Adenovirus terminal protein protects single stranded DNA from digestion by a cellular exonuclease.

Adenovirus 5 DNA-protein complex is isolated from virions as a duplex DNA molecule covalently attached by the 5' termini of each strand to virion protein of unknown function. The DNA-protein complex can be digested with E. coli exonuclease III to generate molecules analogous to DNA replication intermediates in that they contain long single stranded regions ending in 5' termini bound to terminal protein. The infectivity of pronase digested Adenovirus 5 DNA is greatly diminished by exonuclease III digestion. However, the infectivity of the DNA-protein complex is not significantly altered when up to at least 2400 nucleotides are removed from the 3' ends of each strand. This indicates that the terminal protein protects 5' terminated single stranded regions from digestion by a cellular exonuclease. DNA-protein complex prepared from a host range mutant with a mutation mapping in the left 4% of the genome was digested with exonuclease III, hybridized to a wild type restriction fragment comprising the left 8% of the genome, and transfected into HeLa cells. Virus with wild type phenotype was recovered at high frequency.     

Genome-linked protein associated with the 5' termini of bacteriophage phi29 DNA.

A DNA-protein complex was isolated from Bacillus subtilis bacteriophage phi29 by sucrose gradient sedimentation or gel filtration in the presence of agents known to break noncovalent bonds. A 28,000-dalton protein was released from this complex by subsequent hydrolysis of the DNA. The DNA-protein complex was examined for its susceptibility to enzymes which act upon the 5' and 3' termini of DNA molecules. It was susceptible to exonucleolytic degradation from the 3' termini by exonuclease III but not from the 5' termini by lambda exonuclease. Attempts to label radioactively the 5' termini by phosphorylation with T4 polynucleotide kinase were unsuccessful despite prior treatment with alkaline phosphatase or phosphatase treatment of denatured DNA. Removal of the majority of the bound protein by proteolytic digestion did not increase susceptibility. These results suggest that the linked protein is covalently attached to the 5' termini of phi29 DNA.     

Adenovirus DNA replication in vitro: a protein linked to the 5' end of nascent DNA strands.

Soluble nuclear extracts prepared from adenovirus-infected HeLa cells supported adenovirus DNA replication with exogenous DNA-protein complex as template, but protease-treated, phenol-extracted DNA was less active. Replication was enhanced when creatine phosphate and creatine phosphokinase were included in the reaction mixture, rendering the reaction independent of exogenous ATP. Genomic-length, newly synthesized DNA strands were first observed 30 min after initiation of replication and continued to increase in amount for at least 4 h. Thus, the rate of replication is consistent with previous estimates of the rate of replication in vivo. Nascent DNA strands bound to benzoylated, naphthoylated DEAE-cellulose due to their association with protein. The 5' termini of nascent DNA strands were resistant to the 5'- to 3'-specific T7 exonuclease, and the 3' termini of nascent strands were sensitive to the 3'- to 5'-specific exonuclease III. These results suggest that a protein becomes covalently linked to the 5' termini of nascent DNA strands replicated in vitro. Nuclear extracts prepared from adenovirus type 2-infected cells also supported replication of DNA-protein complex prepared from the unrelated type 7 adenovirus. The limited sequence homology between these two viruses at the origin of replication further defines recognition sequences at the origin. These results are discussed in terms of a model for adenovirus DNA replication in which the terminal protein and sequences within the inverted terminal repetition are involved in the formation of an initiation complex that is able to prime DNA replication.     

Genetic analysis of the vaccinia virus I6 telomere-binding protein uncovers a key role in genome encapsidation

The linear, double-stranded DNA genome of vaccinia virus contains covalently closed hairpin termini. These hairpin termini comprise a terminal loop and an A+T-rich duplex stem that has 12 extrahelical bases. DeMasi et al. have shown previously that proteins present in infected cells and in virions form distinct complexes with the telomeric hairpins and that these interactions require the extrahelical bases. The vaccinia virus I6 protein was identified as the protein showing the greatest specificity and affinity for interaction with the viral hairpins (J. DeMasi, S. Du, D. Lennon, and P. Traktman, J. Virol. 75:10090-10105, 2001). To gain insight into the role of I6 in vivo, we generated eight recombinant viruses bearing altered alleles of I6 in which clusters of charged amino acids were changed to alanine residues. One allele (temperature-sensitive I6-12 [tsI6-12]) conferred a tight ts phenotype and was used to examine the stage(s) of the viral life cycle that was affected at the nonpermissive temperature. Gene expression, DNA replication, and genome resolution proceeded normally in this mutant. However, proteolytic processing of structural proteins, which accompanies virus maturation, was incomplete. Electron microscopic studies confirmed a severe block in morphogenesis in which immature, but no mature, virions were observed. Instead, aberrant spherical virions and large crystalloids were seen. When purified, these aberrant virions were found to have normal protein content but to be devoid of viral DNA. We propose that the binding of I6 to viral telomeres directs genome encapsidation into the virus particle.     

Temperate bacteriophages of Streptococcus pneumoniae that contain protein covalently linked to the 5'' ends of their DNA.

We have characterized three temperate bacteriophages of pneumococcus (HB-3, HB-623, and HB-746). Although all the phages belong to the same family, the polypeptide composition of the virions and the DNA restriction endonuclease analysis of their DNAs revealed differences among the three phages. The genomes of these bacteriophages have been isolated as DNA-protein complexes. The protein is specifically associated with the two 5' termini of the DNA as shown by experiments carried out with exonucleases. The protein bound to the DNA in the three phages studied, iodinated in vitro with 125I, has a molecular weight of 23,000 as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Treatment of the complexes with chaotropic agents suggested that the protein is covalently bound to the 5' termini of the DNA. Comparative pulsed-field gel electrophoresis analysis and Southern hybridization of the SmaI restriction fragments of DNAs from one lysogenic bacteria and its parental strain revealed that the prophage genome was integrated in the host chromosome.     

Initiation of adenovirus DNA replication.

In an attempt to study the mechanism of initiation of adenovirus DNA replication, an assay was developed to investigate the pattern of DNA synthesis in early replicative intermediates of adenovirus DNA. By using wild-type virus-infected cells, it was possible to place the origin of adenovirus type 2 DNA replication within the terminal 350 to 500 base pairs from either of the two molecular termini. In addition, a variety of parameters characteristic of adenovirus DNA replication were compared with those obtained in a soluble nuclear extract competent for viral DNA replication. It was observed that in vitro DNA replication, which is dependent on the exogenously added viral DNA-protein complex as its optimal template, occurs in a manner apparently indistinguishable from the situation in virus-infected cells. This includes the presence of proteinaceous material on the molecular termini of newly initiated viral DNA.     

Specific interaction of a protein(s) at or near the termini of adenovirus 2 DNA

When human adenovirus type 2 DNA was extracted from the virions, and the sedimentation profile of the DNA-protein complex in a linear sucrose gradient was analyzed by the nitrocellulose filter binding assay, it was found that 90–95% of adenovirus 2 DNA molecules was tightly associated with the protein(s). The analyses of the binding site of the protein(s) on the DNA by using the restriction endonucleases suggested that the protein(s) interact with the DNA at or near the termini of the viral genome. The stability of the linkage between the DNA and the protein(s) was tested in the presence of various reagents. From the available data, it is suggestive that the protein(s) is linked to the DNA covalently.     

The AAV origin binding protein Rep68 is an ATP-dependent site-specific endonuclease with DNA helicase activity

Genetic studies of adeno-associated virus (AAV) indicate that two AAV genes are required for viral DNA replication: the palindromic terminal repeat, which is the origin for DNA replication, and the rep gene, which codes for a family of at least four viral nonstructural proteins. To determine the biochemical function of the Rep proteins, we have purified the AAV Rep68 protein to apparent homogeneity. We find that it contains a site-specific and strand-specific endonuclease activity that specifically cuts the AAV origin at the terminal resolution site (TRS). The TRS endonuclease requires the presence of ATP for activity and becomes covalently attached to the 5' end at the cut site. In addition to the specific endonuclease activity, Rep68 also contains a DNA helicase activity. These results demonstrate that the large AAV Rep proteins have a direct role in AAV DNA replication; namely, they provide the activities required for the resolution of covalently joined AAV termini.     

Vaccinia virus telomeres: interaction with the viral I1, I6, and K4 proteins

The 192-kb linear DNA genome of vaccinia virus has covalently closed hairpin termini that are extremely AT rich and contain 12 extrahelical bases. Vaccinia virus telomeres have previously been implicated in the initiation of viral genome replication; therefore, we sought to determine whether the telomeres form specific protein-DNA complexes. Using an electrophoretic mobility shift assay, we found that extracts prepared from virions and from the cytoplasm of infected cells contain telomere binding activity. Four shifted complexes were detected using hairpin probes representing the viral termini, two of which represent an interaction with the "flip" isoform and two with the "flop" isoform. All of the specificity for protein binding lies within the terminal 65-bp hairpin sequence. Viral hairpins lacking extrahelical bases cannot form the shifted complexes, suggesting that DNA structure is crucial for complex formation. Using an affinity purification protocol, we purified the proteins responsible for hairpin-protein complex formation. The vaccinia virus I1 protein was identified as being necessary and sufficient for the formation of the upper doublet of shifted complexes, and the vaccinia virus I6 protein was shown to form the lower doublet of shifted complexes. Competition and challenge experiments confirmed that the previously uncharacterized I6 protein binds tightly and with great specificity to the hairpin form of the viral telomeric sequence. Incubation of viral hairpins with extracts from infected cells also generates a smaller DNA fragment that is likely to reflect specific nicking at the apex of the hairpin; we show that the vaccinia virus K4 protein is necessary and sufficient for this reaction. We hypothesize that these telomere binding proteins may play a role in the initiation of vaccinia virus genome replication and/or genome encapsidation.     

The NS-1 polypeptide of minute virus of mice is covalently attached to the 5'' termini of duplex replicative-form DNA and progeny single strands.

When A9 cells are infected with minute virus of mice, a small proportion of the virally coded NS-1 polypeptide becomes covalently attached to newly synthesized viral DNA. Antisera directed against NS-1 will specifically precipitate two forms of monomer duplex replicative-form DNA, multimeric duplex intermediates and progeny single strands, and restriction analysis of the duplex forms in these precipitates reveals that NS-1 is exclusively associated with extended-form conformers of the genomic termini. Pulse-labeled viral DNA, harvested at various times in a highly synchronized infection, can be almost quantitatively precipitated with any one of a series of antisera directed against different protein domains distributed throughout the NS-1 molecule but not with antibodies directed against other viral proteins. In each case the interaction with NS-1 can be shown to involve both termini of duplex DNA and single-strand forms, suggesting that in each case a full-length (83-kilodalton) copy of NS-1 is present. Precipitation of the replicating viral DNA with an antibody directed against a synthetic 16-amino-acid peptide containing the sequence at the extreme carboxy terminus of NS-1 can be quantitatively and specifically inhibited with the immunizing peptide in its unconjugated form, showing that the antibodies responsible for precipitating viral DNA are directed against the NS-1 sequence itself and not against a trace contaminant. Exonuclease digestion studies show that the association effectively blocks the 5' ends of the DNA molecules. Very little (less than 0.1%) of the newly synthesized [35S]methionine-labeled NS-1 made in highly synchronized cells during a 15-min pulse early in infection (6.25 to 6.5 h into the S phase) becomes associated with viral DNA immediately. However, pulse-chase experiments show that later in infection (10 to 13 h into the S phase), when viral DNA replication is reaching its peak, a few percent of the molecules in these preexisting pools of NS-1 do become covalently attached to the newly replicated DNA. Isolated viral DNA-protein complexes labeled with [35S]methionine in this way can be obtained by fractionation of the immunoprecipitated complexes on Sepharose CL4B in sodium dodecyl sulfate. Digestion of the purified complexes with nuclease releases an 83-kilodalton molecule which exactly comigrates with authentic NS-1 in sodium dodecyl sulfate-polyacrylamide gels.     

Polyoma Viral DNA Replicated as a Nucleoprotein Complex in Close Association with the Host Cell Chromatin

Polyoma viral DNA is shown to be replicated in close association with the mouse cell chromatin. Two virus-specific nucleoprotein complexes, designated complex A and B, can be dissociated from the isolated chromatin by gentle homogenization in 0.5 M NaCl. Complex A contains only replicating polyoma (Py) DNA whereas complex B contains only mature Py DNA I. The results show, furthermore, that complex A, containing viral DNA in different stages of replication, and complex B are both nucleoproteins with the same buoyant density. The data presently available suggest that newly synthesized stretches of Py DNA are immediately complexed with mouse cell histones and that complex B becomes the "core" of progeny Py virions. These results suggested that Py-induced replication of the mouse cell chromatin may be necessary to provide replicating Py DNA with histones.     

Hepatitis B virus contains protein attached to the 5′ terminus of its complete DNA strand

Hepatitis B virus DNA contains a tightly bound protein which was not removed by heating to 60°C with 2% SDS, 2% mercaptoethanol. The protein was indirectly demonstrated by the extraction of the DNA-protein complex with phenol before but not after its digestion with proteinase K. The DNA-protein complex had a lower buoyant density than protease-treated or free DNA; it was bound to glass fiber filters; it migrated at a slower rate in gel electrophoresis; and it could be radiolabeled by oxidative iodination. The binding site of the protein was mapped by extraction of restriction endonuclease digests with phenol and analysis of the digests for missing DNA fragments. The protein was localized to a site near the 5′ end of the complete viral DNA strand. It remained attached to this strand after heating with SDS to 90°C or treatment with 0.1 N NaOH, suggesting a covalent linkage. The 5′ end of neither viral DNA strand could be phosphorylated in a reaction with polynucleotide kinase, consistent with attachment of protein to the 5′ ends. The incomplete DNA strand, however, which is the strand elongated by the virion DNA polymerase reaction, did not contain a detectable amount of polypeptide as did the complete strand. The reasons for the apparent block of the 5′ end of the incomplete DNA strand is thus not known. The protein bound covalently to HBV DNA could be involved in the replication of the complete viral DNA strand and/or endonucleolytic generation of linear unit-length DNA pieces from replicative intermediates, although its function and origin are not yet known.     

Fusion of the termini of the murine cytomegalovirus genome after infection.

The genome of murine cytomegalovirus, extracted from extracellular virions, is a linear double-stranded DNA molecule ca. 240 kilobase pairs long. In our initial cloning of subgenomic fragments of the murine cytomegalovirus genome, we obtained a HindIII clone which contained fused HindIII-terminal fragments. By hybridizing this cloned DNA fragment to infected-cell DNA, we identified an intracellular restriction fragment which was the length of the sum of the two authentic termini. This fusion fragment was not present in virion DNA but could be detected as early as 2 h postinfection and reached its highest level shortly after the onset of DNA replication at 16 h postinfection. The prereplicative increase of fused ends was not inhibited by a level of phosphonoacetic acid which effectively shut off viral DNA synthesis, nor was the early conversion from free to fused ends prevented by inhibitors of protein or RNA synthesis. The results are consistent with the fused state of viral DNA being a replicative intermediate and precursor to DNA synthesis.     

Interactions of the RNA polymerase with the viral genome at the 5'- and 3'-ends contribute to 20S RNA narnavirus persistence in yeast

20S RNA narnavirus is a positive strand RNA virus found in the yeast Saccharomyces cerevisiae. The viral genome (2514 nucleotides) only encodes a single protein (p91), the RNA-dependent RNA polymerase and does not have capsid proteins to form intracellular virions. The genomic RNA has no 3' poly(A) tail and perhaps no cap structure at the 5'-end; thus resembling an intermediate of mRNA degradation. The virus, however, escapes the host surveillance and replicates in the yeast cytoplasm persistently. The viral genome is not naked but exists in the form of a ribonucleoprotein complex with p91 in a 1:1 stoichiometry. Here we investigated interactions between p91 and the viral genome. Our results indicate that p91 directly or indirectly interacts with the RNA at the 5'- and 3'-end regions and to a lesser extent at a central part. The 3'-end site is identical to or overlaps with the 3' cis signal for replication identified previously. The 5'-site is at the second stem loop structure from the 5'-end (nucleotides 72-104), and this structure also contains a cis signal for replication. Analysis of mutants in the structure revealed a tight correlation between replication and formation of complexes. These results highlight the importance of ribonucleoprotein complexes for the viral life cycle. We will discuss implications of these findings especially on how the virus escapes from mRNA degradation pathways and resides in the cytoplasm persistently despite the lack of a protective capsid.     

Characterization of a protein covalently linked to the 5' termini of the DNA of Bacillus subtilis phage phi29.

We have isolated a covalent DNA-protein complex from bacteriophage φ29 particles. Polyacrylamide gel electrophoresis and tryptic peptide analysis showed that the protein present in the complex is very similar or identical to p3, an early induced protein essential for viral DNA replication.When the DNA-protein complex is treated with the restriction endonuclease EcoRI, the protein is specifically associated to the two terminal fragments, A and C. The protein is probably linked to the 5′ termini of the DNA since proteinase K-treated DNA is resistant to phosphorylation with polynucleotide kinase, even after treatment with alkaline phosphatase, while it is sensitive to exonuclease III. By electron microscopy the protein is visualized as a dot located at the ends of unit length DNA molecules.Mixed infection of Bacillus subtilis, at 42 °C, with ts² mutants in cistrons 2 and 3 only produces ts 2 progeny. This finding suggests that an inactive protein p3 bound to the DNA of the ts 3 mutant is not replaced by a functional protein and, as a consequence, replication of the ts 3 DNA does not occur.     

Characterization of Moloney murine leukemia virus p12 mutants blocked during early events of infection

Mutations affecting either the N- or C-terminal regions of the Gag protein p12 block replication of Moloney murine leukemia virus (M-MuLV). Viruses carrying mutations in this portion of gag can mediate the assembly and release of virions but are unable to successfully carry out the early phase of the M-MuLV life cycle. Wild-type and mutant viruses were found to synthesize similar levels of linear viral DNA in both cytoplasmic and nuclear fractions, and there were no significant differences in either the density or sedimentation of the viral protein-nucleic acid complexes. Analysis of the termini of the linear viral DNAs showed that the 3' ends of the mutant viral DNA were processed normally by the integrase. Further, the preintegration complexes extracted from the cytoplasm of cells infected with the mutant viruses were competent for integration into target DNA in vitro. Nevertheless, no circular viral DNAs were detected in cells infected by the mutants, and functional proviruses were not formed. These results suggest that p12 has an unexpected role in the early phase of the life cycle and is needed after viral DNA synthesis to deliver the incoming DNA to the correct location and in the appropriate state to permit either circularization or integration of the viral DNA in vivo.     

Replication of adenovirus mini-chromosomes

We have isolated adenovirus origins of DNA replication from both the right and left ends of the genome, which are functional on linear autonomously replicating mini-chromosomes. The mini-chromosomes contain two cloned inverted adenovirus termini and require non-defective adenovirus as a helper. Replicated molecules are covalently attached to protein, and DNA synthesis is initiated at the correct nucleotide even when the origins are not located at molecular ends. The activity of embedded origins leads to the generation of linear minichromosomes from circular or linear molecules. These observations therefore suggest that sequences within the adenovirus origin of replication position the protein priming event at the adenovirus terminus. Experiments investigating the regeneration of deleted viral inverted terminal repeat sequences show a sequence-independent requirement for inverted sequences in this process. This result strongly suggests that repair results from the formation of a panhandle structure by a displaced single strand. On the basis of these observations we propose a model for the generation of adenovirus mini-chromosomes from larger molecules.