Current Drug Targets - Volume 8, Issue 10, 2007

The field of baculovirus research has provided many important and novel insights into virus-host interactions and pathology, DNA virus transcriptional regulation and replication mechanisms, virus/host co-evolution and genome interactions, and basic eukaryotic cellular functions such as apoptosis and nuclear importation. Many of these insights have already proven beneficial in developing the baculovirus expression vector system and derivative protein expression systems for insect cells, and for guiding similar observations in human disease systems. The impact of the insect cell-based protein expression systems for eukaryotic gene expression and rapid characterization of gene products is enormous, and continues to provide a rich avenue of practical research and biotechnology development. All in all, the relatively small research community that has studied baculovirus systems over the years has contributed abundantly to both fundamental and practical science. The baculovirus replication cycle is a relatively complex program of regulated transcription that involves the coordinate activation and/or repression of over 40 genes, some of which are highly productive for their gene products. The highly productive p10 and polyhedrin genes have been harnessed for protein expression in infected cells. However, the complex regulation that leads to their abundant expression has yet to be duplicated outside the context of the infected cells. Such a virus-free system could present a significant advancement for protein expression in insect cells, and can only be achieved with more and better understanding of the baculovirus regulatory cascade. This issue of Current Drug targets focuses attention on current Baculovirus research in an effort to not only review what has been discovered, but to also provide a foundation for further useful discoveries in this biotechnologically relevant system. The article by Herniou and Jehle on baculovirus phylogeny and evolution covers our current state of knowledge about the relatedness of baculoviruses and their co-evolution with their hosts from a sequence analysis perspective, while van Oers and Vlak review the functional relatedness and organization of genes among the baculoviruses. Three articles exemplify the importance of baculovirus research in defining important aspects of cellular function during DNA virus replication. Clem discusses the relationship between baculovirus pathology and apoptosis, which follows upon observations that were among the first to be made for suppression of apoptosis in a virus/host system. Volkman details the role of nuclear actin in baculovirus pathology and assembly, an observation that may have relevance for other nuclear replicating viruses. Braunagel and Summers elucidate the molecular biology of occlusion-derived virus envelope assembly, and in the process define an integral membrane protein trafficking pathway involving a unique importin first identified in this system. Two articles review transcriptional regulation and replication of these viruses. Vanarsdall, Mikhailov, and Rohrmann present our current understanding of DNA replication among the baculoviruses, defining the function of several important gene products in this process. Passarelli and Guarino review the extensive database of information characterizing the activity and function of late and very late genes important for the regulation of baculovirus host range, activation of polyhedron and p10 promoters, and replication of the genome. Finally, two articles focus on baculoviruses as protein expression systems. Shi and Jarvis discuss the characteristics of the N-glycosylation pathway in the baculovirus-insect cell system and some practical solutions to correcting differences between this pathway and that of mammalian cells. Condreay and Kost review the current state of baculovirus expression vector technology as well as the accumulating evidence for baculoviruses as safe and effective gene transduction vectors for animal cells. It is my hope that this issue of Current Drug Targets will enlighten the readers with the many exciting observations that have flowed from this unique and valuable biological system, and will help stimulate continued research interest in this productive experimental model.

The family Baculoviridae represents one of the largest and most diverse groups of viruses and a unique model for studying the forces driving the evolution and biodiversity of double-stranded DNA viruses with large genomes. With the advent of comparative genomics, the phylogenetic relationships of baculoviruses have been put on solid bases. This, as well as improved bioinformatic approaches, has provided a detailed picture of baculovirus phylogeny and evolution. According to the present knowledge, baculoviruses can be classified into at least four evolutionary lineages: the most ancestral dipteran nucleopolyhedroviruses, the hymenopteran nucleopolyhedroviruses and the lepidopteran nucleopolyhedroviruses and granuloviruses. Despite the growing understanding of baculovirus phylogeny and macro-evolution, our knowledge of the micro-evolutionary processes within baculovirus species and virus populations is still limited. Here we present the state of the art on baculovirus phylogeny and evolution.

Baculovirus genomes are covalently closed circles of double stranded-DNA varying in size between 80 and 180 kilobase-pair. The genomes of more than fourty-one baculoviruses have been sequenced to date. The majority of these (37) are pathogenic to lepidopteran hosts; three infect sawflies (Hymenoptera) and one has a mosquito host (Diptera). With this information, general patterns of genome structure and gene content became apparent. Baculovirus open reading frames are tightly packed with minimal intergenic regions and the coding sequences are almost equally distributed over both strands. Baculovirus genes form single transcription units, with early and late transcribed ORFs scattered along the genome. A set of twenty nine core genes is conserved and therefore is characteristic for baculoviruses. Most baculovirus genomes contain multiple homologous regions with repeated sequences and often palindromic motifs, which play a crucial role as enhancers of early transcription and most likely in viral DNA replication. Baculovirus genomes have a certain degree of plasticity, as evidenced from the genomic variations within virus isolates from the field. Recombination events and transposon insertions appear to play a role in the uptake of new genes from co-infecting viruses or from the insect host. This review deals with the structural and functional properties of baculovirus genomes including both conserved and variable genes.

Apoptosis is used by metazoan organisms to dispose of damaged or unnecessary cells during development, tissue homeostasis, and disease. One of the situations where apoptosis is important is in defense against microbial pathogens, especially viruses. The demonstration that apoptosis could be stimulated by baculovirus infection was one of the first examples of apoptosis associated with virus infection, and this system remains one of the most valuable for studying how apoptosis can be a defense against viruses. In addition, studying how baculoviruses regulate apoptosis has led to many important findings in the field of apoptosis research, such as the discovery of P35, a caspase inhibitor that is widely used in studies of apoptosis, and IAP (inhibitor of apoptosis) proteins, which have homologs in cellular genomes that play important roles in regulating apoptosis and cytokinesis. This review highlights the range of apoptotic responses observed between different baculoviruses and different lepidopteran insects, and the diverse baculovirus genes that have evolved to regulate apoptosis.

Baculovirus infection occurs when susceptible insect larvae ingest viral occlusions and occlusion-derived virus is released in the midgut lumen. Midgut columnar epithelial cells (the sole targets) are penetrated when the viral envelopes fuse with microvillar membranes; subsequently, nucleocapsids are transported basally through the microvilli toward the nucleus where replication ensues. Rapid infection of trachael cells (secondary targets) is under heavy selection because midgut cells are sloughed, an effective defense against systemic infection. The unique multiple nucleocapsid per virion trait acquired by some baculoviruses functions in countering this defense. Systemic infection is amplified after infected tracheal cells transmit infection to hemocytes. Tracheal cells serve as the conduit for virus spread through basal laminal barriers. Discordant susceptibilities to infection of midgut cells, tracheal cells and hemocytes may exist within an individual insect; in fully permissive hosts, all are highly susceptible. Viral manipulation of the actin cytoskeleton both during nucleocapsid transport and after viral gene expression is at the core of successful infection and replication. G-actin, normally cytoplasmic, is efficiently localized within the nucleus during early viral gene expression, and nuclear actin polymerizes during late gene expression, concurrent with shut down of host protein synthesis and early viral gene expression. Nuclear G-actin is now considered essential for cellular transcription and nuclear F-actin can affect transcription by binding chromatin-remodeling complexes. A new hypothesis is offered for how viral manipulation of actin influences timing of viral gene transcription, genome processing and packaging.

Study of the biology of the occlusion-derived virus (ODV) of the baculovirus Autographa californica nucleopolyhedrovirus provides opportunities to reveal new discoveries into the mechanism of several cellular pathways. The synchronous pulse of multiple ODV envelope proteins that integrate into the endoplasmic reticulum (ER) and traffic to the nuclear membranes on their way to the ODV envelope provide a unique tool to study the mechanisms of integral membrane protein trafficking from the ER to the outer and inner nuclear membrane. Studies of the formation of virus-induced, intranuclear membrane microvesicles provide insight on mechanisms that alter fluidity and regulate budding of the inner nuclear membrane. Since ODV is specially adapted for primary infection of the insect gut, studies of the structure and function of ODV envelope proteins reveals insights on the mechanism of viral invasion of the gut and this knowledge is fundamental for the development of new strategies for insect control. This review focuses on recent advances in understanding the source of the ODV envelope and the molecular events that sort and traffic integral membrane proteins from the ER to the ODV envelope. The composition of ODV is reviewed, however it is worth noting that the function of many ODV proteins are currently unknown.

In this report, factors involved in baculovirus DNA replication are reviewed. These include factors that are required for DNA synthesis, other factors that have been implicated in genome processing or packaging, and homologs of proteins that are involved in DNA replication or repair in other systems. Conservation of a number of these factors in all baculovirus genomes suggest that many of the observations for specific viral systems may apply to the most if not all members of the Baculoviridae.

Baculoviruses have adapted novel tactics to transcribe their genes during the late stages of replication. These include a DNAdirected RNA polymerase which is evolutionarily divergent from cellular polymerases. The viral RNA polymerase is a multisubunit and multifunctional RNA polymerase that has the ability to recognize late promoters, transcribe the linked genes, and process the transcripts at both 5' and 3' ends. The viral RNA polymerase binds to late viral gene promoter elements that are compact and differ from early viral gene and cellular promoters. Baculoviruses also encode a number of proteins devoted to the synthesis of late transcripts. Many of these are highly conserved among all the baculovirus genomes sequenced to date, suggesting common transcription mechanisms. Although viral late mRNAs resemble host mRNAs, the transcribing/processing machinery is distinct. Characterization of the late gene transcription apparatus will elucidate new viral mechanisms for transcription.

One of the major advantages of the baculovirus-insect cell system is that it is a eukaryotic system that can provide posttranslational modifications, such as protein N-glycosylation. However, this is a vastly oversimplified view, which reflects a poor understanding of insect glycobiology. In general, insect protein glycosylation pathways are far simpler than the corresponding pathways of higher eukaryotes. Paradoxically, it is increasingly clear that various insects encode and can express more elaborate protein glycosylation functions in restricted fashion. Thus, the information gathered in a wide variety of studies on insect protein N-glycosylation during the past 25 years has provided what now appears to be a reasonably detailed, comprehensive, and accurate understanding of the protein Nglycosylation capabilities of the baculovirus-insect cell system. In this chapter, we discuss the models of insect protein N-glycosylation that have emerged from these studies and how this impacts the use of baculovirus-insect cell systems for recombinant glycoprotein production. We also discuss the use of these models as baselines for metabolic engineering efforts leading to the development of new baculovirus- insect cell systems with humanized protein N-glycosylation pathways, which can be used to produce more authentic recombinant N-glycoproteins for drug development and other biomedical applications.

Functional expression of recombinant proteins has become a routine, but critical tool in modern molecular biology. Since their introduction, the use of baculovirus vectors to produce proteins for purification has become one of the most widely-used viral gene delivery systems as expression levels obtained are difficult to match with any other eukaryotic expression system. Extensive engineering to simplify and accelerate the process of recombinant virus construction has made this system acessible to virtually any modern biological laboratory. The utility of baculoviruses has been broadened with the discovery that appropriately modified virus can mediate gene expression in a wide variety of mammalian cell lines, and thus can function as a flexible cell-based assay development tool. The wide range of applications and potential for commercialization of products leads to consideration of a number of aspects of the system.

Cell viability and motility are critical for cancer progression. Among a plethora of mechanisms that regulate these phenotypes, the balance of water and monovalent metal cations plays a pivotal role in the dynamics of focal contacts and cytoskeletal rearrangements at the cell's leading edge. Furthermore, cell survival requires the optimal concentration of water and solutes. This balance is largely maintained by aquaporins (AQPs), a family of 13 small integral plasma membrane proteins whose major function is the transport of water and small solutes across the plasma membrane. We review the recent knowledge about the role of AQPs in cell migration, survival, tumor angiogenesis and metastasis with the focus on therapeutic possibilities to prevent these clinically unfavourable events. The review discusses the inhibition of AQP expression and/or AQP-mediated water influx by acetazolamide, cyclophosphamide, topiramate, thiopental, phenobarbital and propofol. Down-regulation of water transport by these drugs affects cancer cell migration and metastasis. We conclude that AQPs can be considered a point where the mechanisms of survival and motility converge. Therapeutic targeting of AQPs may thus be advantageous for blocking the mechanism common for a number of key cancer phenotypes.

Reducing sugars can react non-enzymatically with amino groups of protein to form Amadori products. These early glycation products undergo further complex reaction such as rearrangement, dehydration, and condensation to become irreversibly cross-linked, heterogeneous fluorescent derivatives, termed advanced glycation end products (AGEs). The formation and accumulation of AGEs have been known to progress at an accelerated rate in diabetes. There is a growing body of evidence that AGEs and their receptor (RAGE) axis is implicated in the pathogenesis of diabetic vascular complications. Indeed, the engagement of RAGE with AGEs is shown to elicit oxidative stress generation and subsequently evoke inflammatory responses in various types of cells, thus playing an important role in the development and progression of diabetic micro- and macroangiopathy. Moreover, administration of a recombinant soluble form of RAGE (sRAGE), has been shown to suppress the development of accelerated atherosclerosis in diabetic apolipoprotein E-null mice. These observations suggest that exogenously administered sRAGE may capture and eliminate circulating AGEs, thus protecting against the AGEs-elicited tissue damage by acting as a decoy receptor. Recently, endogenous sRAGE has been identified in humans. However, there is few comprehensive review about the regulation and role of endogenous sRAGE in diabetes. In the former part of this paper, we review the role of the AGE-RAGE system in the pathogenesis of diabetic vascular complications. Then we summarize in the latter part of this review the kinetics and pathophysiological role of endogenous sRAGE in diabetes. We also discuss the possibility that endogenous sRAGE may be a therapeutic target for the prevention of diabetic vascular complications.