Benedikt B. Kaufer

Two-step Red-mediated recombination for versatile high-efficiency markerless DNA manipulation in Escherichia coli

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.

A self-excisable infectious bacterial artificial chromosome clone of varicella-zoster virus allows analysis of the essential tegument protein encoded by ORF9.

ABSTRACT 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.

Herpesvirus telomeric repeats facilitate genomic integration into host telomeres and mobilization of viral DNA during reactivation

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

A herpesvirus ubiquitin-specific protease is critical for efficient T cell lymphoma formation

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 Mareks 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.

Herpesvirus Genome Integration into Telomeric Repeats of Host Cell Chromosomes

It is well known that numerous viruses integrate their genetic material into host cell chromosomes. Human herpesvirus 6 (HHV-6) and oncogenic Mareks disease virus (MDV) have been shown to integrate their genomes into host telomeres of latently infected cells. This is unusual for herpesviruses as most maintain their genomes as circular episomes during the quiescent stage of infection. The genomic DNA of HHV-6, MDV, and several other herpesviruses harbors telomeric repeats (TMRs) that are identical to host telomere sequences (TTAGGG). At least in the case of MDV, viral TMRs facilitate integration into host telomeres. Integration of HHV-6 occurs not only in lymphocytes but also in the germline of some individuals, allowing vertical virus transmission. Although the molecular mechanism of telomere integration is poorly understood, the presence of TMRs in a number of herpesviruses suggests it is their default program for genome maintenance during latency and also allows efficient reactivation.

Chromosomally integrated HHV-6: impact on virus, cell and organismal biology

HHV-6 integrates its genome into telomeres of human chromosomes. Integration can occur in somatic cells or gametes, the latter leading to individuals harboring the HHV-6 genome in every cell. This condition is transmitted to descendants and referred to as inherited chromosomally integrated human herpesvirus 6 (iciHHV-6). Although integration can occur in different chromosomes, it invariably takes place in the telomere region. This integration mechanism represents a way to maintain the virus genome during latency, which is so far unique amongst human herpesviruses. Recent work provides evidence that the integrated HHV-6 genome can be mobilized from the host chromosome, resulting in the onset of disease. Details on required structural determinants, putative integration mechanisms and biological and medical consequences of iciHHV-6 are discussed.

Viral bacterial artificial chromosomes: generation, mutagenesis, and removal of mini-F sequences.

Maintenance and manipulation of large DNA and RNA virus genomes had presented an obstacle for virological research. BAC vectors provided a solution to both problems as they can harbor large DNA sequences and can efficiently be modified using well-established mutagenesis techniques in Escherichia coli. Numerous DNA virus genomes of herpesvirus and pox virus were cloned into mini-F vectors. In addition, several reverse genetic systems for RNA viruses such as members of Coronaviridae and Flaviviridae could be established based on BAC constructs. Transfection into susceptible eukaryotic cells of virus DNA cloned as a BAC allows reconstitution of recombinant viruses. In this paper, we provide an overview on the strategies that can be used for the generation of virus BAC vectors and also on systems that are currently available for various virus species. Furthermore, we address common mutagenesis techniques that allow modification of BACs from single-nucleotide substitutions to deletion of viral genes or insertion of foreign sequences. Finally, we review the reconstitution of viruses from BAC vectors and the removal of the bacterial sequences from the virus genome during this process.

Marek's Disease Viral Interleukin-8 Promotes Lymphoma Formation through Targeted Recruitment of B Cells and CD4+ CD25+ T Cells

ABSTRACT Mareks disease virus (MDV) is a cell-associated and highly oncogenic alphaherpesvirus that infects chickens. During lytic and latent MDV infection, a CXC chemokine termed viral interleukin-8 (vIL-8) is expressed. Deletion of the entire vIL-8 open reading frame (ORF) was shown to severely impair disease progression and tumor development; however, it was unclear whether this phenotype was due to loss of secreted vIL-8 or of splice variants that fuse exons II and III of vIL-8 to certain upstream open reading frames, including the viral oncoprotein Meq. To specifically examine the role of secreted vIL-8 in MDV pathogenesis, we constructed a recombinant virus, vΔMetvIL-8, in which we deleted the native start codon from the signal peptide encoding exon I. This mutant lacked secreted vIL-8 but did not affect Meq–vIL-8 splice variants. Loss of secreted vIL-8 resulted in highly reduced disease and tumor incidence in animals infected with vΔMetvIL-8 by the intra-abdominal route. Although vΔMetvIL-8 was still able to spread to naïve animals by the natural route, infection and lymphomagenesis in contact animals were severely impaired. In vitro assays showed that purified recombinant vIL-8 efficiently binds to and induces chemotaxis of B cells, which are the main target for lytic MDV replication, and also interacts with CD4+ CD25+ T cells, known targets of MDV transformation. Our data provide evidence that vIL-8 attracts B and CD4+ CD25+ T cells to recruit targets for both lytic and latent infection.

Chromosomally integrated human herpesvirus 6 in heart failure: prevalence and treatment

Human herpesvirus 6 (HHV‐6) A and B are two betaherpesviruses that are associated with many conditions including roseola, drug‐induced hypersensitivity syndrome, limbic encephalitis, and myocarditis. HHV‐6 is integrated in the germline [chromosomically integrated HHV‐6 (ciHHV‐6)] in ∼0.8% of the human population. To date, the prevalence, species distribution, and treatment responses of ciHHV‐6 are unknown for cardiac patients.

Fluorescently Tagged pUL47 of Marek's Disease Virus Reveals Differential Tissue Expression of the Tegument Protein In Vivo

ABSTRACT Mareks disease virus (MDV), a lymphotropic alphaherpesvirus, causes Mareks disease (MD) in chickens. MD is characterized by neurological signs, chronic wasting, and T cell lymphomas that predominate in the visceral organs. MDV replicates in a highly cell-associated manner in vitro and in vivo, with infectious virus particles being released only from feather follicle epithelial (FFE) cells in the skin. Virus produced and shed from FFE cells allows transmission of MDV from infected to naïve chickens, but the mechanisms or roles of differential virus gene expression have remained elusive. Here, we generated recombinant MDV in which we fused enhanced green fluorescent protein (EGFP) to the C terminus of the tegument protein pUL47 (vUL47-EGFP) or pUL49 (vUL49-EGFP). While vUL49-EGFP was highly attenuated in vitro and in vivo, vUL47-EGFP showed unaltered pathogenic potential and stable production of pUL47-EGFP, which facilitated direct analysis of pUL47 expression in cells and tissues. Our studies revealed that pUL47-EGFP is expressed at low levels and localizes to the nucleus during lytic replication in vitro and in lymphocytes in the spleen in vivo, while it is undetectable in tumors. In contrast, pUL47-EGFP is highly abundant and localizes predominantly in the cytoplasm in FFE cells in the skin, where MDV is shed into the environment. We concluded that differential expression and localization of MDV pUL47-EGFP tegument protein is potentially important for the unique cell-associated nature of MDV in vitro and in lymphocytes in vivo, as well as production of free virus in FFE cells.