Long Noncoding RNAs in Cancer Pathways

Herpesvirus particles consist of four morphologically distinct structures, the core, capsid, tegument, and envelope. The inner nucleoprotein core comprising the linear double-stranded DNA genome is included in an icosahedral (T=16) capsid shell of 150 hexons and 12 pentons. The capsid is surrounded by a layer of proteinaceous material designated the tegument which, in turn, is enclosed in an envelope of host cell-derived lipids containing virus-encoded (glyco)proteins. Whereas capsid formation in the nuclei of infected cells is understood in some detail, the mechanisms of tegumentation and envelopment and the intracellular compartments involved have long been disputed. This review focuses on recent findings that demonstrate a rather complex process of herpesvirus maturation including primary envelopment of capsids by budding at the inner leaflet of the nuclear membrane and translocation of capsids into the cytoplasm after loss of the primary envelope by fusion with the outer leaflet of the nuclear membrane. Subsequently, final tegumentation occurs in the cytoplasm and tegumented capsids obtain their final envelope by budding into vesicles of the trans-Golgi network. Tegumentation and envelopment are driven by specific protein-protein interactions that appear, at least in cultured cells, to exhibit a remarkable redundancy.

Herpesvirus Assembly and Egress

Bacterial artificial chromosomes are used to maintain and modify large sequences of different origins in Escherichia coli. In addition to RecA-based shuttle mutagenesis, Red recombination is commonly used for sequence modification. Since foreign sequences, such as antibiotic resistance genes as well as frt- or loxP-sites are often unwanted in mutant BAC clones, we developed a Red-based technique that allows for the scarless generation of point mutations, deletions, and insertion of smaller and larger sequences. The method employs a sequence duplication that is inserted into the target sequence in the first recombination step and the excision of the selection marker by in vivo I-SceI cleavage and the second Red recombination. To allow for convenient and highly efficient mutagenesis without the use of additional plasmids, the E. coli strain GS1783 with a chromosomal encoded inducible Red- and I-SceI-expression was created.

En passant mutagenesis: a two step markerless red recombination system.

In addition to members causing milder human infections, the Coronaviridae family includes potentially lethal zoonotic agents causing severe acute respiratory syndrome (SARS) and the recently emerged Middle East respiratory syndrome. The ∼30-kb positive-stranded RNA genome of coronaviruses encodes a replication/transcription machinery that is unusually complex and composed of 16 nonstructural proteins (nsps). SARS-CoV nsp12, the canonical RNA-dependent RNA polymerase (RdRp), exhibits poorly processive RNA synthesis in vitro, at odds with the efficient replication of a very large RNA genome in vivo. Here, we report that SARS-CoV nsp7 and nsp8 activate and confer processivity to the RNA-synthesizing activity of nsp12. Using biochemical assays and reverse genetics, the importance of conserved nsp7 and nsp8 residues was probed. Whereas several nsp7 mutations affected virus replication to a limited extent, the replacement of two nsp8 residues (P183 and R190) essential for interaction with nsp12 and a third (K58) critical for the interaction of the polymerase complex with RNA were all lethal to the virus. Without a loss of processivity, the nsp7/nsp8/nsp12 complex can associate with nsp14, a bifunctional enzyme bearing 3′-5′ exoribonuclease and RNA cap N7-guanine methyltransferase activities involved in replication fidelity and 5′-RNA capping, respectively. The identification of this tripartite polymerase complex that in turn associates with the nsp14 proofreading enzyme sheds light on how coronaviruses assemble an RNA-synthesizing machinery to replicate the largest known RNA genomes. This protein complex is a fascinating example of the functional integration of RNA polymerase, capping, and proofreading activities.

https://www.pnas.org/content/pnas/111/37/E3900.full.pdf

One severe acute respiratory syndrome coronavirus protein complex integrates processive RNA polymerase and exonuclease activities

The major goals of veterinary vaccines are to improve the health and welfare of companion animals, increase production of livestock in a cost-effective manner, and prevent animal-to-human transmission from both domestic animals and wildlife. These diverse aims have led to different approaches to the development of veterinary vaccines from crude but effective whole-pathogen preparations to molecularly defined subunit vaccines, genetically engineered organisms or chimeras, vectored antigen formulations, and naked DNA injections. The final successful outcome of vaccine research and development is the generation of a product that will be available in the marketplace or that will be used in the field to achieve desired outcomes. As detailed in this review, successful veterinary vaccines have been produced against viral, bacterial, protozoal, and multicellular pathogens, which in many ways have led the field in the application and adaptation of novel technologies. These veterinary vaccines have had, and continue to have, a major impact not only on animal health and production but also on human health through increasing safe food supplies and preventing animal-to-human transmission of infectious diseases. The continued interaction between animals and human researchers and health professionals will be of major importance for adapting new technologies, providing animal models of disease, and confronting new and emerging infectious diseases.

Current Status of Veterinary Vaccines

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.

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

Marek's disease virus (MDV) is an oncogenic herpesvirus that causes various clinical syndromes in its natural host, the chicken. MDV has long been of interest as a model organism, particularly with respect to the pathogenesis and immune control of virus-induced lymphoma in an easily accessible small-animal system. Recent advances in MDV genetics and the determination of the chicken genome sequence, aided by functional genomics, have begun to dramatically increase our understanding not only of lytic MDV replication, but also of the factors and mechanisms leading to latency and tumour formation. This new information is helping to elucidate cellular signalling pathways that have undergone convergent evolution and are perturbed by different viruses, and emphasizes the value of MDV as a comparative biomedical model. Furthermore, the door is now open for rational and efficient engineering of new vaccines against one of the most important and widespread infectious diseases in chickens.

Marek's disease virus: from miasma to model

Marek's disease virus (MDV) encodes a protein exhibiting high amino acid similarity to the US3 protein of herpes simplex virus type 1 and the gene 66 product of varicella-zoster virus The MDV US3 orthologue was replaced with a kanamycin resistance gene in the infectious bacterial artificial chromosome clone BAC20 After transfection of US3-negative BAC20 DNA (20ΔUS3), the resulting recombinant 20ΔUS3 virus exhibited markedly reduced growth kinetics Virus titers on chicken embryo cells were reduced by approximately 10-fold, and plaque sizes were significantly smaller (65% reduction) compared to parental BAC20 virus The defect of the US3-negative MDV was completely restored in a revertant virus (20US3*) expressing a US3 protein with a carboxy-terminal FLAG tag Electron microscopical studies revealed that the defect of the 20ΔUS3 mutant to efficiently spread from cell to cell was concomitant with an accumulation in the perinuclear space of primarily enveloped virions in characteristic vesicles containing several virus particles, which resulted in reduced numbers of particles in the cytoplasm The formation of these vesicles was not observed in cells infected with either parental BAC20 virus or the 20US3* revertant virus The role of the MDV US3 protein in actin stress fiber breakdown was investigated by visualizing actin with phalloidin-Alexa 488 after infection or transfection of a US3 expression plasmid Addition of the actin-depolymerizing drug cytochalasin D to cells transfected or infected with BAC20 resulted in complete inhibition of plaque formation with as little as 50 nM of the drug, while concentrations of nocodazole as high as 50 μM only had a relatively minor effect on MDV plaque formation The results indicated that the MDV US3 serine-threonine protein kinase is transiently involved in MDV-mediated stress fiber breakdown and that polymerization of actin, but not microtubules, plays an important role in MDV cell-to-cell spread

The protein encoded by the US3 orthologue of Marek's disease virus is required for efficient de-envelopment of perinuclear virions and involved in actin stress fiber breakdown.

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.

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

B. Karsten Tischer

Papers

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

En passant mutagenesis: a two step markerless red recombination system.

Marek's disease virus: from miasma to model

The protein encoded by the US3 orthologue of Marek's disease virus is required for efficient de-envelopment of perinuclear virions and involved in actin stress fiber breakdown.

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