C-type viruses

DNA was synthesized with detergent-disrupted virions of several C-type viruses and used to measure the extent of homology among the genomes of these viruses by molecular hybridization. The DNA was reacted with viral RNA under conditions which permit saturation of most if not all complementary nucleotide sequences in the RNA. This technique provides a quantitative estimate of the extent of homology among viral RNAs and is superior to current procedures that measure the fraction of DNA hybridized to an excess of viral RNA. The genomes of RD-114 and Crandell virus are at least 85% related, whereas there is no detectable homology among the genomes of RD-114 virus, feline sarcoma-leukemia viruses, murine leukemia virus, avian sarcoma virus, and visna virus. The genome of feline sarcoma-leukemia viruses, like that of RD-114 virus, is at least partly homologous to DNA from normal cats, suggesting that normal cats harbor endogenous genes coding for components of at least two classes of C-type viruses.