Biomedical subjects
Publications and source records attributed to E R Stout.
The DNAs encapsidated by parvoviruses show distinctly different patterns with respect to the ratio of plus-to-minus strands and sequence heterogeneity at the ends. A kinetic model, based on differential rates of hairpin transfer at 3' termini, is described and shown to account for all known parvoviral DNA distributions.
Sequence analyses of the left and right termini of LuIII virus show they are nonidentical imperfect palindromes of 122 and 211 nucleotides, respectively. The left terminus of the minus strand of LuIII DNA, uniquely in the flip conformation, can assume a T-shaped structure. The right terminus of the minus strand of LuIII DNA can assume a U-shaped structure, and it exists in either the flip or flop conformation. The termini of LuIII shared a high degree of sequence homology and showed conserved secondary structure with those of the rodent parvoviruses MVMp and H-1. LuIII, like adeno-associated virus, encapsidates equal amounts of plus- and minus-strand DNA. However, the sequence data for LuIII virus demonstrate that identical termini are not required for this encapsidation pattern.
Laboratory rabbits from various commercial and private sources were found to have high serum antibody titers specific for lapine parvovirus (LPV). By both immunofluorescence and hemagglutination inhibition assays, 75% of these sera were positive for LPV. This finding, together with the recovery of LPV from kidneys of neonatal rabbits, suggested that LPV infection is common in commercially available rabbits in the United States. It was concluded that use of infected rabbits could interfere with research in which rabbit cell cultures or in vitro immunologic assays are used.
The distribution of terminal-sequence orientations in the viral DNA of bovine parvovirus (BPV), an autonomous parvovirus, was studied by end labeling and restriction enzyme digestion and also by cloning. The left (3') end of the minus strand of BPV was found in two alternative sequence orientations (designated as flip and flop, which are reverse complements of each other), with a 10-fold excess of flip. This is in contrast to the autonomous rodent parvoviruses which encapsidate minus-strand DNA with only the flip orientation at this end. The right (5') end of the minus strand of BPV contained both sequence orientations with equal frequencies, as in the rodent parvoviruses. Sequence inversions were also detected at both ends of the plus strand, which makes up about 10% of the encapsidated BPV DNA. Each terminus of BPV DNA had a characteristic ratio of flip to flop forms, and this ratio was restored in the progeny DNA resulting from transfection with genomic clones of different defined terminal conformations. Replicative-form DNA showed the same distribution of terminal-sequence orientations as the reannealed plus and minus virion DNAs, suggesting that the distribution of flip and flop forms observed in virion DNA is not due to selective encapsidation, but rather to the specific distribution of replicative forms. The current replication model for autonomous parvoviruses, which was based on the available data for the rodent parvoviruses, cannot account for the observed distribution of BPV DNA. An alternative model is suggested.
Genomic clones of the autonomous parvovirus bovine parvovirus (BPV) were constructed by blunt-end ligation of reannealed virion plus and minus DNA strands into the plasmid pUC8. These clones were stable during propagation in Escherichia coli JM107. All clones tested were found to be infectious by the criteria of plaque titer and progressive cytopathic effect after transfection into bovine fetal lung cells. Sequencing of the recombinant plasmids demonstrated that all of the BPV inserts had left-end (3')-terminal deletions of up to 34 bases. DNA isolated from progeny virions arising from transfected infectious clones was found to be indistinguishable from wild-type DNA by restriction enzyme analysis. Defective genomes could also be detected in the progeny DNA even though the infection was initiated with homogenous, cloned DNA. Full-length genomic clones with 3' flip and 3' flop conformations were constructed and were found to have equal infectivity. Analysis of low-molecular-weight DNA isolated from lysates of cells transfected with these clones demonstrated that rescue and replication of BPV DNA could be detected 3 to 8 days after transfection. Expression of capsid proteins from transfected genomes was demonstrated by hemagglutination, indirect immunofluorescence, and immunoprecipitation of [35S]methionine-labeled cell lysates. Use of appropriate antiserum for immunoprecipitation showed the synthesis of BPV capsid and noncapsid proteins after transfection. Independently, a series of genomic clones with increasingly larger 3'-terminal deletions was prepared from separately subcloned 3'-terminal fragments. Transfection of these clones into bovine fetal lung cells revealed that deletions of up to 34 bases at the 3' end lowered but did not abolish infectivity, while deletions of greater than 52 bases were lethal. End-label analysis showed that the 34-base deletion was repaired to wild-type length in the progeny virus.
We have investigated the interaction between bovine parvovirus (BPV) capsid and non-capsid proteins and restriction fragments of the BPV genome by a combined DNA hybridization and immunodetection assay. 32P-labelled DNA was bound to nitrocellulose membranes bearing lysates of mock-infected and virus-infected cells whose proteins had been separated by SDS-polyacrylamide gel electrophoresis. The position of bound DNA was determined by autoradiography. The proteins on the membrane were still accessible to specific antibodies, allowing confirmation of the DNA-binding species by an immunodetection reaction. In 0.2 M-NaCl, BPV capsid proteins VP2 (72,000 daltons) and VP3 (62,000 daltons) bound the 0 to 16 map unit EcoRI fragment of BPV DNA which contained label in either the minus or plus strand. At higher salt concentration (0.5 M), only VP2 still bound DNA. Within this fragment, the capsid protein binding was restricted to those nucleotides between map units 0 and 4. No binding to capsid proteins was seen with the fragment spanning the middle of the genome and minor binding to VP3 was seen with the 5' end. Binding to the BPV non-capsid protein NP-1 was observed with the 0 to 16 map unit fragment when label was in the virion strand and to other possibly BPV-coded proteins when label was in the plus strand. The NP-1 binding was localized to map units 4 to 16. We did not detect binding to the BPV homologue(s) of the autonomous parvovirus non-capsid protein NS1, due in part to its low concentration in the cell lysates used. Points of the parvovirus replication cycle at which DNA-binding proteins may serve controlling functions are discussed.
Two nonstructural proteins of bovine parvovirus (BPV) with apparent molecular sizes of 75,000 and 83,000 daltons have been detected. The proteins were immunoprecipitated from lung cells infected with various isolates of BPV and from in vitro translations of infected cell mRNA. These proteins were expressed as nuclear phosphoproteins and were synthesized early in infection, before the peak of capsid protein synthesis. Early in infection, the 75-kilodalton-size species could be resolved into two bands of equal intensity, but later in infection, the lower-molecular-size form predominated. Antibodies directed against bacterial fusion proteins encoding amino acid sequences from a highly conserved region of the NS-1 polypeptides of two other parvoviruses, minute virus of mice and the human virus B19, gave specific nuclear fluorescence with BPV-infected cells, although the antibodies failed to immunoprecipitate any viral proteins. The noncapsid proteins appear to be homologous to the previously characterized NS-1 proteins of other autonomous parvoviruses.
Parvoviral genomes have been searched for sequences which may code for the nuclear transport of viral proteins. Sequences similar to those which regulate the nuclear transport of T antigen and yeast mating type protein were detected within the sequences coding for capsid and non-capsid proteins.
We determined the complete nucleotide sequence of bovine parvovirus (BPV), an autonomous parvovirus. The sequence is 5,491 nucleotides long. The terminal regions contain nonidentical imperfect palindromic sequences of 150 and 121 nucleotides. In the plus strand, there are three large open reading frames (left ORF, mid ORF, and right ORF) with coding capacities of 729, 255, and 685 amino acids, respectively. As with all parvoviruses studied to date, the left ORF of BPV codes for the nonstructural protein NS-1 and the right ORF codes for the major parts of the three capsid proteins. The mid ORF probably encodes the major part of the nonstructural protein NP-1. There are promoterlike sequences at map units 4.5, 12.8, and 38.7 and polyadenylation signals at map units 61.6, 64.6, and 98.5. BPV has little DNA homology with the defective parvovirus AAV, with the human autonomous parvovirus B19, or with the other autonomous parvoviruses sequenced (canine parvovirus, feline panleukopenia virus, H-1, and minute virus of mice). Even though the overall DNA homology of BPV with other parvoviruses is low, several small regions of high homology are observed when the amino acid sequences encoded by the left and right ORFs are compared. From these comparisons, it can be shown that the evolutionary relationship among the parvoviruses is B19 in equilibrium with AAV in equilibrium with BPV in equilibrium with MVM. The highly conserved amino acid sequences observed among all parvoviruses may be useful in the identification and detection of parvoviruses and in the design of a general parvovirus vaccine.
The replication of the autonomous parvovirus, bovine parvovirus (BPV), has been studied in virus-infected cells. Gel electrophoresis was used to determine the effect of aphidicolin, a specific inhibitor of DNA polymerase alpha, and L-canavanine, an inhibitor of protein synthesis, on viral DNA replication. Synchronized cell cultures were infected with 32P-labelled or unlabelled BPV in the presence or absence of aphidicolin and L-canavanine. Cells were harvested at various times post-infection, and DNA was electrophoresed and blotted. When aphidicolin was added to cells at the time of infection, then removed 8 h later, BPV replicative form DNA (RF) synthesis began within 2 h after its removal. This preceded the peak of cellular DNA synthesis by 2 h, unlike an uninhibited infection, when viral RF synthesis follows the peak of S phase by 2 to 4 h. Furthermore, if aphidicolin was added at any point during the replication cycle, BPV DNA synthesis stopped. This effect was shown to be completely reversible and indicated that aphidicolin did not disrupt the replication apparatus required for viral DNA synthesis. L-Canavanine inhibited synthesis of the virus-specific proteins NP-1 and VP3 and synthesis of BPV DNA. Upon removal of L-canavanine, viral protein synthesis was detected by 30 min followed by viral DNA synthesis. These results indicate that a specific S phase function other than cellular DNA synthesis is required for initiation of BPV DNA synthesis, that DNA polymerase alpha plays a major role in BPV DNA replication in vivo, and that these inhibitors can be used to inhibit reversibly various stages of BPV DNA replication.
A phosphorylated protein (NP-1) with an Mr of 28,000 has been detected in nuclei of bovine parvovirus (BPV)-infected cells in association with chromatin. No protein in this size range was detected after infection of appropriate cells with several autonomous rodent parvoviruses although the BPV-specific protein is similar in size to noncapsid proteins associated with rabbit parvovirus or adeno-associated virus infection. Structural homology between NP-1 and a BPV capsid protein could be detected by electrophoretic analysis of the products of proteolysis with chymotrypsin. This protein can be detected after in vitro translation of RNA from BPV-infected cells and BPV-specific RNA. Homology between the in vivo- and in vitro-synthesized species was shown by the similarity of the chymotryptic products.
Since parvoviruses apparently do not possess a DNA polymerase activity, one or more of the host cell DNA polymerases must be responsible for replicating the single-stranded DNA genome. We have focused on determining which polymerase, alpha, beta, or gamma (pol alpha, pol beta, or pol gamma, respectively), is responsible for the first step in bovine parvoviral DNA replication: conversion of the single-stranded DNA genome to a parental replicative form (RF). In this study, we used aphidicolin, a specific inhibitor of DNA pol alpha, to assay for the requirement of pol alpha activity in parental RF formation in vivo. Synchronized cell cultures were infected with bovine parvovirus with or without aphidicolin, and the products of viral replication were separated on agarose gels and identified by Southern blot analysis. We found that complete inhibition of viral DNA synthesis resulted when 20 microM aphidicolin was present throughout the infection. In addition, viral DNA synthesis was inhibited by as little as 1 microM aphidicolin, whereas lower concentrations (0.1 and 0.01 microM) resulted in partial inhibition of the replication process. Using 32P-labeled bovine parvovirus as the input virus we differentiated parental RF from daughter RF and progeny DNA synthesis. We conclude that DNA pol alpha is required for the production of RF during bovine parvovirus replication in vivo and that this requirement is most likely for the conversion of bovine parvovirus input single-stranded DNA to parental RF. These results do not rule out a possible role for DNA pol gamma in the first step, nor do they rule out a role for pol alpha or pol gamma in later stages of the replication cycle.
DNA polymerase gamma, purified from fetal bovine liver, replicated virion single-stranded DNA from bovine parvovirus to a unit-length double-stranded DNA molecule. This product was not nicked and was covalently linked to the 3' hairpin primer. The reaction was inhibited by dideoxythymidine 5'-triphosphate, but was unaffected by ATP or aphidicolin. Double-stranded viral DNA was not a functional template for purified DNA polymerase gamma.
A physical map has been constructed for bovine parvovirus (BPV) replicative form (RF) DNA synthesized in vitro. Analysis of restriction enzyme digestion products for 12 enzymes by neutral gel electrophoresis allowed the mapping of 26 cleavage sites between the 3' and 5' termini of the in vitro RF DNA. Cleavage sites for these enzymes were not detected within the 3'-terminal hairpin. HindIII, SalI, SmaI, SstII and XorII did not cleave BPV DNA, while 12 other enzymes cleaved the genome at one to four sites. Eleven additional enzymes were shown to cleave the genome at 8 to 20 sites. Comparison of the restriction site locations for BPV with those of other autonomous parvoviruses suggests that there may be conservation within the genome of certain restriction sites.
Total cytoplasmic RNA from bovine parvovirus (BPV)-infected cells or BPV-specific RNA selected by hybridization to cloned BPV genomic sequences were translated in a message-dependent rabbit reticulocyte lysate. Immunoprecipitation, using immunoglobulin G from rabbits injected with purified BPV, resulted in the detection of [35S]methionine-labeled polypeptides with MrS of 80,000, 72,000, 62,000, and 60,000. These in vitro translation products had the same mobility on sodium dodecyl sulfate-polyacrylamide gels as that of the four proteins found in purified virions. The three largest polypeptides had amino acid sequence homology, as judged by serological methods and partial proteolysis with Staphylococcus aureus V8 protease. Additional noncapsid proteins with MrS of 25,000, 27,000, and 31,000 were also detected as translation products of these RNAs. All of the above species were immunoprecipitated by immunoglobulin G from a calf which was naturally infected with BPV. All four capsid proteins but only one of the lower-molecular-weight polypeptides were detected after the immunoprecipitation of BPV-infected cells. The results presented here indicate that the BPV genome codes for four capsid proteins and a noncapsid protein which may be structurally related to the capsid proteins.
We have developed a nuclear lysate system from infected, synchronized cells capable of synthesizing unit-length parvoviral DNA in vitro. It was necessary to supplement the nuclear lysates with the polyamines, spermidine and spermine, to prevent degradation of template and product DNAs. In this system RF, RI, and single-stranded progeny DNAs were synthesized. Label incorporated in viral RF DNA in vivo appeared first in RI DNA and then in single-stranded DNA during incubation in vitro. By sedimentation the product DNAs were identical to those found in infected cells. Their viral identity was confirmed by hybridization. The addition of ribonucleotides, RNase, or alpha-amanitin did not affect parvoviral DNA synthesis in this system. The results with the specific inhibitors of mammalian DNA polymerases, aphidicolin, N-ethylmaleimide, and 2',3'-dideoxythymidine 5'-triphosphate indicated that DNA polymerase alpha was required for synthesis of parvoviral DNA in the nuclear lysates. This requirement was confirmed by experiments with antibody to bovine DNA polymerase alpha.
Transcription of the genome of the nondefective parvovirus BPV was examined in nuclei isolated from synchronized bovine fetal spleen cells. The relative levels of total RNA polymerase and RNA polymerase I, II, and III activities in nuclei isolated from BPV-infected and mock-infected cells were found to be similar throughout the course of infection. Hybridization of RNA synthesized in isolated nuceli indicated that BPV-specific RNA synthesis began during the period of 8 to 12 h postinfection and proceeded linearly until at least 20 h postinfection. By 20 h postinfection, 5% of the total RNA synthesized in nuclei from infected cells was virus specific. BPV-specific RNA synthesis was inhibited by 95% in the presence of 0.1 microgram of alpha-amanitin per ml, suggesting that the viral genome is transcribed by cellular RNA polymerase II.
We have examined four of the nondefective parvoviruses for an associated DNA polymerase. Virions were purified from neuraminidase-treated infected-cell lysates by isopycnic centrifugation in CsCl or from infected cell material by CaCl(2) precipitation and centrifugation through sucrose into CsCl. Preparations of bovine parvovirus or Kilham rat virus obtained by the former procedure contained DNA polymerase activity but were not free of contaminating cellular proteins. The latter method produced viral preparations free of contaminating cellular proteins, and no DNA polymerase activity was detected in light infectious particles of H-1, LuIII, bovine parvovirus, or Kilham rat virus. Examination of levels of each cellular DNA polymerase in these preparations from each step of both purification procedures revealed that DNA polymerase beta had a greater tendency to copurify with bovine parvovirus and Kilham rat virus than did DNA polymerases alpha or gamma. Disruption of infectious virions obtained by the second purification method with detergents and sonic treatment did not result in the detection of a DNA polymerase activity. The biological activity and purity of each of the four different viruses obtained by the latter procedure were determined by hemagglutination and infectivity assays, polyacrylamide gel electrophoresis, and electron microscopy. In each case, the virions banding at a density of 1.39 to 1.41 g/cm(2) in CsCl were infectious and contained only the virion structural proteins. DNA polymerase activity was not detected in any of these preparations, and we have concluded that a virion-associated DNA polymerase is not required for productive infection with the nondefective parvoviruses.