The genome of a very virulent Marek's disease virus

Here we present the first complete genomic sequence, with analysis, of a very virulent strain of Marek's disease virus serotype 1 (MDV1), Md5. The genome is 177,874 bp and is predicted to encode 103 proteins. MDV1 is colinear with the prototypic alphaherpesvirus herpes simplex virus type 1 (HSV-1) within the unique long (UL) region, and it is most similar at the amino acid level to MDV2, herpesvirus of turkeys (HVT), and nonavian herpesviruses equine herpesviruses 1 and 4. MDV1 encodes 55 HSV-1 UL homologues together with 6 additional UL proteins that are absent in nonavian herpesviruses. The unique short (US) region is colinear with and has greater than 99% nucleotide identity to that of MDV1 strain GA; however, an extra nucleotide sequence at the Md5 US/short terminal repeat boundary results in a shorter US region and the presence of a second gene (encoding MDV097) similar to the SORF2 gene. MD5, like HVT, encodes an ICP4 homologue that contains a 900-amino-acid amino-terminal extension not found in other herpesviruses. Putative virulence and host range gene products include the oncoprotein MEQ, oncogenicity-associated phosphoproteins pp38 and pp24, a lipase homologue, a CxC chemokine, and unique proteins of unknown function MDV087 and MDV097 (SORF2 homologues) and MDV093 (SORF4). Consistent with its virulent phenotype, Md5 contains only two copies of the 132-bp repeat which has previously been associated with viral attenuation and loss of oncogenicity.     

Regions of the terminal repetitions of the herpes simplex virus type 1 genome. Relationship to immunoglobulin switch-like DNA sequences

Several recombinant clones isolated from a mouse genomic library were previously shown to hybridize with a SmaI fragment located in the terminal repetition of the S component of herpes simplex virus DNA. We report here the nucleotide sequence of the related regions in two mouse clones, TGL19 and TGL35, as well as that of the SmaI fragment of HSV-1. The mouse DNA clones have a core of repetitive sequences 80% homologous to a tandem repeat (reiteration II) in the viral fragment. The regions of homology are in turn related to immunoglobulin class-switch sequences, due mostly to the presence of the pentamer TGGG(G), involved in class-switch recombination. These results suggest that the HSV genome has recombination sequences identical to those of the host cell and provide a possible explanation for the high frequency of recombination events observed in this region of the viral genome.     

Comparison of the DNA sequence and secondary structure of the herpes simplex virus L/S junction and the adeno-associated virus terminal repeat

The defective parvovirus Adeno-associated virus (AAV) is absolutely dependent upon coinfection with either Adenovirus or Herpes Simplex Virus (HSV) for its multiplication. We have compared the terminal repeats of HSV-1F strain DNA with the terminal 200 nucleotides of AAV DNA. Our findings demonstrate similarities between portions of the HSV inverted repeats found at the L/S junction and the termini of AAV. By computer analysis we have determined potential secondary folding patterns for both genomes. The following points can be made about the a, b, and c repeats in HSV: (1) Regions b and c are complementary over a significant portion of their length. (2) The ends of a can fold back on themselves to form large secondary structures. Moreover, when the b and c homology is used to align the ends of a, the b/a and c/a junctions are within 1 base of each other. (3) The short direct repeats within a are essentially a large loop with little secondary structure. The potential implications of this structure are discussed and a model for HSV DNA replication is presented.     

The a sequence is dispensable for isomerization of the herpes simplex virus type 1 genome.

The herpes simplex virus type 1 (HSV-1) genome consists of two components, L (long) and S (short), that invert relative to each other during productive infection to generate four equimolar isomeric forms of viral DNA. Recent studies have indicated that this genome isomerization is the result of DNA replication-mediated homologous recombination between the large inverted repeat sequences that exist in the genome, rather than site-specific recombination through the terminal repeat a sequences present at the L-S junctions. However, there has never been an unequivocal demonstration of the dispensability of the latter element for this process using a recombinant virus whose genome lacks a sequences at its L-S junctions. This is because the genetic manipulations required to generate such a viral mutant are not possible using simple marker transfer, since the cleavage and encapsidation signals of the a sequence represent essential cis-acting elements which cannot be deleted outright from the viral DNA. To circumvent this problem, a simple two-step strategy was devised by which essential cis-acting sites like the a sequence can be readily deleted from their natural loci in large viral DNA genomes. This method involved initial duplication of the element at a neutral site in the viral DNA and subsequent deletion of the element from its native site. By using this approach, the a sequence at the L-S junction was rendered dispensable for virus replication through the insertion of a second copy into the thymidine kinase (TK) gene of the viral DNA; the original copies at the L-S junctions were then successfully deleted from this virus by conventional marker transfer. The final recombinant virus, HSV-1::L-S(delta)a, was found to be capable of undergoing normal levels of genome isomerization on the basis of the presence of equimolar concentrations of restriction fragments unique to each of the four isomeric forms of the viral DNA. Interestingly, only two of these genomic isomers could be packaged into virions. This restriction was the result of inversion of the L component during isomerization, which prevented two of the four isomers from having the cleavage and encapsidation signals of the a sequence in the TK gene in a packageable orientation. This phenomenon was exploited as a means of directly measuring the kinetics of HSV-1::L-S(delta)a genome isomerization. Following infection with virions containing just the two packaged genomic isomers, all four isomers were readily detected at a stage in infection coincident with the onset of DNA replication, indicating that the loss of the a sequence at the L-S junction had no adverse effect on the frequency of isomerization events in this virus. These results therefore validate the homologous recombination model of HSV-1 genome isomerization by directly demonstrating that the a sequence at the L-S junction is dispensable for this process. The strategy used to remove the a sequence from the HSV-1 genome in this work should be broadly applicable to studies of essential cis-acting elements in other large viral DNA molecules.     

Short, duplicated sequence indicative of the recombinogenicity of the junction between a unique and an inverted repeat sequence in the S component of the herpes simplex virus type 1 genome.

A herpes simplex virus type 1 (HSV-1) strain, B3, was found to have a short duplication on the left junction between the unique sequence (US) and the inverted repeat sequence (RS) in the S component of the genome DNA. A short region of RS contiguous to the left US-RS junction was duplicated in B3. Based on the nucleotide sequences in and around the US-RS junctions of B3 and other HSV-1 strains, a concept of junction stretch was proposed. The organization of junction stretch is RS side 5'-(G or A stretch)AGC-3' US side. Introduction of the concept of junction stretch led to a definition of the structure in and around the US-RS junction, in the form common to HSV-1 strains. The right end of US in the HSV-1 genome was the A of the ATG initiation codon of gene US12, and thus the ATG triplet may act as a buffer to prevent expansion of RS, as is the case with HSV-2. The duplication in B3 was generated by a crossover event between a point on RS and the US side end of the left junction stretch. These observations suggest that the US side end of the junction stretch possesses the property of recombinogenicity, responsible for generation of the duplication in strain B3 and also for the formation of the US-RS junction of HSV.     

Complete Genome Sequence of a Recombinant Marek's Disease Virus Field Strain with One Reticuloendotheliosis Virus Long Terminal Repeat Insert

Marek''s disease virus (MDV) Chinese strain GX0101, isolated in 2001 from a vaccinated flock of layer chickens with severe tumors, was the first reported recombinant MDV field strain with one reticuloendotheliosis virus (REV) long terminal repeat (LTR) insert. GX0101 belongs to very virulent MDV (vvMDV) but has higher horizontal transmission ability than the vvMDV strain Md5. The complete genome sequence of GX0101 is 178,101 nucleotides (nt) and contains only one REV-LTR insert at a site 267 nt upstream of the sorf2 gene. Moreover, GX0101 has 5 repeats of a 217-nt fragment in its terminal repeat short (TRS) region and 3 repeats in internal repeat short (IRS) region, compared to the other 10 strains with only 1 or 2 repeats in both TRS and IRS.     

Recombinogenic properties of herpes simplex virus type 1 DNA sequences resident in simian virus 40 minichromosomes.

In a previous work, it was demonstrated that the bacterial transposon Tn5 is capable of undergoing sequence inversion via recombination between its duplicated IS50 elements when replicated by the herpes simplex virus type 1 (HSV-1) origin oris but not by the simian virus 40 (SV40) origin orisv. Further analysis of the latter phenomenon indicated that this lack of recombination was the result of topological constraints imposed by the SV40 minichromosome, such that recombination events could be readily detected in Tn5 derivatives in which the IS50 elements were arranged in a direct rather than inverted orientation. With this information, a second set of experiments were carried out to examine how the highly recombinogenic sequences which mediate the inversion of the long (L) and short (S) components of the HSV-1 genome behave in an SV40 minichromosome. Tandem copies of the L-S junction of the HSV-1 genome were observed to promote deletions in an SV40 shuttle plasmid at a frequency that was considerably greater than that of duplicated bacterial plasmid vector DNA. However, the presence of superinfecting HSV-1 did not enhance the frequency of these recombination events. These results support our previous findings that HSV-1 genome isomerization is mediated by a homologous recombination mechanism which is intimately associated with the act of viral DNA synthesis. Moreover, they demonstrate that the sequences which comprise the L-S junction appear to be inherently recombinogenic and, therefore, do not contain specific signals required for HSV-1 genome isomerization.     

The genome of turkey herpesvirus

Here we present the first complete genomic sequence of Marek's disease virus serotype 3 (MDV3), also known as turkey herpesvirus (HVT). The 159,160-bp genome encodes an estimated 99 putative proteins and resembles alphaherpesviruses in genomic organization and gene content. HVT is very similar to MDV1 and MDV2 within the unique long (UL) and unique short (US) genomic regions, where homologous genes share a high degree of colinearity and their proteins share a high level of amino acid identity. Within the UL region, HVT contains 57 genes with homologues found in herpes simplex virus type 1 (HSV-1), six genes with homologues found only in MDV, and two genes (HVT068 and HVT070 genes) which are unique to HVT. The HVT US region is 2.2 kb shorter than that of MDV1 (Md5 strain) due to the absence of an MDV093 (SORF4) homologue and to differences at the UL/short repeat (RS) boundary. HVT lacks a homologue of MDV087, a protein encoded at the UL/RS boundary of MDV1 (Md5), and it contains two homologues of MDV096 (glycoprotein E) in the RS. HVT RS are 1,039 bp longer than those in MDV1, and with the exception of an ICP4 gene homologue, the gene content is different from that of MDV1. Six unique genes, including a homologue of the antiapoptotic gene Bcl-2, are found in the RS. This is the first reported Bcl-2 homologue in an alphaherpesvirus. HVT long repeats (RL) are 7,407 bp shorter than those in MDV1 and do not contain homologues of MDV1 genes with functions involving virulence, oncogenicity, and immune evasion. HVT lacks homologues of MDV1 oncoprotein MEQ, CxC chemokine, oncogenicity-associated phosphoprotein pp24, and conserved domains of phosphoprotein pp38. These significant genomic differences in and adjacent to RS and RL regions likely account for the differences in host range, virulence, and oncogenicity between nonpathogenic HVT and highly pathogenic MDV1.     

Activation of the human immunodeficiency virus long terminal repeat by herpes simplex virus type 1 is associated with induction of a nuclear factor that binds to the NF-kappa B/core enhancer sequence.

It has been previously shown that herpes simplex virus type 1 (HSV-1) infection of HeLa cells results in augmentation of gene expression directed by the human immunodeficiency virus (HIV) long terminal repeat (LTR). This effect is presumably mediated by protein interactions with the LTR. We have used two different assays of DNA-protein interactions to study the HSV-induced activation of the HIV LTR. Activation of the HIV LTR is associated with increased protein binding to LTR sequences in a region including the NF-kappa B/core enhancer and the Sp1 binding sequences as monitored by an exonuclease protection assay. Gel retardation assays demonstrated that HSV-1 infection resulted in the induction of a nuclear factor(s) that binds to the NF-kappa B/core enhancer sequence. In addition to the activation of the HIV LTR, HSV induction of NF-kappa B activity may be important for the regulation of HSV gene expression during a herpesvirus infection.     

Nucleotide sequence and structural features of a novel US-a junction present in a defective herpes simplex virus genome.

Defective genomes generated during serial propagation of herpes simplex virus type 1 (Justin) consist of tandem reiterations of sequences that are colinear with a portion of the S component of the standard viral genome. We determined the structure of the novel US-a junction, at which the US sequences of one repeat unit join the a sequences of the adjacent repeat unit. Comparison of the nucleotide sequence at this junction with the nucleotide sequence of the corresponding US region of the standard virus genome indicated that the defective genome repeat unit arose by a single recombinational event between an L-S junction a sequence and the US region. The recombinational process might have been mediated by limited sequence homology. The sequences retained within the US-a junction further define the signal for cleavage and packaging of viral DNA.     

Murine mammary tumor virus pol-related sequences in human DNA: characterization and sequence comparison with the complete murine mammary tumor virus pol gene.

Sequences in the human genome with homology to the murine mammary tumor virus (MMTV) pol gene were isolated from a human phage library. Ten clones with extensive pol homology were shown to define five separate loci. These loci share common sequences immediately adjacent to the pol-like segments and, in addition, contain a related repeat element which bounds this region. This organization is suggestive of a proviral structure. We estimate that the human genome contains 30 to 40 copies of these pol-related sequences. The pol region of one of the cloned segments (HM16) and the complete MMTV pol gene were sequenced and compared. The nucleotide homology between these pol sequences is 52% and is concentrated in the terminal regions. The MMTV pol gene contains a single long open reading frame encoding 899 amino acids and is demarcated from the partially overlapping putative gag gene by termination codons and a shift in translational reading frame. The pol sequence of HM16 is multiply terminated but does contain open reading frames which encode 370, 105, and 112 amino acid residues in separate reading frames. We deduced a composite pol protein sequence for HM16 by aligning it to the MMTV pol gene and then compared these sequences with other retroviral pol protein sequences. Conserved sequences occur in both the amino and carboxyl regions which lie within the polymerase and endonuclease domains of pol, respectively.     

The herpes simplex virus type 1 (HSV-1) a sequence serves as a cleavage/packaging signal but does not drive recombinational genome isomerization when it is inserted into the HSV-2 genome.

We inserted the terminal repeat (a sequence) of herpes simplex virus type 1 (HSV-1) strain KOS into the tk gene of HSV-2 strain HG52 in order to assess the ability of the HSV-1 a sequence to provoke genome isomerization events in an HSV-2 background. We found that the HSV-1 a sequence was cleaved by the HSV-2 cleavage/packaging machinery to give rise to novel genomic termini. However, the HSV-1 a sequence did not detectably recombine with the HSV-2 a sequence. These results demonstrate that the viral DNA cleavage/packaging system contributes to a subset of genome isomerization events and indicate that the additional recombinational inversion events that occur during infection require sequence homology between the recombination partners.     

The DNA replication origins of herpes simplex virus type 1 strain Angelotti

The nucleotide sequences of the origins of DNA replication (ori) of the S- and L-component (oriS, oriL) of the herpes simplex virus type 1 (HSV-1) standard genome were determined from HSV-1 strain Angelotti (ANG). In contrast to other HSV-1 strains, the ANG oriS sequence revealed an insertion of an TA-dinucleotide in an otherwise very conserved but imperfect palindromic sequence of 47 bp. The oriL sequence of the standard ANG genome was found to be identical to that of an ANG class II defective genome which exhibits a duplication of a 144 bp palindrome. A model is presented to explain the origination of the amplified ANG oriL sequences from the parental genome with a single copy of oriL via illegitimate recombination. Alignment of the ori sequences of HSV, adeno- and papovaviruses unveiled that the HSV ori region can be subdivided into two distinct sites of homology to the DNA initiation signals of papova- and adenoviruses, suggesting that the HSV origins of replication comprise elements for DNA replication by both, cellular and virus-encoded DNA polymerases.     

The structure of Marek's disease virus DNA: amplification of repeat sequence in IRs and TRs

The structure of Marek's disease virus (MDV) DNA was investigated by using Southern blot hybridization analysis. A heterogeneous region was observed in the inverted repeats region, IRs and TRs, as well as in the TRL and IRL. The results of DNA sequencing of the heterogeneous region showed that the heterogeneity of IRS and TRS was due to amplification of a 178-bp repeat sequence. Amplification of IRS and TRS was found in viral DNA from both pathogenic and nonpathogenic strains. The structure of DNA from the latent MDV genome present in established lymphoblastic cells was also determined. Amplification of the 132-bp repeat sequence in IRL and TRL was not found in latent MDV DNA of established lymphoblastic cells, whereas amplification of the 178-bp repeat sequence in IRS and TRS was found in the same DNA.     

The alpha sequence of the cytomegalovirus genome functions as a cleavage/packaging signal for herpes simplex virus defective genomes.

Although herpes simplex virus (HSV) 1 and human cytomegalovirus (CMV) differ remarkably in their biological characteristics and do not share nucleotide sequence homology, they have in common a genome structure that undergoes sequence isomerization of the long (L) and short (S) components. We have demonstrated that the similarity in their genome structures extends to the existence of an alpha sequence in the CMV genome as previously defined for the HSV genome. As such, the alpha sequence is predicted to participate as a cis-replication signal in four viral functions: (i) inversion, (ii) circularization, (iii) amplification, and (iv) cleavage and packaging of progeny viral DNA. We have constructed a chimeric HSV-CMV amplicon (herpesvirus cis replication functions carried on an Escherichia coli plasmid vector) substituting CMV DNA sequences for the HSV cleavage/packaging signal in a test of the ability of this CMV L-S junction sequence to provide the cis signal for cleavage/packaging in HSV 1-infected cells. We demonstrate that the alpha sequence of CMV DNA functions as a cleavage/packaging signal for HSV defective genomes. We show the structure of this sequence and provide a functional demonstration of cross complementation in replication signals which have been preserved over evolutionary time in these two widely divergent human herpesviruses.     

Protection from herpes simplex virus type 1 lethal and latent infections by secreted recombinant glycoprotein B constitutively expressed in human cells with a BK virus episomal vector.

The herpes simplex virus type 1 (HSV-1) glycoprotein B (gB-1) gene, deleted of 639 nucleotides that encode the transmembrane anchor sequence and reconstructed with the extramembrane and intracytoplasmic domains, was cloned under control of the Rous sarcoma virus long terminal repeat in the episomal replicating vector pRP-RSV, which contains the origin of replication and early region of the human papovavirus BK as well as a cDNA for a mutant mouse dihydrofolate reductase that is resistant to methotrexate. gB-1 (0.15 to 0.25 pg per cell per 24 h) was constitutively secreted into the culture medium of pRP-RSV-gBs-transformed human 293 cells. Treatment of transformed cells with methotrexate at high concentrations (0.6 to 6 microM) increased gB-1 production 10- to 100-fold, because of an amplification of the episomal recombinant. Mice immunized with secreted gB-1 produced HSV-1- and HSV-2-neutralizing antibodies and were protected against HSV-1 lethal, latent, and recurrent infections. Constitutive expression of secreted gB-1 in human cells may establish a system to develop diagnostic material and a subunit vaccine for HSV infections.     

Epstein-Barr virus DNA. X. Direct repeat within the internal direct repeat of Epstein-Barr virus DNA.

The 3,360-base-pair internal direct repeat (IR) in Epstein-Barr virus DNA separates the short and long unique DNA domains. IR has a single BamHI site. The juncture between the short unique domain and IR has been mapped by restriction endonucleases and is less than 2,600 nucleotides before the BamHI site in IR. The junction between IR and the long unique domain has been sequenced and is approximately 650 nucleotides after the BamHI site in IR. Thus, relative to the start of IR at the juncture with the short unique domain, the last repeat is at least 90 base pairs short of being complete. There is homology between the 250-nucleotide fragments to the left and the right of the unique BamHI site in IR. A 35-base-pair sequence of the left fragment is directly repeated within the right fragment, once fully and once partially. The implications of these findings are discussed.     

Proviral genome of radiation leukemia virus: molecular cloning of biologically active proviral DNA and nucleotide sequence of its long terminal repeat.

The proviral genome of a leukemogenic and thymotropic C57BL/Ka mouse retrovirus, RadLV/VL3(T+L+), was cloned as a biologically active PstI insert in the bacterial plasmid pBR322. Its restriction map was compared with those, already known, of two nonthymotropic and nonleukemogenic viruses of the same mouse strain: the ecotropic BL/Ka(B) virus and the xenotropic constituent of the radiation leukemia virus complex. Differences were observed around the gag-pol gene junction, in the pol gene, and in the env gene. Moreover, the nucleotide sequence of the RadLV/VL3(T+L+) long terminal repeat revealed the existence of two copies of a 43-base-pair sequence, of which BL/Ka(B) possesses only one copy.     

Nucleotide sequences at recombinational junctions present in pseudorabies virus variants with an invertible L component

The genome of pseudorabies virus (PrV) consists of two components--a noninvertible long (L) and an invertible short (S) component. The S component is bracketed by inverted repeats. In some variant strains of PrV (which have a selective growth advantage in certain cell lines), a sequence normally present at the left end of the L component has been translocated to the right end of the L component next to the inverted repeat. Consequently, these strains have acquired a genome with an L component that is bracketed by inverted repeats and that also inverts. We have determined the restriction maps and have analyzed the nucleotide sequences of those parts of the genome of three strains with invertible L components that contain the translocated segment of DNA. The results were as follows. The translocated fragments were derived in all cases from the extreme left end of the L component only. The sizes of the translocated fragments were similar, ranging from 1.3 to 1.4 kilobase pairs. The junction between the L and S components in these strains was the same as that in standard viral concatemeric DNA. The translocation of sequences from the left end of the genome next to the inverted repeats was always accompanied by a deletion of sequences from the right end of the L component. The sizes of the deleted fragments varied considerably, ranging from 0.8 to 2.3 kilobase pairs. Sequence homology at the points of recombination, i.e., at the junction between the right end and the left end of the L component, existed sometimes but not always. A model depicting how a virus with a class 2 genome (such as PrV) may acquire a genome with characteristics of a class 3 genome (such as herpes simplex virus) is presented.