Peter Brunovskis

Nanoparticles of compacted DNA transfect postmitotic cells.

Charge-neutral DNA nanoparticles have been developed in which single molecules of DNA are compacted to their minimal possible size. We speculated that the small size of these DNA nanoparticles may facilitate gene transfer in postmitotic cells, permitting nuclear uptake across the 25-nm nuclear membrane pore. To determine whether DNA nanoparticles can transfect nondividing cells, growth-arrested neuroblastoma and hepatoma cells were transfected with DNA/liposome mixtures encoding luciferase. In both models, growth-arrested cells were robustly transfected by compacted DNA (6,900–360-fold more than naked DNA). To evaluate mechanisms responsible for enhanced transfection, HuH-7 cells were microinjected with naked or compacted plasmids encoding enhanced green fluorescent protein. Cytoplasmic microinjection of DNA nanoparticles generated a ∼10-fold improvement in transgene expression as compared with naked DNA; this enhancement was reversed by the nuclear pore inhibitor, wheat germ agglutinin. To determine the upper size limit for gene transfer, DNA nanoparticles of various sizes were microinjected into the cytoplasm. A marked decrease in transgene expression was observed as the minor ellipsoidal diameter approached 25 nm. In summary, suitably sized DNA nanoparticles productively transfect growth arrested cells by traversing the nuclear membrane pore.

Marek's Disease Virus (MDV) Encodes an Interleukin-8 Homolog (vIL-8): Characterization of the vIL-8 Protein and a vIL-8 Deletion Mutant MDV

ABSTRACT Chemokines induce chemotaxis, cell migration, and inflammatory responses. We report the identification of an interleukin-8 (IL-8) homolog, termed vIL-8, encoded within the genome of Mareks disease virus (MDV). The 134-amino-acid vIL-8 shares closest homology to mammalian and avian IL-8, molecules representing the prototype CXC chemokine. The gene for vIL-8 consists of three exons which map to theBamHI-L fragment within the repeats flanking the unique long region of the MDV genome. A 0.7-kb transcript encoding vIL-8 was detected in an n-butyrate-treated, MDV-transformed T-lymphoblastoid cell line, MSB-1. This induction is essentially abolished by cycloheximide and herpesvirus DNA polymerase inhibitor phosphonoacetate, indicating that vIL-8 is expressed with true late (γ2) kinetics. Baculovirus-expressed vIL-8 was found to be secreted into the medium and shown to be functional as a chemoattractant for chicken peripheral blood mononuclear cells but not for heterophils. To characterize the function of vIL-8 with respect to MDV infection in vivo, a recombinant MDV was constructed with a deletion of all three exons and a soluble-modified green fluorescent protein (smGFP) expression cassette inserted at the site of deletion. In two in vivo experiments, the vIL-8 deletion mutant (RB1BvIL-8ΔsmGFP) showed a decreased level of lytic infection in comparison to its parent virus, an equal-passage-level parent virus, and to another recombinant MDV containing the insertion of a GFP expression cassette at the nonessential US2 gene. RB1BvIL-8ΔsmGFP retained oncogenicity, albeit at a greatly reduced level. Nonetheless, we have been able to establish a lymphoblastoid cell line from an RB1BvIL-8ΔsmGFP-induced ovarian lymphoma (MDCC-UA20) and verify the presence of a latent MDV genome lacking vIL-8. Taken together, these data describe the identification and characterization of a chemokine homolog encoded within the MDV genome that is dispensable for transformation but may affect the level of MDV in vivo lytic infection.

Meq: An MDV-specific bZIP transactivator with transforming properties

The principal cause of chicken T-lymphomas and their accompanying demyelinating disease is infection with Marek’s disease virus (MDV) (Calnek et al. 1997), a herpesvirus. Mareks disease can be prevented by vaccination with an antigenically related nonpathogenic herpesvirus, HVT (turkey herpesvirus). MDV is among the most potent oncogenic herpesviruses, and induces tumors as early as 4 weeks post-inoculation. As such, the virus is likely to encode a direct-acting oncogene or transforming gene. To search for a possible candidate(s), early studies focused on genes expressed in tumor cells or transformed cells. In general, in these cells where there is no virus production, the transcriptional activities are confined to the repeat regions only, which, based on BamHI digestion map, span the BamH, -I2, -Q2, -L, and -A fragments (Ross 1999). Within this region, most of the open reading frames are short and their existence as proteins not conclusively resolved. The exceptions are pp38, ICP4, and Meq, for which the protein products have been clearly identified.It should be noted that most of these studies were carried out using the entire population of tumor cells and cell lines; it is not clear whether all these proteins are expressed in the same or different cell populations. This is an important issue, as in any given latent state, there is usually a fraction of cells, spontaneously releasing viruses, which may contribute to the detection of some lytic gene products. In surveying through the literature, Meq, the focus of this chapter, emerges as one that is most consistently expressed in all tumors and transformed cell lines, both at the transcript and at the protein level. Meq has a structure resembling nuclear oncogenes and, as will be described in detail below, shared properties, characteristic of oncogenes.

RETROTRANSPOSITION AND HERPESVIRUS EVOLUTION

One of the more interesting developments in herpesvirus evolution concerns the acquisition of novel, non-ubiquitous herpesvirus genes. A number of these are related to known cellular genes. How did herpesviruses acquire such genes? Our recent demonstration of retrovirus integration into herpesviruses suggests a potentially important role for retrotransposition in herpesvirus evolution and in the acquisition of novel genes, cellular in origin. Herpesvirus genome development has been characterized by a number of structural and evolutionary properties that support this proposal. We first discuss the evidence for retroviral integration into herpesviruses. The functional significance of this phenomenon is presently unclear. However, in the broader context of retrotransposition, a number of attractive features serve to explain the capture of structural and regulatory elements throughout herpesvirus evolution. These possibilities are discussed in detail.

Transcriptional Analysis of Marek's Disease Virus Glycoprotein D, I, and E Genes: gD Expression Is Undetectable in Cell Culture

ABSTRACT The various alphaherpesviruses, including Mareks disease virus (MDV), have both common and unique features of gene content and expression. The entire MDV Us region has been sequenced in our laboratory (P. Brunovskis and L. F. Velicar, Virology 206:324–338, 1995). Genes encoding the MDV glycoprotein D (gD), glycoprotein I (gI), and glycoprotein E (gE) homologs have been found in this region, although no gG homolog was found. In this work, transcription of the tandem MDV gD, gI, and gE genes was studied and found to have both unique characteristics and also features in common with other alphaherpesviruses. MDV gD could not be immunoprecipitated from MDV GA-infected duck embryo fibroblast cells by antisera reactive to its TrpE fusion proteins, while gI and gE could be. When the gD gene was subjected to in vitro-coupled transcription-translation, the precursor polypeptide was produced and could be immunoprecipitated by anti-gD. Northern blot, reverse transcriptase PCR, and RNase protection analyses have shown that (i) no mRNA initiating directly from the gD gene could be detected; (ii) a large but low-abundance 7.5-kb transcript spanning five genes, including the one encoding gD, was seen on longer exposure; and (iii) transcription of the gI and gE genes formed an abundant bicistronic 3.5-kb mRNA, as well as an abundant 2.0-kb gE-specific mRNA. Therefore, the MDV gD gene expression is down-regulated at the transcription level in MDV-infected cell culture, which may be related to the cell-associated nature of MDV in fibroblast cells. Compared to the highly gD-dependent herpes simplex virus and the other extreme of the varicella-zoster virus which lacks the gD gene, MDV is an intermediate type of alphaherpesvirus.