Mutagenesis of the Herpesvirus Saimiri Terminal Repeat Region Reveals Important Elements for Virus Production

Mutagenesis of the Herpesvirus Saimiri Terminal Repeat Region Reveals Important Elements for Virus Production

All herpesviruses contain linear, double-stranded DNA genome surrounded by a 100-nm diameter icosahedral protein capsid core consisting of 162 capsomers. (eds), Advances in Leucaemia Research. 1981. Within the TR unit, the region around the cleavage site of the genome appears both necessary and sufficient for virus production. This large size hampers handling and systematic mutagenesis of the virus genome using standard modern molecular biology techniques. This would be the seventh. We have sequenced the entire genome of herpesvirus papio 2 (HVP-2; This broad spectrum of pathogenicity is an interesting feature of the genus Simplexvirus, and comparison of different strains may lead to the identification of simplexvirus genes responsible for neurovirulence.

Van Der Pol B, Warren T, Taylor SN, Martens M, Jerome KR, Mena L, Lebed J, Ginde S, Fine P, Hook EW 3rd (2012) Journal of Clinical Microbiology 50: 3466-3471. Information published on this site is not intended as a substitute for medical advice. That inversion could occur by recombination between different concatemers is suggested by the observation that concatemers generated from different HSV-1 replication templates frequently recombine (54). Sequence analysis of the human and mouse CMV genomes revealed a similar genetic organization and a coding capacity for presumably more than 220 polypeptides (3–5). The viral genomes typically persist in many copies as extrachromosomal nonintegrating episomes and acquire cellular histones to form regular nucleosome-like structures (9, 29, 39). For EHV2 86/67, EHV2 G9/92, and EHV5 2-141/67, 1,617,084, 7,239,808, and 2,411,370 reads were obtained, with the majority (84, 91, and 94%) aligned (using Tanoti) with the sequences at coverage values of 1,812, 8,776, and 3,085 reads per nucleotide, respectively. pHVS15+ efficiently forms plaques upon transfection of permissive owl monkey kidney (OMK) cells, while pHVS15+ΔTR fails to do so (19).

The formation of viral replication compartments in herpes simplex virus forces the host chromatin to the margins of the cell. This redistribution of host chromosomes can be followed by a histone 2B-green fluorescent protein (H2B-GFP) fusion (15). We have observed this phenomenon within OMK cells infected by HVS (not shown) and therefore used H2B-GFP as a surrogate marker for the formation of viral replication compartments. indicates that other cells may also be the primary target or site of latency. 1). Each 25uL reaction contained 1X TaqMan Universal Master Mix, TaqMan probe (10pmoL), and 1uL of DNA template. Helices are rather simple structures formed by stacking repeated components with a constant relationship (amplitude & pitch) to one another – note that if this simple constraint is broken a spiral forms rather than a helix – unsuitable for containing a virus genome.

Lane 1: circular pUC18 plasmid in absence of the nuclease. There are 85 genes, as shown previously, including analyses of HHV-6A GS, plus HHV-7, a distant roseolovirus (13, 21, 22). Of these, some (typical examples are shown) have relocalized the H2B-GFP to the periphery of the nucleus, indicative of replication compartment formation. For contrast, the cell transfected with H2B-GFP alone was the one that most closely resembled the peripheral localization seen in infected cells; most uninfected cells exhibit a more-diffuse H2B-GFP distribution within the nucleus. In order to identify the regions responsible for this defect, we have constructed recombinant BACs in order to complement the deletion of the TRs. Recombinants were made by introducing regions of the TRs into pHVS15+ΔTR, using a RecA-based markerless recombination system described previously (14, 19). To control for second-site mutations during the recombination process, it would be ideal to reconstruct wild-type virus for each mutant; however, the large size of the TR makes it impossible to reconstruct these viruses within the current recombination system.

For each complementing virus, we have therefore made and tested two independent recombinants, as the best alternative to revertants. We constructed a recombinant MCMV containing an additional, ectopic cleavage site and found that it was cleaved efficiently. 2A. Hereby it tethers the viral episome to cellular mitotic chromosomes via an interaction with histone H1 (8). Comparisons of CCV with salmonid herpesviruses appear useful in this respect, since the fossil record indicates that the three main subgroups of euteleosts (salmoniforms, neoteleosts, and ostariophysans, the latter including catfish) diverged around 130 million years ago (1). 2B). The selective association of pac2 with concatemer ends from a variety of herpesviruses led to the proposal that pac2 may define packaging directionality by mediating initiation of concatemer packaging (49) in a manner analogous to that described for bacteriophage λ (14).

Functional equivalence of ectopic and natural cleavage sites should give rise to an equal ratio of the two genome types. For this, the total RNA isolated from HVT-infected CEFs was hybridized with individual miRNA probes. Briefly, 4 ng of each probe (specific activity, 35,000 cpm/ng) was hybridized overnight at 42°C with 20 to 25 μg of total RNA in hybridization buffer and then digested with an RNase A-T1 mixture for 30 min at 37°C. Enzyme sites used to subclone regions are as follows: Bg, BglII; E, EcoRV; F, FspI; N, NotI; B, BssHII; P, PstI; S, SmaI; and A, ApaI. Where letters are struck through, the enzyme site was destroyed in the cloning process. Where noncompatible enzymes are adjacent in the scheme, or enzyme sites are lost, a short artificial linking sequence is present. Positions within the terminal repeat sequence (GenBank accession no.

K03361) are shown for the junctions with the ends of the unique region of the genome (16) and for the smallest region sufficient to mediate packaging. The nucleotide positions of the ends of the L-DNA (GenBank accession no. X64346) are also indicated. “J” represents the sequence to the right of the terminal cleavage site. The left of the cleavage site is indicated by ApaI sites. Unless you have had breast surgery, do not count on perfectly symmetrical breasts. ).

The pKSO-gpt plasmid was linearized with NotI between the two loxP sites and inserted in the NotI site of plasmid pHE′ΔE. Histone acetylation, which is commonly found in context with actively transcribed genes, had previously been investigated with three different antibodies, and investigation revealed that only the H DNA and the latently transcribed stpC/tip promoter were wrapped up with acetylated histones, while the lytic gene promoters, as well as the orf73/lana promoter, remained free of histone acetylation (1). The ability of the construct to produce virus is indicated by − (unable), +++ (near that of wild type), ++ (slightly retarded), or + (substantially retarded), as described for Fig. 3A. (B) Pulsed-field gel of the BAC DNA preparation used in the study, digested with NheI. For each construct, two independently generated recombinants were made and are shown here to be identical. To assess the phenotype of each recombinant, 1 μg of DNA for each BAC was transfected in duplicate into a well of a six-well dish of 80% confluent OMK cells using a Lipofectin-peptide transfection reagent (12, 20).

The cells were incubated in Dulbecco’s modified Eagle medium supplemented with l-glutamine, penicillin and streptomycin, and 2% fetal calf serum (10% fetal calf serum was used to culture OMK cells). The mean times taken to destroy the cell sheets are presented in Fig. All 50 PCR-negative samples were also EBER in situ hybridization negative. (A) Lytic efficiency of recombinant BACs. For each recombinant BAC, one microgram of two independently generated recombinant BACs was transfected in duplicate into OMK cells. The mean time taken to lyse the cell sheet of a well of a six-well plate is shown. Error bars represent one standard deviation above and below the mean.
Mutagenesis of the Herpesvirus Saimiri Terminal Repeat Region Reveals Important Elements for Virus Production

Three recombinant BACs (pHVS15+ΔTR, pHVSΔTR-NS, and pHVSΔTR-NN) failed to form plaques or lyse the cells, so they are not shown here. (B) Schematic and nucleotide resolution scheme comparing HVS and EBV TRs and their cleavage. The EBV genome has been reversed from its conventional orientation so that the EBV and HVS genomes are colinear. Boxes indicate C-rich regions repeated within the TR unit that are adjacent to the cleavage site (vertical arrow/line). The broken box indicates a near repeat of this C-rich region. Boxes enclose predicted components of pac1 and pac2 that have wild-type sequences (solid borders) or contain mutations (dashed borders). The FspI restriction site used to delete the right end of the genome is indicated.

The control plasmid pcDNA-HA-MH was generated by deleting the ORF73 coding sequence from pcDNA-HA73MH by digestion with BamHI and religation. Membranes were routinely reprobed with labeled SalHV-1 DNA in order to locate all SalHV-1 DNA fragments. [4]; GenBank accession no. No exogenous nuclease was added, and to prevent endogenous nuclease activity, 6 μl of 0.5 M EDTA was added prior to incubation on ice for 4 h. The correct sequence for each ectopic site was confirmed by Sanger dideoxy sequencing of DNA isolated from virions as previously described (21). Total RNAs from HVT-infected CEFs (lanes 1) and uninfected CEFs (lanes 2) were separated on a 15% denaturing polyacrylamide gel and probed with [γ-32P]ATP-radiolabeled antisense oligonucleotides to the indicated miRNAs. The resulting plasmid was named pVM7 and expressed ORF57 with a FLAG tag on its C terminus.

The minimum requirement of the BACS closest to wild type in efficiency (pHVSΔTR-NP+1TR) is a single TR (cloned using NotI) and the right end of the L-DNA plus the first 80 bp of the TR. When the TR is subcloned with BssHII (pHVSΔTR-NS+BTR), the productivity of the BAC is slightly impaired. This disrupts the 14-bp element of a set of three tandem sequence duplications (4). Since the BACs containing smaller parts of the TR (pHVSΔTR-NS+BNTR and pHVSΔTR-NS+NΔPTR) have the same functionality, a single functional element that contributes to virus production must lie across the BssHII site. Since pHVSΔTR-NN fails to make virus, we can conclude that the region shared by both of these partial TRs (position 588 to 1044) is both necessary and sufficient for packaging. It contains the site of genome linearization (962/3) but lacks the tandem duplications and the ORF73 binding sites (1118 to 1166) previously identified within the TR (17). Deletion of the junction at the right end of the L-DNA (pHVSΔTR-FS+1TR and pHVSΔTR-FP+1TR) initially resulted in extremely small and slow-growing plaques, but after a while, some plaques spread much more rapidly and dominated the destruction of the cell sheet.

The FspI site used to make this construct is just 8 bp from the H/L-DNA junction (Fig. 3B), so the critical element must be very close to the flank. The solution requires serious doctor without guarantees operation. The translocation, however, also introduced an internal, alternative cleavage site similar to those of the two MCMV mutants described here. Ia and Ib). Three different human HVS-transformed CBL lines, untreated or treated with TSA (1 μg/ml) for different periods of time, were used for whole-RNA extraction using Trizol reagent (Invitrogen). This is consistent with observations for murine gammaherpesvirus 68 amplicons, where a single TR is sufficient for packaging, while two TRs give a higher packaging efficiency (10).

The possible requirement for the right end of the genome offers an interesting comparison with the generation of EBV termini (21). There, an element from the junction of unique sequence and TR at the equivalent end of the genome is duplicated on one terminus of the linear viral DNA. Figure 3B compares the structures of the TRs of HVS and EBV. In both cases, two copies of the C-rich elements at the ‘left’ terminus of the linearized genome are duplicated elsewhere within the repeat unit, albeit with a single base insertion for EBV. Also, the sequence from the junction at the left flank of the TR is important for packaging/linearization, and the C-rich element is repeated here. In EBV, the addition of the element from the TR’s left flank onto the terminus invokes a recombination-based model for conversion from a circular genome to a linear one. Histologic examination of tonsil tissue revealed sheets of large cells with folded or cleaved nuclei and vesicular chromatin (centroblastic polymorphous appearance).

However, the parallels between EBV and HVS suggest that there may be some mechanistic similarity between the TR cleavage processes of the two viruses. Using flanking regions might also represent a common mechanism that prevents genome cleavage at multiple sites within the TR. In order to assess the nature of the virus produced by the transfected BACs, equal titers of the duplicates of the two most productive viruses were used to infect OMK cells. After 40 h, the cells were lysed and genomic DNA extracted. One percent of this DNA was electroporated into Electromax DH10B E. coli (Invitrogen), to recover circular BACs. For each virus, eighteen individual colonies were picked, and plasmid DNA was isolated using standard small-scale methods and analyzed by digestion and pulsed-field gel electrophoresis (Fig.

4). Most of the rescued BACs were observed to be intact, with the exception of variations in the size of the TR band—the rescues have been ordered in Fig. After amplification, 5-μl aliquots of the PCR products were separated by electrophoresis on 1.5% agarose gels and visualized with ethidium bromide and UV light. A minority of BACs have undergone deletions or rearrangements that affect the digestion pattern of the unique region of the genome, as shown to the right of the intact BACs in Fig. For immunofluorescence analysis transfected or infected OMK cells were fixed in 4% paraformaldehyde. Restriction fragments of SalHV-1 DNA or cosmids were transferred from a 0.6% agarose gel and probed with radiolabeledBamHI K, U, or T at 75°C. Analysis of DNA from BACs recovered from OMK cells infected with recombinant virus.

To determine whether the 230-kb GPCMV DNA formed in the presence of BDCRB is protected from nuclease, infected cells were cultured in the presence or absence of BDCRB and hypotonically lysed. (A) Substitutions and nested deletions on the pac1 side of the cleavage site with predicted right ends aligned to the right. The original size of the TR band is indicated by an arrow, and the variable sized band containing the TR array is indicated by a bracket. 1B, top panel). “L” is a DNA size standard, which is a mixture of the ‘lambda mono-cut mix’ and ‘BstEII lambda’ (both from New England Biolabs). The numbers in parentheses after the virus names indicate the identities of the specific BAC clones from which the viruses originated. Presumably, the tendency to increase TR numbers is a consequence of single-TR BACs being less efficient at packaging, being well below the packaging capacity of the virion.

The distribution of repeat numbers at each end of the genome suggests a ‘head-full’ packaging mechanism for the virus, with a fixed size observed for the genome in the virion (16), consistent with studies on EBV-based amplicons, which were packaged most efficiently at the size closest to an apparent maximum of 180 kb (5). This contrasts with rescued episomes, which have no apparent optimal number of repeats. Indeed, rescue of pHVS15+ episomes from infected cells showed a similar variation in TR size. The recircularization of HVS presumably occurs by recombination between TR units on opposite flanks. Unless some mechanism favors repeats nearer the termini, this will naturally result in a variable number of TR units. The study of the latency requirements of HVS is both complicated and simplified by these observations. The tendency of HVS to change TR numbers makes the establishment of latent episomes with specific TR numbers difficult, preventing us from comparing the viral context with observations that three to four repeat units are sufficient to replicate plasmid vectors (2, 8).

However, this also means that a single modified TR unit should produce an equivalent array, simplifying the construction of HVS with a mutant TR array, allowing the analysis of precisely modified TRs in the future. b). In summary, these microarray data show that the latency-related transcriptional nonpermissiveness of large parts of the coding viral genome is reflected at the chromatin level. Comparison of the HVS TR with sequences within the EBV TR leads us to propose some mechanistic similarities between the cleavage of the two virus genomes, despite the different natures of the termini. In addition, we have shown that in the course of lytic replication and genome circularization, single TRs are amplified into multimers.

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