Electron microscopic demonstration of the presence of amplified sequences at the 5''-ends of the polyoma virus late mRNAs.

Electron microscopic techniques were used to examine the structure of the leader sequences at the 5'-ends of the late polyoma virus mRNAs. The three late mRNA's were partially purified and hybridized to an E. coli plasmid containing two polyoma virus genomes inserted in tandem. The hybrids were spread by the cytochrome c-formamide technique and visualized in the electron microscope. These studies revealed that whereas the body of a given mRNA molecule can hybridize with only one of the two corresponding body sequences in the two adjacent viral genomes, the leader of the same mRNA molecule can hybridize with both copies of the leader sequence-specific DNA. The mVP1 and mVP3 RNA species thus generated hybrids containing two loops, while mVP2 molecules formed hybrids containing one loop. Hence, the leaders of the three polyoma virus late mRNA species must contain two or more repeats of a sequence transcribed from a unique DNA segment. Length measurements showed that most leaders in the late mRNA's consist of at least 200 nucleotides and some contain up to 500 nucleotides, whereas the basic repeat sequence contains about 60 nucleotides.     

Hairpin-loop formation by inverted repeats in supercoiled DNA is a local and transmissible property

Short inverted repeat sequences adopt hairpin stem-loop type structures in supercoiled closed circular DNA molecules, demonstrated by S1 nuclease cleavage. Fine mapping of cleavage frequencies is in good agreement with expected cleavage patterns based upon the interaction between an unpaired loop and a sterically bulky enzyme molecule. Whilst the topological properties of underwound DNA circles depend ultimately upon reduced linkage, necessarily a global molecular property, hairpin loop formation is an essentially local property. Thus molecular size is unimportant for the S1 hypersensitivity of the Co1E1 inverted repeat. Furthermore, a 440 bp Sau3AI, EcoRI fragment of Co1E1 which contains the inverted repeat has been cloned into pBR322 whereupon it exhibits S1 cleavage similar to Co1E1 in the supercoiled recombinant molecule. The effect is therefore both local and transmissible. Direct competition, between inverted repeats in the recombinant, coupled with close examination of flanking sequences, enables some simple 'rules' for base pairing in hairpin loops to be formulated. Whilst limited G-T and A-C base pairing appears not to be destabilising, A-G, T-C or loop outs are highly destabilising.     

Polyoma virus defective DNAs. II. Physical map of a molecule with rearranged and reiterated sequences (D74).

The structure of the polyoma virus defective species D74 (74% the size of full-length polyoma virus DNA) has been determined and compared with that of polyoma virus A2 DNA. D74 appears to be composed entirely of viral DNA sequences. (No host DNA sequences have been detected.) It is made up of three DNA segments, each about 24, 24 and 27% in size. The two 24% segments appear to be identical and the 27% segment contains one copy of all the sequences found in the 24% fragments as well as a duplication of some of the sequences. When related to the physical map of A2 DNA, each segment is found to be composed of viral sequences from 1 to about 19 map units, 67 to 69 map units and 70 to 72 map units.Three features found in other polyoma virus defective species (Lund et al., 1977) are also present in D74. (1) Sequences from the region around 67 map units are linked to other (non-contiguous) viral sequences. (2) Sequences at about 72 map units are linked to sequences at 1 map unit. (3) Multiple copies of sequences from around the origin of viral DNA replication are present. From studies on other polyoma defective molecules (Griffin &; Fried, 1975; Lund et al., 1977), the origin of DNA replication for polyoma virus has been defined to lie within the sequences from 67 to 72 map units. Since D74 replicates efficiently but lacks the sequences between 69 to 70 map units, the origin of DNA replication appears to be further defined as lying within 67 and 69 map units and/or 70 to 72 map units.     

The infectivity of adenovirus genomes lacking DNA sequences from their left-hand termini.

Deletions extending various distances into the left-hand terminal DNA sequences of the adenovirus type 2 (Ad2) genome were generated in a plasmid containing a cloned fragment spanning from 0 to 4.9 map units. The altered Ad2 DNA sequences were introduced into viral genomes by ligating a plasmid-derived fragment, which included the sequences extending to 3.8 map units, to the 3.8-100 map unit fragment generated by XbaI cleavage of the DNA of the Ad5 variant, d1309 (N.Jones and T.Shenk, Cell 17 683-689, 1979). The infectivity of the ligation products was studied by transfection of line 293 cells. Genomes lacking 11, 40, or 51 nucleotides from their left-hand termini, or containing an additional 18dG residues linked to this position were infectious, and analysis of the progeny virus genomes demonstrated that the structure of these modified termini had been restored to normal. In contrast, genomes from which the first 160 base pairs (bp), including the entire 102 bp left hand inverted terminal repeat (ITR), had been removed were non-infectious. The results indicate that the ITRs present at the opposite ends of transfecting DNA molecules are able to interact in vivo, and enable the production of viable viruses containing corrected left-hand terminal sequences. Possible mechanisms for this interaction are discussed.     

Sequence and hairpin structure of an inverted repeat series at termini of the Physarum extrachromosomal rDNA molecule

The termini of the 61 kb palindromic rDNA molecules of Physarum polycephalum possess a series of multiple inverted repeats in which are located specific single-strand gaps and tightly attached protein. After treating rDNA with S1 nuclease, we have cloned several 5 kb Eco RI terminal restriction fragments. Sequencing of more than 800 nucleotides from the end of one such clone reveals the presence of six to ten tandemly repeated units averaging 140 +/- 4 bp in length and flanked by Hae III sites. Each 140 nucleotide repeat unit can form thermodynamically stable hairpin structures based on complex internal palindromic components. When the specific gap sequence CCCTA is present, it is located near the apex of a hairpin component. These secondary structures are formed in growing plasmodia, as seen in electron micrographs of native rDNA molecules, which also reveal apparent recombination forms involving rDNA ends and noncontiguous DNA segments. Recombination initiated at terminal single-strand hairpin loops can result in genetic exchange of ribosomal gene sequences and can lead to completion of 5' nucleotide sequences at ends of newly replicated rDNA molecules.     

Mutational analysis of the influenza virus cRNA promoter and identification of nucleotides critical for replication

Replication of the influenza A virus virion RNA (vRNA) requires the synthesis of full-length cRNA, which in turn is used as a template for the synthesis of more vRNA. A "corkscrew" secondary-structure model of the cRNA promoter has been proposed recently. However the data in support of that model were indirect, since they were derived from measurement, by use of a chloramphenicol acetyltransferase (CAT) reporter in 293T cells, of mRNA levels from a modified cRNA promoter rather than the authentic cRNA promoter found in influenza A viruses. Here we measured steady-state cRNA and vRNA levels from a CAT reporter in 293T cells, directly measuring the replication of the authentic influenza A virus wild-type cRNA promoter. We found that (i) base pairing between the 5' and 3' ends and (ii) base pairing in the stems of both the 5' and 3' hairpin loops of the cRNA promoter were required for in vivo replication. Moreover, nucleotides in the tetraloop at positions 4, 5, and 7 and nucleotides forming the 2-9 base pair of the 3' hairpin loop were crucial for promoter activity in vivo. However, the 3' hairpin loop was not required for polymerase binding in vitro. Overall, our results suggest that the corkscrew secondary-structure model is required for authentic cRNA promoter activity in vivo, although the precise role of the 3' hairpin loop remains unknown.     

Mung bean nuclease cleavage of a dA + dT-rich sequence or an inverted repeat sequence in supercoiled PM2 DNA depends on ionic environment

We have determined the nucleotide sequences around two alternative sites cleaved in supercoiled PM2 DNA by single-strand-specific mung bean nuclease in different ionic environments. In 10 mM Tris-HC1 (pH 7.0, 37 degrees C), the major site is a dA+dT-rich sequence which maps with a known early denaturation region at 0.75 map units. About 30 cleavages occurred in a 135 bp region. Cleavages were largely excluded at (dA)n . (dT)n (n = 3-7) sequences. Cleavage patterns of this type have not been previously observed in dA+dT-rich sequences. With the addition of 0.1 M NaC1 the major alternative site occurred in a hyphenated inverted repeat sequence 500 bp away (0.70 map units) and did not map to an early denaturation region. One major and 4 minor cleavages occurred in the region between the repeats, suggesting that a hairpin containing at most a 12 bp stem and 10 base loop is recognized. The basis for nuclease recognition of the dA+dT-rich sequence is not clear. The differences in the sequences and cleavage patterns at the alternative sites indicate that their secondary structures differ.     

Polyoma virus defective DNAs. I. Physical maps of a related set of defective molecules (D76, D91, D92).

Three related polyoma virus species, designated D92 (92% the size of full-length polyoma virus DNA), D91 (91%) and D76 (76%) have been analysed and their structures compared with that of polyoma virus A2 DNA. Three independent methods (restriction endonuclease cleavage, depurination fingerprinting and DNA-DNA hybridization) were used in the analysis.The defective DNAs appear to be: (1) entirely composed of viral sequences (no host DNA sequences were detected): (2) made up in part of long continuous sequences of DNA which appear identical to sequences of A2 DNA (D92 contains continuous sequences from 1 to 72 map units on the physical map of A2 DNA; that is, it contains the entire late region and part of the early region of the viral DNA. D91 and D76 contain those same sequences except for a 1% deletion around 18 map units): (3) made up in part of rearranged viral sequences.Several interesting features were noted about the rearranged sequences present in the defective DNAs. Sequences from the region around 67 map units were found linked to other (non-contiguous) regions of the DNA. Sequences from about 72 map units were linked to sequences from about 1 map unit. Multiple copies of sequences from 67 to 72 map units (from around the origin of DNA replication) were found (4 copies in D91 and D92, and 2 copies in D76).     

Electron microscopic study of equine herpesvirus type 1 DNA.

Electron microscopic studies of equine herpesvirus DNA revealed that single strands that were allowed to reanneal formed single-stranded loops with double-stranded stems only at one end of the molecule. These observations support restriction enzyme analyses which indicate that the 92-megadalton DNA molecule exists as a long region of unique sequences covalently linked to a short region. The short region is comprised of an internal unique sequence, which forms the loop during reannealing of single strands, and two terminal inverted repeat sequences that bracket the unique sequence and form the double-stranded stem structure observed upon reannealing of single strands. Measurements of the unique sequence and terminal inverted repeat subgenomic sequences indicate a size of 6.4 megadaltons for each and thus fix the size of the short region at approximately 19.2 megadaltons.     

A nearby inverted repeat of the terminal sequence of herpes simplex virus DNA.

When herpes simplex virus DNA is digested with λ-exonuclease, annealed, and then mounted for observation in the electron microscope, two types of molecules are seen. One type is circular DNA which forms because the enzyme has revealed the terminal repetition. The other type is full length linear duplex DNA with a short single-stranded loop on one end. This latter type, which apparently represents a fold-back reaction, forms because there is a nearby inverted repeat of the terminal sequence of herpes simplex virus DNA.     

Hairpin loop structure of African swine fever virus DNA.

The ends of African swine fever virus genome are formed by a 37 nucleotide-long hairpin loop composed, almost entirely, of incompletely paired A and T residues. The loops at each DNA end were present in two equimolar forms that, when compared in opposite polarities, were inverted and complementary (flip-flop), as in the case of poxvirus DNA. The hairpin loops of African swine fever and vaccinia virus DNAs had no homology, but both DNAs had a 16 nucleotide-long sequence, close to the hairpin loops, with an homology of about 80%. An analysis of African swine fever virus replicating DNA showed head-to-head and tail-to-tail linked molecules that may be replicative intermediates.     

Genomic structure of human polyoma virus JC: nucleotide sequence of the region containing replication origin and small-T-antigen gene

The nucleotide sequence of the region of human polyoma virus JC DNA between 0.5 and 0.7 map units from a unique EcoRI cleavage site was determined and compared with those of the corresponding regions of another human polyoma virus, BK, and simian virus 40 DNAs. Within this region consisting of 945 base pairs, we located the origin of DNA replication near 0.7 map units, the entire coding region for small T antigen, and the splice junctions for large-T-antigen mRNA. The deduced amino acid sequences for small T antigen and the part of large T antigen markedly resembled those of polyoma virus BK and simian virus 40. The results strongly suggest that polyoma virus JC has the same organization of early genome as polyoma virus BK and simian virus 40 on the physical map, with the EcoRI site as a reference point.     

Polyoma infected cells contain at least three spliced late RNAs.

Poly(A)-containing polyoma cytoplasmic RNA was hybridized with linear double-stranded polyoma DNA and RNA displacement loops (R-loops) were formed. The structures visualized in the electron microscope are consistent with the conclusion that there are at least three late polyoma specific RNAs and that the leader sequences at the 5' ends of these viral RNAs are not coded immediately adjacent to the bodies of the RNAs. Measurements carried out on the R-loop structures have provided the locations on the physical map of polyoma DNA, for the bodies and leaders of the RNAs and the length of the bodies, leaders and the corresponding intervening DNA sequences.     

Functional mapping and DNA sequence of an equine herpesvirus 1 origin of replication.

The genome of equine herpesvirus 1 (EHV-1) defective interfering (DI) particle DNA originates from discrete regions within the standard (STD) EHV-1 genome: the left terminus (0.0 to 0.04 map units) and the inverted repeats (0.78 to 0.79 and 0.83 to 0.87 map units of the internal inverted repeat; 0.91 to 0.95 and 0.99 to 1.00 map units of the terminal inverted repeat). Since DI DNA must contain cis-acting DNA sequences, such as replication origins, which cannot be supplied in trans by the STD EHV-1 virus, regions of the EHV-1 genome shown to be in DI DNA were assayed for the presence of a viral origin of DNA replication. Specifically, STD EHV-1 DNA fragments encompassing the genomic regions present in DI particle DNA were inserted into the vector pAT153, and individual clones were tested by transfection assays for the ability to support the amplification and replication of plasmid DNA in EHV-1-infected cells. The Sma-1 subfragment of the internal inverted repeat sequence (0.83 to 0.85 map units) was shown to contain origin of replication activity. Subcloning and BAL 31 deletion analysis of the 2.35-kilobase-pair (kbp) Sma-1 fragment delineated a 200-bp fragment that contained origin activity. The origin activities of all EHV-1 clones which were positive by the transfection assay were confirmed by methylation analysis by using the methylation-sensitive restriction enzymes DpnI and MboI. DNA sequencing of the 200-bp fragment which contained an EHV-1 origin of replication indicated that this region has significant homology to previously characterized origins of replication of human herpesviruses. Furthermore, comparison of known origin sequences demonstrated that a 9-bp sequence, CGTTCGCAC, which is conserved among all origins of replication of human lytic herpesviruses and which is contained within the 18-bp region in herpes simplex virus type 1 origins shown by others to be protected by an origin-binding protein (P. Elias, M. E. O'Donnell, E. S. Mocarski, and I. R. Lehman, Proc. Natl. Acad. Sci. USA 83:6322-6326) is also conserved across species in the EHV-1 origin of replication.     

Efficient resolution of replicated poxvirus telomeres to native hairpin structures requires two inverted symmetrical copies of a core target DNA sequence.

The terminal hairpin sequences of the linear double-stranded DNA genome of the leporipoxvirus Shope fibroma virus (SFV) has been cloned in Saccharomyces cerevisiae and in recombination-deficient Escherichia coli as a palindromic insert within circular plasmid vectors. This sequence configuration is equivalent to the inverted repeat structure detected as a telomeric replicative intermediate during poxvirus replication in vivo. Previously, it has been shown that when circular plasmids containing this palindromic insert were transfected into SFV-infected cells, efficient replication and resolution generated linear minichromosomes with bona fide viral hairpin termini (A. M. DeLange, M. Reddy, D. Scraba, C. Upton, and G. McFadden, J. Virol. 59:249-259, 1986). To localize the minimal target DNA sequence required for efficient resolution, a series of staggered unidirectional deletions were constructed at both ends of the inverted repeat. Analyses of the resolution efficiencies of the various clones indicate that up to 240 base pairs (bp) centered at the symmetry axis were required for maximal resolution to minichromosomes. To investigate the role of the AT-rich central axis sequences, which in SFV include 8 nonpalindromic bp, a unique AflII site at the symmetry axis was exploited. Bidirectional deletions extending from this AflII site and insertions of synthetic oligonucleotides into one of the deletion derivatives were constructed and tested in vivo. The efficiency with which these plasmids resolved to linear minichromosomes with hairpin termini has enabled us to define the minimal target DNA sequence as two inverted copies of an identical DNA sequence between 58 and 76 bp in length. The nonpalindromic nucleotides, which, after resolution, constitute the extrahelical residues characteristic of native poxviral telomeres, were not required for resolution. The close resemblance of the SFV core target sequence to the analogous region from the orthopoxvirus vaccinia virus is consistent with a conserved mechanism for poxviral telomere resolution.     

Polyoma Virus DNA: Complete Nucleotide Sequence of the Gene Which Codes for Polyoma Virus Capsid Protein VP1 and Overlaps the VP2/VP3 Genes

The nucleotide sequence of part of the late region of the polyoma virus genome was determined. It contains coding information for the major capsid protein VP1 and the C-terminal region of the minor proteins VP2 and VP3. In the sequence with the same polarity as late mRNA's, all coding frames are blocked by termination codons in a region around 48 units on the physical map. This is the region where the N-terminus of VP1 and the C-termini of VP2 and VP3 have been located (T. Hunter and W. Gibson, J. Virol. 28:240-253, 1978; S. G. Siddell and A. E. Smith, J. Virol. 27:427-431, 1978; Smith et al., Cell 9:481-487, 1976). There are two long uninterrupted coding frames in the late region of polyoma virus DNA. One lies at the 5' end of the sequence and contains potential coding sequences for VP2 and VP3. The other contains 383 consecutive sense codons starting with the ATG at nucleotide position 1,218, extends from 47.5 to 25.8 units counterclockwise on the physical map, and is located where the VP1 gene has been mapped. The VP1 gene overlaps the genes for proteins VP2/VP3 by 32 nucleotides and uses a different coding frame. From the DNA sequence, the amino acid sequence of VP1 was predicted. The proposed VP1 sequence is in good agreement with other data, namely, with the partial N-terminal amino acid sequence and the total amino acid composition. The VP1 coding frame terminates with a TAA codon at 25.8 map units. This is followed by an AATAAA sequence, which may act as a processing signal for the viral late mRNA's. When both nucleotide and amino acid sequences are compared with their counterparts in the related simian virus 40, extensive homologies are found over the entire region of the two viral genomes. Maximum homology appears to occur in those regions which code for the C-termini of the VP1 proteins. The overlap region of VP1 with VP2/VP3 of polyoma virus is shorter by 90 nucleotides than is that of simian virus 40 and shows very limited homology with the simian virus 40 sequence. This leads to the suggestion that the overlap segments of both viruses have been freed from stringency imposed on drifting during evolution and that proteins VP2 and VP3 of polyoma virus may have been truncated by the appearance of a termination codon within the sequence.