key: cord-0809392-nb4437qs
authors: Omel’yanchuk, L. V.; Yudina, O. S.
title: Drosophila melanogaster as a model for studying the function of animal viral proteins
date: 2011-07-16
journal: Russ J Genet
DOI: 10.1134/s1022795411040090
sha: a1e2a99eb903f9e37a119320d21c890e965f0222
doc_id: 809392
cord_uid: nb4437qs

Studies in which Drosophila melanogaster individuals carrying transgenes of animal viruses were used to analyze the action of animal viral proteins on the cell are reviewed. The data presented suggest that host specificity of viruses is determined by their proteins responsible for the penetration of the virus into the cell, while viral proteins responsible for interactions with the host cell are much less host-specific. Due to this, the model of Drosophila with its developed system of searching for genetic interactions can be used to find intracellular targets for the action of viral proteins of the second group.

Drosophila melanogaster is a well studied model organism containing (according to the Drosophila genome project) 10000-12000 genes [1] . Many of these genes are similar to genes of other animals as well as to human genes. It was found that 70% of Droso phila genes have human homologs [2] and 75% of human disease associated genes have homologs in Drosophila [3] . Irrespective of currently available large collections of mutant strains, it becomes more conve nient to study the function of Drosophila genes with the use of rather elaborate transgenic constructs [4] . The system of ectopic expression of Gal4 UAS genes consists of two components: a driver (P element con taining the yeast Gal4 protein coding region under the control of the genomic enhancer located close to the integrated element) and a target transgene under the control of the Gal4 specific promoter UAS (upstream activated system) [5] . Drosophila has a set of drivers (hs Gal4) able to activate UAS transgenes in various tissues at different stages of development. There is also a possibility to activate the tissue specific transgene conditionally using an external stimulus and even to form a feedback loop for autoactivation of the trans gene (UAS Gal4). These possibilities, initially devel oped for genetic analysis of Drosophila genes, can also be used to analyze the functions of foreign genes.

Viral genomes can maximally encode four to sev eral hundred proteins (the genome of Mimivirus is 1.2 megabases in size and contains 1262 reading frames corresponding to 911 proteins [6] ), although most typical is the influenza virus encoding 11 proteins [7] . Viral genomes encode viral envelope proteins responsible for interactions of the virus with the host cell at the stage of penetration into the cell. At the same time, it is commonly known that in addition to these proteins serving the structural function viral genomes contain genes responsible for finer aspects of interaction with host cells: cell proliferation (in fact, isolation of oncogenes was made on the basis of this property) and cell apoptosis. In this work, we review the results and methodical approaches of studies in which Drosophila was used to analyze human viral infections.

Drosophila was successfully used to study viral genes and proteins inhibiting programmed death (apoptosis) of host cells. Animal viruses are known to carry genes able to prevent apoptosis of host cells [8] [9] [10] [11] [12] [13] [14] [15] [16] . On this basis, Hay et al. [17] cloned the gene P35 of the Autographa californica baculovirus in the pGMR (glass multimer reporter) vector able to pro vide transgene overexpression in the eye. The effect of transgene overexpression in embryogenesis was exam ined in individuals containing an insertion of this vec tor and additionally carrying the hs glass transgene capable of activating the pGMR transgene in all tissues under the action of heat shock. Heat shock induction in such individuals resulted in the lack of apoptosis in late embryos, while it is normally observed in wild type individuals [18] . To study apoptosis of eye cells, activation of the pGMR transgene was used. Inhibition of apoptosis characteristic of normal development was demonstrated on the level of eye morphology of an adult individual and on the imaginal disc level. Inhibi OMEL'YANCHUK, YUDINA tion of apoptosis induced by X irradiation was also demonstrated. At present, constructs based on P35 serve as a commonly accepted test for classifying cell death as apoptosis.

Drosophila was successfully used to study HIV infec tion. The authors of the work [19] cloned the HIV tat gene in the pCasPeR vector containing the hs promoter capable of heat shock activation of transgene expression in all tissues. Activation of the transgene in oogenesis resulted in an essential increase (from 1 to 10%) of the share of eggs with the phenotype of fused dorsal appendages. The authors found that the Tat protein affects the localization of the Gurken and Kinesin pro teins, i. e., it alters both the dorsoventral and anteropos terior axes of embryos. Activation of transgene expres sion in spermatogenesis showed colocalization of Tat with alfa tubulin. Furthermore, the authors established in an in vitro experiment that Tat inhibits the assembly of microtubules. The study of the viral protein in accor dance to the commonly accepted scheme showed that it functions in the process of viral RNA translation [20] . The study with the use of the Drosophila model made it clear that in addition to this function the protein is also able to change the cell structure.

To study the role of the HIV nef gene in HIV infec tion, Lee et al. [21] cloned DNA of this gene and DNA of its mutant form (G2A substitution altering the myris tolation site of the protein) into the pUAST vector and activated the transgene with the use of drivers 1096 Gal4 and ap Gal4 in wing imaginal discs. Expression of the wild type gene led to abnormalities in the wing shape, while no abnormalities were observed in the mutant. Using BrdU incorporation and detection of mitoses with the help of antibodies against the phosphorylated H3 his tone form, it was found that ectopic expression of HIV nef itself and its mutant form had no influence on wing cell proliferation. Yet, ectopic expression increased the level of apoptosis, which was seen with the use of both acridinorange detection and antibodies against Drosophila Drice caspase. The authors found that genes involved in the JNK (June N terminal kinase) signaling pathway (bsk, hep) genetically interact with HIV nef (both UAS con structs and point mutations) under conditions of overex pression, and the activity of the puc gene, which is a target for the action of bsk, is enhanced in the presence of an additional amount of the HIV nef gene product. Since the activity of the JNK signaling pathway in Drosophila is characterized by a complicated spatial regulation (for example, dorsal and thorax closures regulated by JNK are complicated events of morphologcal alterations of an embryo or a growing pupa), the authors also checked cel lular autonomy of the action of HIV nef with the help of the mosaic analysis. Then the authors established that HIV nef, but not its mutant form, inhibits expression of the antimicrobial peptide Dpt of Drosophila. The protein Relish (transcription factor NF kB) of Drosophila, which controls Dpt transcription, is translocated from the cyto plasm into the nucleus in the case of microbial infections. HIV nef expression inhibits this change in the localization of Relish. The authors also studied the question as to whether inhibition of Drosophila immunity is JNK dependent and showed that the effects of HIV nef on immunity and on JNK are independent of one another. Thus, despite a small length (206 a.a.), this viral protein has two different targets in animal cells.

Leulier et al. [22] cloned cDNA of the HIV vpu gene and its mutant form (Ser52 and Ser56 substituted by Arg), which does not permit phosphorylation of the product, into the pUAST vector. The transgene was expressed with the use of the yolk GAL4 driver activating expression in the fat body. In human cells, HIV vpu is found in the phosphorylated and nonphosphorylated forms. After treatment with alkaline phosphatase, the difference in the mobility of the proteins disappears. The authors showed that in Drosophila cells the protein behaves analogously, i. e., it can be phosphorylated. This fact by itself is a strong argument in favor of using Droso phila as a model object for studying viral infections. In Drosophila, responsible for resistance to fungal infections is the Toll signaling pathway controlling expression of the Drs and Mtk peptides [23] . Induction of these peptides is essentially disturbed in individuals with HIV vpu expres sion, but is not disturbed in the nonphosphorylatable mutant. In support of this, the authors showed that the dominant Tl 10B mutation of the Toll signaling pathway, which causes connstitutive activation of Drs and Mtk, does not produce this effect against the background of Vpu overexpression. At the same time, the authors found that the resistance to gram positive bacteria associated with the Imd signaling activity does not change in the case of HIV vpu overexpression.

The data on the ability of the Drosophila model to reveal new aspects of HIV-host cells interactions were considered by Spresser and Carlson [24] . The authors proposed to study additionally genes CCR3, Vpr, CA150, Pol gamma, GL1, P TEFb, and Spt 4, 5, 6, since they have homologs in the Drosophila genome and may well be of interest for studying HIV infections.

Drosophila was also used as a model object for studying infections caused by other viruses. To study the functional role of the 3a gene of the SARS (Severe Acute Respiratory Syndrome) coronavirus encoding a 274 a.a. protein (expression of the protein was con firmed for patients infected by this virus), a transgenic Drosophila strain containing a genomic insertion of the gene of this protein in the pUAST vector was gen erated in the work [25] . Ectopic expression of the 3a protein in Drosophila under the action of the eye driver GMR Gal4 caused the dominant phenotype of the eyes. It was found that ectopic expression of 3a influ ences the vesicular cell traffic and induces cell apopto sis that can be modulated by the level of cellular cyto chrome C and by the activity of caspases destroying cellular structures during apoptosis. As shown by genetic screening, the phenotype resulting from ectopic expression is modified by heterozygous muta tions at 78 loci of Drosophila. Thus, the use of trans genic Drosophila individuals was proved to be efficient for studying the function of viral genes.

The Epstein-Barr virus causes infectious mononu cleosis, lymphomas, and nasopharyngeal carcinoma. The BZLF1 protein functions as a transcriptional acti vator of expression of early genes of the virus and con trols the transition from the latent form to the lytic form. BZLF1 also interferes with the cell cycle regula tion, intercellular signaling, and transcription. To study the function of BZLF1, Adamson et al. cloned cDNA of the gene into the pGMR vector [26] . The authors also cloned cDNA of the mutant form of the A185L gene, which alters the DNA binding domain of BZLF1 and leads to an experimentally recorded bind ing defect. Several insertions differing in the level of expression of GMR BZLF1 and UAS BZLF1 trans genes were obtained.

The authors showed that insertions of the con structs obtained into the Drosophila genome bring about morphological defects in the Drosophila eye as a result of the disappearance of ommatidia. Morpholog ical defects of the eye for normal and mutant BZLF1 did not differ, thus confirming that the viral protein acts not through transcription regulation, as was expected from the molecular function of the protein, but in some other manner [26] . The study of cell divi sions in transgenic individuals showed a reduction in the number of dividing cells in the second mitotic wave in the eye imaginal disc and an intensification of cell apoptosis. This is in good agreement with the data obtained for human cells that demonstrate a cell cycle arrest under the action of BZLF1.

Then Adamson et al. [26] checked, using antibod ies against the Drosophila eye protein Elav, whether BZLF1 acts on differentiating photoreceptors of the eye. It turned out that the protein had no influence on the cells of the photoreceptors at this, later, stage. In addition, the authors activated the UAS BZLF1 transgene with the use of the sev Gal4 driver (it acts later than elav) and confirmed the lack of the eye phe notype. It was further found that in the case of BZLF1 overexpression the eye conic cells lose Cut protein expression. The protein Shaven (encoded by the sv gene) normally activates the expression of cut in these cells by binding with its promoter. Using anti Shaven antibodies, the authors demonstrated that BZLF1 expression affects the localization of Shaven and the level of its expression. The authors assumed that the virul protein influences the process of Cut transactiva tion with the help of Shaven. By comparing the eye morphology in sv/+ and BZLF1/+; sv/+ individuals, the authors revealed genetic interactions between sv and BZLF1. On the basis of this finding, the authors proceeded to studying human homologs of Shaven: proteins Pax2, Pax5, and Pax8. Since the Epstein-Barr virus infects B cells of the human immune sys tem, in which Pax5 is functional, rather than the Pax2 and Pax8 homologs, the authors tried to elucidate whether the proteins BZLF1 and Pax5 form a com plex. It was found that these proteins are able to form a complex and that the formation of a complex causes binding of BZLF1 with mitotic chromosomes. Thus, the authors of this work succeeded in discovering and characterizing the human virul protein with the employment of the methods of molecular genetics of Drosophila. The results obtained and the existence of homology between the human and Drosophila proteins enabled the authors to return to studying the action of the human proteins.

Embryonic infection by the human cytomegalovirus (HCMV) leads to microencephalia, microgyria, and hydrocephalus [27] . Transgenic constructs were obtained in the work [28] , of which one contained the Major Immediate Early promoter (MIEP) of HCMV attached to the coding region of the YFP reporter. Another con struct contained two viral genes, IE72 and IE86, under the control of the hs promoter of the pCasPeR vector. The promoter activity was detectable only in early embryos at the stage of blastoderm. This fact agrees well with the data on the expression of this promoter in mouse embryos and is a strong argument in favor of using Droso phila as a model object for studying viral infections. Moreover, the viral proteins under study were found to be localized in the nucleus. Such a localization is expected for transcription factors. Overexpression of the construct containing viral genes leads to the death of 20-30% of embryos with a phenotype called by the authors "frag mented embryo." Normally, DE cadherin in Drosophila binds to the Arm protein. Both proteins are major com ponents of intercellular contacts (adherence junctions). While the localization of DE cadherin in transgenic embryos overexpressing a cassette of viral transgenes remains unchanged, the pattern of Arm expression rep resenting a system of bands changes up to becoming uni form. The authors concluded that the viral transgenes alter the composition of adherence junctions in embryos.

The rabies virus, able to infect different mammals, is considered to have the widest range of hosts among ani mal viruses. At the same time, the action of many animal viruses is species specific [29] . The question arises as to whether the range of hosts is limited by genes determin ing the penetration of the virus into the cell or whether viral genes responsible for virus-host interactions also OMEL'YANCHUK, YUDINA make a contribution to this limitation. The data consid ered in this review show that viral genes of the latter group can act on a broad diversity of organisms, including Drosophila. In particular, the Vpu protein of HIV can be phosphorylated in Drosophila cells, and the cytomega lovirus promoter (MIEP) functions only in embryonic tissues of Drosophila, which coincides with the phase of its action in mammals (see above). These facts cannot be accidental, and they indicate that the class of viral genes responsible for the interaction of the virus with host cells is similar to house keeping genes that perform the same functions in all animals. This means that host specificity is mainly a result of species specificity of proteins responsible for the penetration of the virus into the cell.

The range of intracellular targets for genes of the second type is rather wide: cell apoptosis, assembly of microtubules, JNK signaling, cellular immunity, intercellular contacts, and vesicular traffic. It is hard to imagine that this class of viral proteins influencing all this diversity of cell functions occurs in viruses de novo. It is most likely that genes of this type are adopted by a persisting virus from the host genome, as can be often seen from homology of the primary struc tures. On the other hand, the study of the functions of viral proteins in Drosophila can help in revealing the units of regulation of intracellular processes that have not yet been identified in direct studies.

Recall that Adamson et al. [26] succeeded in discov ering and characterizing the human virul protein by the methods of molecular genetics of Drosophila, and, pro ceeding from the results obtained and on the basis of homology of the human and Drosophila proteins, they could return to studying the action of the virus on the human protein. Thus, Drosophila can be used as an inter mediate object for studying human viral infections. What tasks can be solved at this stage? First, ectopic expression of transgenes in Drosophila is possible at any stage of development and in any tissue. This permits one to find a tissue and a developmental stage having targets for the action of viral proteins. Of course, there are other objects more similar to humans (e. g., mouse) that can be used in such studies, but the generation of a transgenic mouse is incomparable in costs with the generation of a transgenic Drosophila fly. Second, Drosophila has an elaborate sys tem of genetic screenings for discovering genetic interac tions. Cases of application of this approach can be found in the works [21, 25] . Genetic methods permit a more complete characterization of the spectrum of targets for the action of viral proteins. They also make it possible to discover weak interactions that can be revealed only by the modern methods detecting physical binding of mac romolecules.

Around the Genomes: The Drosophila Genome Project

Developmental Control of Cell Cycle Regulators: A Fly's Perspective

A Systematic Analysis of Human Disease Associated Gene Sequences in Drosophila melanogaster

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The 19 Kilodalton Adenovirus E1B Transforming Protein Inhibits Programmed Cell Death and Prevents Cytoly sis by Tumor Necrosis Factor Alpha

A 14.700 MW Protein from the E3 Region of Adenovirus Inhibits Cytolysis by Tumor Necrosis Factor

The 10.400 and 14.500 Dalton Proteins Encoded by Region E3 of Adenovirus Function Together to Protect Many but not All Mouse Cell Lines Against Lysis by Tumor Necrosis Factor

The E1B 19K Protein of Group C Adenoviruses Prevents Cytolysis by Tumor Necrosis Factor of Human Cells but not Mouse Cells

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Nef Induces Apoptosis by Activating JNK Signaling Pathway and Inhibits NF B Dependent Immune Responses in Droso phila

Directed Expression of the HIV 1 Accessory Protein Vpu in Drosophila Fat Body Cells Inhibits Toll Dependent Immune Responses

Two Distinct Pathways Can Control Expression of the Drosophila Antimicrobial Peptide Metchnikowin

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In vivo Func tional Characterization of the SARS Coronavirus 3a Protein in Drosophila

Modeling Early Epstein-Barr Virus Infection in Droso phila melanogaster: The BZLF1 Protein

Outcome of Symptomatic Congenital Cytomegalovirus Infection, Results of Long Term Longitudinal Follow Up

Human Cytomegalovirus Immedi ate Early Gene Expression Disrupts Embryogenesis in Transgenic Drosophila

Understanding Viruses