Red recombination using PCR-amplified selectable markers is a well-established technique for mutagenesis of large DNA molecules in Escherichia coli. The system has limited efficacy and versatility, however, for markerless modifications including point mutations, deletions, and particularly insertions of longer sequences. Here we describe a procedure that combines Red recombination and cleavage with the homing endonuclease I-SceI to allow highly efficient, PCR-based DNA engineering without retention of unwanted foreign sequences. We applied the method to modification of bacterial artificial chromosome (BAC) constructs harboring an infectious herpesvirus clone to demonstrate the potential of the mutagenesis technique, which was used for the insertion of long sequences such as coding regions or promoters, introduction of point mutations, scarless deletions, and insertion of short sequences such as an epitope tag. The system proved to be highly reliable and efficient and can be adapted for a variety of different modifications of BAC clones, which are fundamental tools for applications as diverse as the generation of transgenic animals and the construction of gene therapy or vaccine vectors.

In order to facilitate the generation of mutant viruses of varicella-zoster virus (VZV), the agent causing varicella (chicken pox) and herpes zoster (shingles), we generated a full-length infectious bacterial artificial chromosome (BAC) clone of the P-Oka strain. First, mini-F sequences were inserted into a preexisting VZV cosmid, and the SuperCos replicon was removed. Subsequently, mini-F-containing recombinant virus was generated from overlapping cosmid clones, and full-length VZV DNA recovered from the recombinant virus was established in Escherichia coli as an infectious BAC. An inverted duplication of VZV genomic sequences within the mini-F replicon resulted in markerless excision of vector sequences upon virus reconstitution in eukaryotic cells. Using the novel tool, the role in VZV replication of the major tegument protein encoded by ORF9 was investigated. A markerless point mutation introduced in the start codon by two-step en passant Red mutagenesis abrogated ORF9 expression and resulted in a dramatic growth defect that was not observed in a revertant virus. The essential nature of ORF9 for VZV replication was ultimately confirmed by restoration of the growth of the ORF9-deficient mutant virus using trans-complementation via baculovirus-mediated gene transfer. Varicella-zoster virus (VZV), the causative agent of chicken pox (varicella) and shingles (herpes zoster), is a highly cellassociated herpesvirus both in vitro and in vivo (7, 23). Similar to the situation with a closely related alphaherpesvirus, Marek's disease virus (MDV), VZV spreads directly from cell to cell, presumably utilizing the machinery involved in adherens junction modeling and architecture. Infectious virus is released into the environment only after lytic VZV replication in the skin and respiratory mucous membranes of infected individuals (2, 43). Efficient and timely coordinated interaction between the tegument, a proteinaceous structure surrounding the icosahedral nucleocapsid, and envelope membrane (glyco) proteins plays a crucial role in the secondary envelopment and spread of herpesviruses in general and VZV in particular (35). It has been shown for related viruses, such as herpes simplex virus, that the inner layer of tegument is tightly associated with the nucleocapsid, whereas the outer layer of tegument provides the link to envelope proteins (34, 36). The current model of herpesviral tegumentation predicts that the inner layer of tegument is added to the nucleocapsid in the nucleus and deenveloped particles in the cytoplasm, while proteins representing the outer layer are thought to accumulate at cytoplasmic membranes where they make contact with their respective partners, either other tegument proteins or membrane proteins or both (34-36). Through these intricately regulated interactions, final secondary envelopment of particles is facilitated and, ultimately, infectious virus is produced and released.Determination of the role of individual tegument or membrane (glyco)proteins in the replication cycle of various herpesviru...

Herpesvirus telomeric repeats facilitate virus integration into host telomeres, a process which is required for the establishment of virus latency.

Human herpesvirus 6A (HHV-6A) and 6B (HHV-6B) are ubiquitous betaherpesviruses that infects humans within the first years of life and establishes latency in various cell types. Both viruses can integrate their genomes into telomeres of host chromosomes in latently infected cells. The molecular mechanism of viral integration remains elusive. Intriguingly, HHV-6A, HHV-6B and several other herpesviruses harbor arrays of telomeric repeats (TMR) identical to human telomere sequences at the ends of their genomes. The HHV-6A and HHV-6B genomes harbor two TMR arrays, the perfect TMR (pTMR) and the imperfect TMR (impTMR). To determine if the TMR are involved in virus integration, we deleted both pTMR and impTMR in the HHV-6A genome. Upon reconstitution, the TMR mutant virus replicated comparable to wild type (wt) virus, indicating that the TMR are not essential for HHV-6A replication. To assess the integration properties of the recombinant viruses, we established an in vitro integration system that allows assessment of integration efficiency and genome maintenance in latently infected cells. Integration of HHV-6A was severely impaired in the absence of the TMR and the virus genome was lost rapidly, suggesting that integration is crucial for the maintenance of the virus genome. Individual deletion of the pTMR and impTMR revealed that the pTMR play the major role in HHV-6A integration, whereas the impTMR only make a minor contribution, allowing us to establish a model for HHV-6A integration. Taken together, our data shows that the HHV-6A TMR are dispensable for virus replication, but are crucial for integration and maintenance of the virus genome in latently infected cells.

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The herpesvirus ubiquitin-specific protease (USP) family, whose founding member was discovered as a protease domain embedded in the large tegument protein of herpes simplex virus 1 (HSV-1), is conserved across all members of the Herpesviridae. Whether this conservation is indicative of an essential function of the enzyme in vivo has not yet been established. As reported here, USP activity is conserved in Marek's disease virus (MDV), a tumorigenic alphaherpesvirus. A single amino acid substitution that abolishes the USP activity of the MDV large tegument protein diminishes MDV replication in vivo, and severely limits the oncogenic potential of the virus. Expression of the USP transcripts in MDV-transformed cell lines further substantiates this hypothesis. The herpesvirus USP thus appears to be required not only to maintain a foothold in the immunocompetent host, but also to contribute to malignant outgrowths.chicken ͉ deubiquitinating enzyme ͉ herpes ͉ Marek's disease virus T he ubiquitin-proteasome system controls cytosolic proteolysis, certain aspects of transcription, antigen presentation via major histocompatibility complex (MHC) class I products, and the trafficking of surface-exposed receptors (1-5). As for many other posttranslational modifications, both ubiquitin conjugation and its reversal by ubiquitin-specific proteases (USPs) determine the biological outcome of the reaction. The enzyme families that catalyze ubiquitin conjugation and removal are quite diverse (6-9). Consequently, bioinformatic analysis is not always adequate to identify novel USPs. To target such enzymes biochemically, we developed activity-based probes for USPs and enzymes that act on ubiquitin-like modifiers (10). These probes are equipped with an affinity handle to allow retrieval and identification of the enzymes targeted by the electrophilic warhead installed at the probe's carboxyl terminus.Through the use of one of these probes, HA-ubiquitin vinylmethylester (HA-UbVME), we identified the large tegument protein of herpes simplex virus 1 (HSV-1), viral protein (VP) 1/2, encoded by the unique-long (U L ) 36 gene, as the source of an active USP. Its sequence showed no obvious similarity to known eukaryotic USPs and failed to yield an obvious signature of residues diagnostic of known cysteine protease families. Nonetheless, sequence comparisons across the Herpesviridae show the presence of a few absolutely conserved residues (Cys, Asp, His, Glu), all of which are consistent with involvement in a potential thiol protease active site. We have, meanwhile, confirmed both the mechanism of action of such ubiquitin-based probes (11)(12)(13)(14)(15) and the identity of the viral cysteine protease domain as an authentic USP by crystallographic analysis of the homologous segment of the murine cytomegalovirus (MCMV) M48 protein (16). Notwithstanding the conservation of the identified USP activity in all herpesviruses, we do not know whether this activity makes a contribution to the replicative success and pathogenicity of herpesviruses in vivo...

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