Search

Byline: V. S. Mikhailov (1,2) Keywords: baculoviruses; DNA replication Abstract: The review describes the current state of studying the baculovirus DNA replication. The structural organization of replication initiation sites and replication intermediates are considered. Attention is focused on virus replication factors, including DNA polymerase, helicase, IE-1, LEF-1, LEF-2, and LEF-3. Author Affiliation: (1) Kol'tsov Institute of Developmental Biology, Russian Academy of Sciences, Moscow, 117808, Russia (2) Oregon State University, Corvallis, Oreg., 97331, United States Article History: Registration Date: 12/10/2004

Research has been conducted on Gal/GlcNAc-6-O-sulfotransferases (GSTs). The development of baculovirus expression system for several cloned GSTs and the use of thin-layer chromatography and polymer dot-blot assay in investigating the kinetic parameters of GSTs are described.

Byline: S. N. Beljelarskaya (1) Keywords: baculoviruses; nuclear polyhedrosis virus; Spodoptera frugiperda cells; polyhedrin; vector DNA; recombinant proteins; expression Abstract: The review considers the biology of baculoviruses, construction of transfer vectors for the baculovirus expression system (BES), selection of recombinant baculoviruses, approaches to expression of multimeric proteins, and BES potentialities and prospects. Author Affiliation: (1) Engelhardt Institute of Molecular Biology, Russian Academy of Sciences, Moscow, 119991, Russia Article History: Registration Date: 09/10/2004

A study that identifies new inhibitors of apoptosis (IAP) in viruses, nematodes and yeasts. IAP are proteins that prevent cell death during cell development or through viral infection, drugs, tumor necrosis factors and over-expression of caspases. The first isolated IAP genes were the baculovirus genes CpIAP and OpIAP that are used by the virus in reproduction in host cells. All IAP genes have baculoviral IAP repeats and also a RING-finger domain and a zinc-binding motif.

The genetically-altered strain of Nucleopolyhedrovirus (SeMNPV) that has been isolated from Spodoptera exigua could recombine with parental viruses to produce new genotypes if these viruses are introduced into natural populations of the virus. The new genotypes could replace the natural strains and they could also have a different biological activity. It is therefore prudent the effect of SeMNPV before they are used as natural insecticides for S. exigua.

Apoptosis can be a potent weapon against viral infection and consequently has selected for viruses carrying antiapoptosis genes. Two baculovirus proteins, IAP and p35, can prevent insect cells from dying in response to infection. p35, which interferes with members of the Ced-3 family of cysteine proteases, can also function in mammalian cells. We investigated the ability of IAP from Orgyia pseudo-tsugata nuclear polyhedrosis virus to prevent death of mammalian cells. IAP was transiently expressed in mammalian cells and its ability to block cell death caused by expression of interleukin-1[Beta] converting enzyme (ICE), FADD, or the ICE homologues ICH-1 and ICE-Lap3, was investigated. IAP strongly inhibited ICE- and ICH-1-induced cell death but protected only partially against death by overexpression of FADD and not at all against death due to enforced ICE-Lap3 expression. These results demonstrate that a baculoviral IAP protein can functionally interact with conserved components of the apoptosis machinery in mammalian cells.

JAK2, a member of the Janus kinase super-family was found to interact functionally with Raf-1, a central component of the ras/mitogen-activated protein kinase signal transduction pathway. Interferon-[Gamma] and several other cytokines that are known to activate JAK2 kinase were also found to stimulate Raf-1 kinase activity toward MEK-1 in mammalian cells. In the baculovirus coexpression system, Raf-1 was activated by JAK2 in the presence of [p21.sup.ras]. Under these conditions, a ternary complex of [p21.sup.ras], JAK2, and Raf-1 was observed. In contrast, in the absence of [p21.sup.ras], coexpression of JAK2 and Raf-1 resulted in an overall decrease in the Raf-1 kinase activity. In addition, JAK2 phosphorylated Raf-1 at sites different from those phosphorylated by [pp60.sup.v-src]. In mammalian cells treated with either erythropoietin or interferon-[Gamma], a small fraction of Raf-1 coimmunoprecipitated with JAK2 in lysates of cells in which JAK2 was activated as judged by its state of tyrosine phosphorylation. Taken together, these data suggest that JAK2 and [p21.sup.ras] cooperate to activate Raf-1.

Baculovirus inhibitors of apoptosis (IAPs) act in insect cells to prevent cell death. Here we describe three mammalian homologs of IAP, MIHA, MIHB, and MIHC, and a Drosophila IAP homolog, DIHA. Each protein bears three baculovirus IAP repeats and an N-terminal ring finger motif. Apoptosis mediated by interleukin 1[Beta] converting enzyme (ICE), which can be inhibited by Orgyia pseudotsugata nuclear polyhedrosis virus IAP (OpIAP) and cowpox virus crmA, was also inhibited by MIHA and MIHB. As MIHB and MIHC were able to bind to the tumor necrosis factor receptor-associated factors TRAF1 and TRAF2 in yeast two-hybrid assays, these results suggest that IAP proteins that inhibit apoptosis may do so by regulating signals required for activation of ICE-like proteases.

The Autographa californica multiple nuclear polyhedrosis virus (ACMNPV) may be useful in introducing genes into mammalian cells. Its effectivity as a vector was observed when AMNPV was loaded with a reporter gene and allowed to infect different mammalian cell lines. While most of the cell lines afterwards expressed the transferred gene in limited amounts, only hepatocytes were found to have produced high amounts of beta-galactosidase.

The vaccinia virus VH1 gene encodes a phosphatase which is highly similar to eukaryotic p80(cdc25), a cell cycle-regulating phosphatase. Homologous VH1 sequences were identified in the racoonpox virus, the smallpox variola virus and other orthopoxviruses as well as one baculovirus. Sequence analysis, protein expression and Western blot analysis showed that the proteins encoded by the VH1 analogs have conserved active sites.

The T-urf13 gene, which encodes the URF13 protein in the mitochondria of male-sterile maize carrying the Texas cytoplasm (cms-T), was cloned in a baculovirus expression system. URF13 interacts with fungal T toxin or methomyl to permeabilize the plasma membranes of insect cells. In addition, URF13 is toxic when expressed in either cell cultures or insect larvae, even without T toxin or methomyl. The results indicate that male sterility and toxin sensitivity in cms-T maize may be due to independent mechanisms.

Full length and truncated baculovirus 2 (BCL2) found in insect cells was overexpressed in a study done to determine its structure mechanism of action. Results show that the overexpression of the full-length but not the truncated BCL2 extended the survival of the baculovirus-infected cells by preventing virus-induced deoxyribonucleic acid cleavage. These results suggest that BCL2 uses hydrophobic domains together with the deleted domains to associate with subcellular membranes.

The gene coding for juvenile hormone esterase (jhe) was inserted into the genome of a Baculovirus containing a synthetic viral promoter. JHE was detected 12 hours after infection of Spondoptera frugiperda cells. Immunoblotting showed that the JHE produced was heterogenous, due probably to differential glycosylation. The recombinant gene was also expressed in vivo, however, no significant differences in weight, development or time of mortality was detected between larvae infected with jhe-expressing or -non-expressing virus.

Research reveals that a genomic parasite in artificial nucleopolyhedrovirus cell cultures is maintained through cell propagation in insects. Results show that a few-polyhedra mutant of nucleopolyhedroviruses is able to persisit in equilibrium with the wild-type strain during serial rounds of infection.