The First Endogenous Herpesvirus,Identified in the Tarsier Genome,and Novel Sequences from Primate Rhadinoviruses and Lymphocryptoviruses

Herpesviridae is a diverse family of large and complex pathogens whose genomes are extremely difficult to sequence. This is particularly true for clinical samples, and if the virus, host, or both genomes are being sequenced for the first time. Although herpesviruses are known to occasionally integrate in host genomes, and can also be inherited in a Mendelian fashion, they are notably absent from the genomic fossil record comprised of endogenous viral elements (EVEs). Here, we combine paleovirological and metagenomic approaches to both explore the constituent viral diversity of mammalian genomes and search for endogenous herpesviruses. We describe the first endogenous herpesvirus from the genome of the Philippine tarsier, belonging to the Roseolovirus genus, and characterize its highly defective genome that is integrated and flanked by unambiguous host DNA. From a draft assembly of the aye-aye genome, we use bioinformatic tools to reveal over 100,000 bp of a novel rhadinovirus that is the first lemur gammaherpesvirus, closely related to Kaposi''s sarcoma-associated virus. We also identify 58 genes of Pan paniscus lymphocryptovirus 1, the bonobo equivalent of human Epstein-Barr virus. For each of the viruses, we postulate gene function via comparative analysis to known viral relatives. Most notably, the evidence from gene content and phylogenetics suggests that the aye-aye sequences represent the most basal known rhadinovirus, and indicates that tumorigenic herpesviruses have been infecting primates since their emergence in the late Cretaceous. Overall, these data show that a genomic fossil record of herpesviruses exists despite their extremely large genomes, and expands the known diversity of Herpesviridae, which will aid the characterization of pathogenesis. Our analytical approach illustrates the benefit of intersecting evolutionary approaches with metagenomics, genetics and paleovirology.     

BAC system: A novel method for manipulation of herpesvirus genomes based on bacterial genetics

Although methods for reverse genetics of herpesviruses have been established in early 1980s, the steps are laborious and time-consuming. In 1997, Dr. Koszinwski's group reported a novel approach for the construction of herpesvirus mutants, based on cloning the viral genome as a bacterial artificial chromosome (BAC) in E. coli. This technique allows the maintenance of viral genomes as plasmid in E. coli and the reconstitution of viral progeny by transfection of the BAC plasmid into eukaryotic cells. Any genetics modification of the viral genome in E. coli using bacterial genetics is possible, thereby facilitating the introduction of mutagenesis into herpesvirus genome. This 'BAC system' has opened new avenues for reverse and forward genetics of herpesviruses in basic research and in vector development for human therapy. Here we describe the principle of the 'BAC system' in herpesvirus researches.     

Significant association of multiple human cytomegalovirus genomic Loci with glioblastoma multiforme samples

Viruses are appreciated as etiological agents of certain human tumors, but the number of different cancer types induced or exacerbated by viral infections is unknown. Glioblastoma multiforme (GBM)/astrocytoma grade IV is a malignant and lethal brain cancer of unknown origin. Over the past decade, several studies have searched for the presence of a prominent herpesvirus, human cytomegalovirus (HCMV), in GBM samples. While some have detected HCMV DNA, RNA, and proteins in GBM tissues, others have not. Therefore, any purported association of HCMV with GBM remains controversial. In most of the previous studies, only one or a select few viral targets were analyzed. Thus, it remains unclear the extent to which the entire viral genome was present when detected. Here we report the results of a survey of GBM specimens for as many as 20 different regions of the HCMV genome. Our findings indicate that multiple HCMV loci are statistically more likely to be found in GBM samples than in other brain tumors or epileptic brain specimens and that the viral genome was more often detected in frozen samples than in paraffin-embedded archival tissue samples. Finally, our experimental results indicate that cellular genomes substantially outnumber viral genomes in HCMV-positive GBM specimens, likely indicating that only a minority of the cells found in such samples harbor viral DNA. These data argue for the association of HCMV with GBM, defining the virus as oncoaccessory. Furthermore, they imply that, were HCMV to enhance the growth or survival of a tumor (i.e., if it is oncomodulatory), it would likely do so through mechanisms distinct from classic tumor viruses that express transforming viral oncoproteins in the overwhelming majority of tumor cells.     

Improving gene annotation of complete viral genomes

Gene annotation in viruses often relies upon similarity search methods. These methods possess high specificity but some genes may be missed, either those unique to a particular genome or those highly divergent from known homologs. To identify potentially missing viral genes we have analyzed all complete viral genomes currently available in GenBank with a specialized and augmented version of the gene finding program GeneMarkS. In particular, by implementing genome-specific self-training protocols we have better adjusted the GeneMarkS statistical models to sequences of viral genomes. Hundreds of new genes were identified, some in well studied viral genomes. For example, a new gene predicted in the genome of the Epstein–Barr virus was shown to encode a protein similar to α-herpesvirus minor tegument protein UL14 with heat shock functions. Convincing evidence of this similarity was obtained after only 12 PSI-BLAST iterations. In another example, several iterations of PSI-BLAST were required to demonstrate that a gene predicted in the genome of Alcelaphine herpesvirus 1 encodes a BALF1-like protein which is thought to be involved in apoptosis regulation and, potentially, carcinogenesis. New predictions were used to refine annotations of viral genomes in the RefSeq collection curated by the National Center for Biotechnology Information. Importantly, even in those cases where no sequence similarities were detected, GeneMarkS significantly reduced the number of primary targets for experimental characterization by identifying the most probable candidate genes. The new genome annotations were stored in VIOLIN, an interactive database which provides access to similarity search tools for up-to-date analysis of predicted viral proteins.     

Machinery to support genome segment inversion exists in a herpesvirus which does not naturally contain invertible elements

In many herpesviruses, genome segments flanked by inverted repeats invert during DNA replication. It is not known whether this inversion is a consequence of an inherently recombinagenic replicative mechanism common to all herpesviruses or whether the replication enzymes of viruses with invertible segments have specifically evolved additional enzymatic activities to drive inversion. By artificially inserting a fusion of terminal sequences into the genome of a virus which normally lacks invertible elements (murine cytomegalovirus), we created a genome composed of long and short segments flanked by 1,359- and 543-bp inverted repeats. Analysis of genomic DNA from this virus revealed that inversion of both segments generates equimolar amounts of four isomers during the viral propagation necessary to produce DNA for analysis from a single viral particle. We conclude that a herpesvirus which naturally lacks invertible elements is able to support efficient segment inversion. Thus, the potential to invert is probably inherent in the replication machinery of all herpesviruses, irrespective of genome structure, and therefore genomes with invertible elements could have evolved simply by acquisition of inverted repeats and without concomitant evolution of enzymatic activities to mediate inversion. Furthermore, the recombinagenicity of herpesvirus DNA replication must have some importance independent of genome segment inversion.     

Complete viral RNA genome sequencing of ultra-low copy samples by sequence-independent amplification

RNA viruses are the causative agents for AIDS, influenza, SARS, and other serious health threats. Development of rapid and broadly applicable methods for complete viral genome sequencing is highly desirable to fully understand all aspects of these infectious agents as well as for surveillance of viral pandemic threats and emerging pathogens. However, traditional viral detection methods rely on prior sequence or antigen knowledge. In this study, we describe sequence-independent amplification for samples containing ultra-low amounts of viral RNA coupled with Illumina sequencing and de novo assembly optimized for viral genomes. With 5 million reads, we capture 96 to 100% of the viral protein coding region of HIV, respiratory syncytial and West Nile viral samples from as little as 100 copies of viral RNA. The methods presented here are scalable to large numbers of samples and capable of generating full or near full length viral genomes from clone and clinical samples with low amounts of viral RNA, without prior sequence information and in the presence of substantial host contamination.     

Viral population analysis and minority-variant detection using short read next-generation sequencing

RNA viruses within infected individuals exist as a population of evolutionary-related variants. Owing to evolutionary change affecting the constitution of this population, the frequency and/or occurrence of individual viral variants can show marked or subtle fluctuations. Since the development of massively parallel sequencing platforms, such viral populations can now be investigated to unprecedented resolution. A critical problem with such analyses is the presence of sequencing-related errors that obscure the identification of true biological variants present at low frequency. Here, we report the development and assessment of the Quality Assessment of Short Read (QUASR) Pipeline (http://sourceforge.net/projects/quasr) specific for virus genome short read analysis that minimizes sequencing errors from multiple deep-sequencing platforms, and enables post-mapping analysis of the minority variants within the viral population. QUASR significantly reduces the error-related noise in deep-sequencing datasets, resulting in increased mapping accuracy and reduction of erroneous mutations. Using QUASR, we have determined influenza virus genome dynamics in sequential samples from an in vitro evolution of 2009 pandemic H1N1 (A/H1N1/09) influenza from samples sequenced on both the Roche 454 GSFLX and Illumina GAIIx platforms. Importantly, concordance between the 454 and Illumina sequencing allowed unambiguous minority-variant detection and accurate determination of virus population turnover in vitro.     

A Modified RNA-Seq Approach for Whole Genome Sequencing of RNA Viruses from Faecal and Blood Samples

To date, very large scale sequencing of many clinically important RNA viruses has been complicated by their high population molecular variation, which creates challenges for polymerase chain reaction and sequencing primer design. Many RNA viruses are also difficult or currently not possible to culture, severely limiting the amount and purity of available starting material. Here, we describe a simple, novel, high-throughput approach to Norovirus and Hepatitis C virus whole genome sequence determination based on RNA shotgun sequencing (also known as RNA-Seq). We demonstrate the effectiveness of this method by sequencing three Norovirus samples from faeces and two Hepatitis C virus samples from blood, on an Illumina MiSeq benchtop sequencer. More than 97% of reference genomes were recovered. Compared with Sanger sequencing, our method had no nucleotide differences in 14,019 nucleotides (nt) for Noroviruses (from a total of 2 Norovirus genomes obtained with Sanger sequencing), and 8 variants in 9,542 nt for Hepatitis C virus (1 variant per 1,193 nt). The three Norovirus samples had 2, 3, and 2 distinct positions called as heterozygous, while the two Hepatitis C virus samples had 117 and 131 positions called as heterozygous. To confirm that our sample and library preparation could be scaled to true high-throughput, we prepared and sequenced an additional 77 Norovirus samples in a single batch on an Illumina HiSeq 2000 sequencer, recovering >90% of the reference genome in all but one sample. No discrepancies were observed across 118,757 nt compared between Sanger and our custom RNA-Seq method in 16 samples. By generating viral genomic sequences that are not biased by primer-specific amplification or enrichment, this method offers the prospect of large-scale, affordable studies of RNA viruses which could be adapted to routine diagnostic laboratory workflows in the near future, with the potential to directly characterize within-host viral diversity.     

Identification of microRNAs of the herpesvirus family

Epstein-Barr virus (EBV or HHV4), a member of the human herpesvirus (HHV) family, has recently been shown to encode microRNAs (miRNAs). In contrast to most eukaryotic miRNAs, these viral miRNAs do not have close homologs in other viral genomes or in the genome of the human host. To identify other miRNA genes in pathogenic viruses, we combined a new miRNA gene prediction method with small-RNA cloning from several virus-infected cell types. We cloned ten miRNAs in the Kaposi sarcoma-associated virus (KSHV or HHV8), nine miRNAs in the mouse gammaherpesvirus 68 (MHV68) and nine miRNAs in the human cytomegalovirus (HCMV or HHV5). These miRNA genes are expressed individually or in clusters from either polymerase (pol) II or pol III promoters, and share no substantial sequence homology with one another or with the known human miRNAs. Generally, we predicted miRNAs in several large DNA viruses, and we could neither predict nor experimentally identify miRNAs in the genomes of small RNA viruses or retroviruses.     

Vaccination of mice with bacteria carrying a cloned herpesvirus genome reconstituted in vivo

Bacterial delivery systems are gaining increasing interest as potential vaccination vectors to deliver either proteins or nucleic acids for gene expression in the recipient. Bacterial delivery systems for gene expression in vivo usually contain small multicopy plasmids. We have shown before that bacteria containing a herpesvirus bacterial artificial chromosome (BAC) can reconstitute the virus replication cycle after cocultivation with fibroblasts in vitro. In this study we addressed the question of whether bacteria containing a single plasmid with a complete viral genome can also reconstitute the viral replication process in vivo. We used a natural mouse pathogen, the murine cytomegalovirus (MCMV), whose genome has previously been cloned as a BAC in Escherichia coli. In this study, we tested a new application for BAC-cloned herpesvirus genomes. We show that the MCMV BAC can be stably maintained in certain strains of Salmonella enterica serovar Typhimurium as well and that both serovar Typhimurium and E. coli harboring the single-copy MCMV BAC can reconstitute a virus infection upon injection into mice. By this procedure, a productive virus infection is regenerated only in immunocompromised mice. Virus reconstitution in vivo causes elevated titers of specific anti-MCMV antibodies, protection against lethal MCMV challenge, and strong expression of additional genes introduced into the viral genome. Thus, the reconstitution of infectious virus from live attenuated bacteria presents a novel concept for multivalent virus vaccines launched from bacterial vectors.     

Rapid identification of essential and nonessential herpesvirus genes by direct transposon mutagenesis

Herpesviruses are important pathogens in animals and humans. The large DNA genomes of several herpesviruses have been sequenced, but the function of the majority of putative genes is elusive. Determining which genes are essential for their replication is important for identifying potential chemotherapy targets, designing herpesvirus vectors, and generating attenuated vaccines. For this purpose, we recently reported that herpesvirus genomes can be maintained as infectious bacterial artificial chromosomes (BAC) in Escherichia coli. Here we describe a one-step procedure for random-insertion mutagenesis of a herpesvirus BAC using a Tn1721-based transposon system. Transposon insertion sites were determined by direct sequencing, and infectious virus was recovered by transfecting cultured cells with the mutant genomes. Lethal mutations were rescued by cotransfecting cells containing noninfectious genomes with the corresponding wild-type subgenomic fragments. We also constructed revertant genomes by allelic exchange in bacteria. These methods, which are generally applicable to any cloned herpesvirus genome, will facilitate analysis of gene function for this virus family.     

Specificity of cleavage in replicative-form DNA of bovine herpesvirus 1.

The linear double-stranded DNA genome of herpesvirus as it is present in infectious virions needs to be circularized after infection of host cells and before DNA replication. Replicative-form genomes have to be cleaved into linear unit-length molecules during virion maturation and are most probably the substrate for inversion of the short segment relative to the long segment of the bovine herpesvirus 1 (BHV-1) genome. Those regions of the BHV-1 genome which are functionally involved in these processes have been analyzed at the molecular level by cloning and sequencing the genomic termini, the fusion of both termini from replicative-form molecules, and the junction between the short and the long genome segment. On the basis of the simple genome arrangement of BHV-1, it was inferable that the cleavage of replicative-form genomes by a hypothetical BHV-1 terminase activity may be specified by a sequence at the left end of UL (An element), which is located proximal to a reiterated beta element that makes up the cleavage site itself. The relationship of those elements in BHV-1 and the comparison to similar regions of other herpesviruses indicate consensus sequence elements which are functionally important for cleavage and isomerization of viral DNA during maturation of virions.     

The Barrier to Autointegration Factor: Interlocking Antiviral Defense with Genome Maintenance

Intrinsic defenses targeting foreign DNA are one facet of the cellular armament tasked with protecting host genomic integrity. The DNA binding protein BAF (barrier to autointegration factor) contributes to multiple aspects of genome maintenance and intercepts retrovirus, poxvirus, and herpesvirus genomes during infection. In this gem, we discuss the unique position BAF occupies at the virus-host interface and how both viral and cellular mechanisms may regulate its capacity to act as a pro- or antiviral effector targeting viral DNA.     

Assessment of the base sequence homology between the two subtypes of equine herpesvirus 1.

The magnitude of the genetic relatedness of the two antigenic subtypes of equine herpesvirus 1 (EHV-1) was determined by DNA-DNA reassociation kinetics. Denatured, labeled viral DNA from one EHV-1 subtype was allowed to reassociate in the presence or absence of the unlabeled heterologous viral DNA. The initial rate of reassociation of either labeled viral DNA was increased by the presence of the heterologous viral DNA to an extent indicating 10 to 20% homology between the two EHV-1 genomes. Similar estimates of the amount of homology between the genomes of the two EHV-1 subtypes were obtained by determining the maximum fraction of labeled viral DNA that could be made resistant to S1 nuclease by hybridization with a large molar excess of the unlabeled, heterologous viral DNA. Analysis of the thermal stability of the subtype 1-subtype 2 heteroduplex DNA indicated approximately 30% base pair mismatching within the hybrid DNA molecules. Cross-hybridization of 32P-labeled virion DNA to nitrocellulose blots of restriction endonuclease cleavage fragments of each EHV-1 subtype DNA indicated that the observed homology between the two viruses was nonuniformly distributed with the viral genome. No homology could be detected between the DNA of either EHV-1 subtype and that of a strain of equine cytomegalovirus (EHV-2). The data suggest that the two biotypes of EHV-1 have arisen by divergent evolution from a common progenitor herpesvirus.     

Differential distributions of mononucleotide repeat sequences in 256 viral genomes and its potential implications

Mononucleotide repeats (MNRs) have been systematically investigated in the genomes of eukaryotic and prokaryotic organisms. However, detailed information on the distribution of MNRs in viral genomes is limited. In this study, we examined the distributions of MNRs in 256 fully sequenced virus genomes which showed extensive variations across viral genomes, and is significantly influenced by both genome size and CG content. Furthermore, the ratio of the observed to the expected number of MNRs (O/E ratio) appears to be influenced by both the host range and genome type of a particular virus. Additionally, the densities and frequencies of MNRs in genic regions are lower than in non-coding regions, suggesting that selective pressure acts on viral genomes. We also discuss the potential functional roles that these MNR loci could play in virus genomes. To our knowledge, this is the first analysis focusing on MNRs in viruses, and our study could have potential implications for a deeper understanding of virus genome stability and the co-evolution that occurs between a virus and its host.     

Deletion mapping of Sindbis virus DI RNAs derived from cDNAs defines the sequences essential for replication and packaging

Defective-interfering (DI) genomes of a virus contain sequence information essential for their replication and packaging. They need not contain any coding information and therefore are a valuable tool for identifying cis-acting, regulatory sequences in a viral genome. To identify these sequences in a DI genome of Sindbis virus, we cloned a cDNA copy of a complete DI genome directly downstream of the promoter for the SP6 bacteriophage DNA dependent RNA polymerase. The cDNA was transcribed into RNA, which was transfected into chicken embryo fibroblasts in the presence of helper Sindbis virus. After one to two passages the DI RNA became the major viral RNA species in infected cells. Data from a series of deletions covering the entire DI genome show that only sequences in the 162 nucleotide region at the 5' terminus and in the 19 nucleotide region at the 3' terminus are specifically required for replication and packaging of these genomes.     

Abduction of Chemokine Elements by Herpesviruses

Chemokines play a key role in orchestrating leukocytic recruitment during inflammatory responses, including those to viral infections. Chemokines are soluble cytokines which mediate their effects through specific G protein-coupled, seven-transmembrane receptors which are expressed on a wide range of cells, including monocytes, T-cells, dendritic cells, and NK cells. Analyses of herpesvirus genomes have revealed that these viral pathogens encode their own versions of both chemokines and chemokine receptors. Viral genes encoding chemokine elements were likely to have been acquired from the host genome and have been remodeled during virus evolution to presumably optimize function or acquire new properties not displayed by their cellular homologues. Virus-encoded chemokines and chemokine receptors are important players in the continuing confrontation between viruses and their mammalian hosts. Detailed characterization of these elements will provide a better understanding of how the immune system responds to viral infection and may suggest new antiviral drug targets and new avenues for the development of antiviral therapies. We will review here the chemokine elements encoded by herpesviruses and how they may aid viral infection and propagation.