Insect response to alphavirus infection—Establishment of alphavirus persistence in insect cells involves inhibition of viral polyprotein cleavage

Virus Research 150 (2010) 73–84

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Insect response to alphavirus infection—Establishment of alphavirus persistence in insect cells involves inhibition of viral polyprotein cleavage Usharani Mudiganti, Raquel Hernandez, Dennis T. Brown ∗ Department of Molecular and Structural Biochemistry, North Carolina State, University, Raleigh, NC 27695, USA

a r t i c l e

i n f o

Article history: Received 28 July 2009 Received in revised form 24 February 2010 Accepted 25 February 2010 Available online 7 March 2010 Keywords: Arbovirus Sindbis Insect cells Microarray Persistent infection

a b s t r a c t Alphavirus persistence in the insect vector is an essential element in the vector–host transmission cycle of the virus and provides a model to study the biochemical and molecular basis for virus–vector coexistence. The prototype alphavirus Sindbis (SV) establishes persistent infections in invertebrate cell cultures which are characterized by low levels of virus production. We hypothesized that antiviral factors may be involved in decreasing the virus levels as virus persistence is established in invertebrate cells. Transcription profiles in Drosophila S2 cells at 5 days post-infection with SV identified families of gene products that code for factors that can explain previous observations seen in insect cells infected with alphaviruses. Genomic array analysis identified up-regulation of gene products involved in intracellular membrane vesicle formation, cell growth rate changes and immune-related functions in S2 cells infected with SV. Transcripts coding for factors involved in different aspects of the Notch signaling pathway had increased in expression. Increased expression of ankyrin, plap, syx13, unc-13, csp, rab1 and rab8 may aid in formation of virus containing vesicles and in intracellular transport of viral structural proteins. Possible functions of these gene products and relevant hypotheses are discussed. We confirmed the up-regulation of a widespectrum protease inhibitor, Thiol-ester containing Protein (TEP) II. We report inhibition of the viral polyprotein cleavage at 5 days post-infection (dpi) and after superinfection of SV-infected cells at 5 dpi. We propose that inefficient cleavage of the polyprotein may, at least in part, lead to reduced levels of virus seen as persistence is established. © 2010 Elsevier B.V. All rights reserved.

1. Introduction Viruses belonging to Alphavirus genus (∼30 species family: Togaviridae) cause diseases with high fever, encephalitis, arthritis and hemorrhagia leading to death in birds and mammals. Female Aedes aegypti mosquitoes act as vectors transmitting the virus between vertebrate hosts (Strauss and Strauss, 1994). Infection of mosquitoes is food-borne and via transovarial transmission (Mitchell et al., 1992) which maintains the virus in nature, without the involvement of vertebrates. Following virus infection, mosquitoes survive the initial acute phase of infection and become persistently infected for life, with varying levels of virus production as well as viral clearance in various organs and tissues (Bowers et al., 1995, 2003). SV encodes a positive-sense, RNA genome of 11,703 nt, with a poly-A tail and 5 cap. Four nonstructural proteins (nsPs)—nsP1, 2, 3 and 4 are produced by SV, initially as nonstructural (ns) polyproteins nsP123 and nsP1234 (Strauss and Strauss, 1994). Protease

∗ Corresponding author at: Campus Box 7622, NC State University, 128 Polk Hall, Raleigh, NC 27695-7622, USA. Tel.: +1 919 515 5765; fax: +1 919 515 2047. E-mail address: dennis [email protected] (D.T. Brown). 0168-1702/$ – see front matter © 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.virusres.2010.02.016

activity encoded by nsP2 is utilized to produce the intermediate ns polyproteins and individual nsPs (de Groot et al., 1990; Ding and Schlesinger, 1989; Hardy and Strauss, 1989; Hardy et al., 1990). Information on dynamics of viral RNA replication has been obtained from studies involving infection of mammalian cells with polyprotein cleavage defective mutants of SV and a closely related virus, Semliki Forest Virus (SFV) or viral replicons. Three species of viral RNA are produced by the viral replicase-transcriptase complex(minus)-RNA that is complementary to genomic RNA, (plus)-RNA that forms the genome of the progeny virus and subgenomic RNA (sgRNA) that is translated to produce viral structural proteins. nsP123 and nsP4 synthesize (minus)-RNA with high efficiency (Shirako and Strauss, 1994). In BHK21, Vero and HeLa cells (minus)RNA synthesis ceases by 4 h post-infection (hpi), and (plus)-RNA and sgRNA are synthesized through out the infection cycle (Sawicki and Sawicki, 1980). At later stages of infection nsP123 is cleaved to produce nsP1, nsP23, nsP4 (Shirako and Strauss, 1990) that can synthesize (minus)-RNA and (plus)-RNA. Final cleavage products form the mature replication complex with nsP1, nsP2, nsP3 and nsP4 that can synthesize only (plus)-RNA (Lemm et al., 1994, 1998; Shirako and Strauss, 1994; Sawicki and Sawicki, 1994). Although our understanding of alphavirus RNA replication in invertebrate cells is very limited, experiments to study the affect of defec-

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tive cleavage of ns polyproteins/polyprotein intermediates on virus production gave similar results in vertebrate and invertebrate cells (Kim et al., 2004); suggesting that the functions of different cleavage intermediates and fully cleaved products are comparable in vertebrate and invertebrate cells. Vertebrate and invertebrate cells show major differences in physiological changes upon SV infection. Virus maturation takes place by envelopment at plasma membrane in vertebrate cells (Brown, 1980). In mosquito and drosophila cells however, virus matures primarily at the membranes of intracellular vesicles. Vesicles filled with mature virus are seen both in drosophila S2 cells (Mudiganti et al., 2006) and mosquito cells (Miller and Brown, 1992) infected with SV. Insect cells start to show this unique and interesting intracellular change, i.e., development of vesicles in infected cells, starting as soon as 6 hpi, (Gliedman et al., 1975) up to several days after infection (Mudiganti et al., 2006). Previous studies using mosquitoes (Brown and Condreay, 1986; Bowers et al., 1995) and C7-10, U4.4, C6/36 mosquito cell lines (Condreay and Brown, 1986; Karpf et al., 1997a,b; Karpf and Brown, 1998) described specific insect cellular responses to SV infection. Drosophila (fruit fly) and drosophila cell lines can be successfully infected with SV, resulting in persistent infections (Bras-Herreng, 1973, 1975, 1976; Mudiganti et al., 2006). Persistently infected mosquito cells do not show any defective interfering particles or truncated viral RNA (Igarashi and Stollar, 1976), excluding the involvement of viral interference during establishment of persistence. Uninfected cells exposed to virus-free cell supernatants of persistently infected cells could not be infected (Riedel and Brown, 1979) and infected cultures excluded superinfection even though very few cells in the culture contained viral structural proteins (Riedel and Brown, 1977). Viral RNA levels also decreased in cells upon treatment with the media from persistently infected cells (Condreay and Brown, 1988), suggesting the presence of an antiviral factor in SV-infected insect cell cultures. Mosquito cells infected with SV produce the same amounts of virus, independent of the multiplicities of infection (moi’s—ranging from 0.00005 to 50) used for infection (Karpf et al., 1997a). All these observations led us to hypothesize that intracellular factors must be responsible for controlling virus production as SV persistence is established. In this study, we utilized a genomic approach to identify the gene products that are differentially expressed during development of SV persistence in insect cells. At 5 dpi, both mosquito (U4.4) and drosophila (S2) cell cultures tested in our lab showed reduction in virus levels (Karpf et al., 1997a; Mudiganti et al., 2006) to the levels (100-fold decreased) that are characteristic of persistent infection. This time point would show the gene expression changes critical for establishment of persistence rather than the immediate response of the cells to virus infection during acute infection (1 or 2 dpi). As the cells were persistently infected, we considered 1.5–2-fold changes in gene expression to be significant (see results). At 5 dpi, increased levels of gene products coding for Notch pathway components, cytoskeletal proteins and a potential JAK-STAT pathway component were identified in SV-infected S2 cells. Our array experiments showed increased levels of Thiol-ester containing Protein II (TEPII) (gene ID—CG7052) in S2 cells infected with SV, at 5 dpi. As TEPII is a wide-spectrum protease inhibitor, we probed for inhibition of viral protease activity in SV-infected cells at 5 dpi. We identified that cleavage of ns polyprotein is inhibited at 5 dpi in both S2 and U4.4 cells. Inhibition of cleavage of ns polyprotein correlated with reduced levels of SV production at 5 dpi (Mudiganti et al., 2006). This finding reveals a possible mechanism of establishment of SV persistence in insect host.

2. Materials and methods 2.1. Cell lines Schneider’s 2 (S2) drosophila cells (ATCC #CRL-1963, Manassas, VA) derived from 20 to 24-h-old embryos were used. Cells were grown in Schneider’s drosophila medium with 20% FBS (Sigma JRH, Lenexa, KS), 2 mM l-glutamine, in 75 cm2 culture flasks at 23 ◦ C in a humidified growth chamber with 10% CO2 . Cells were split 1:3, every 4 days. Aedes albopictus U4.4 cells were subcloned in our laboratory from the original Aedes albopictus cell line isolates of Singh (Singh, 1967) and were grown in Mitsuhashi Maramorosch medium (M and M) (Mitsuhashi and Maramorosch, 1964) at 28 ◦ C supplemented with 20% FBS (Hyclone, Logan, UT). 2.2. Virus and infections For infections, 6 × 106 S2 or U4.4 cells were resuspended in serum-free media to induce formation of monolayers. Infections were done with SVHR (Sindbis Virus-Heat Resistant) at a multiplicity of infection (moi) of 100 (for dot blots Fig. 2) or 1000 [for Western blots (Fig. 3) and immunoprecipitation (IP) experiments (Fig. 4)] by rocking for 1 h at RT. At the end of 1 h, the inoculum was removed and infected cells were grown in media containing serum. Mock infections were done simultaneously, with 1× PBS (phosphate buffered saline) (0.1 M NaCl, 2.7 mM KCl, 1.5 mM KH2 PO4 , 8.5 mM Na2 HPO4 , 1 mM MgCl2 , 0.1 mM CaCl2 , pH 7.4) containing 3% FBS. Both SV-infected and mock-infected cells were split on day 3 after infection and used on day 5 after infection. Cells with low passage numbers (between 10 and 25) were used for all infections. Superinfections were done by infecting the SV-infected cells at 5 dpi, at 100 moi (for dot blots) or 1000 moi (for Western blots and IPs). 2.3. Array analysis For array hybridization 3–4 × 107 S2 cells were infected with SVHR at 100 moi. Both SV-infected and mock-infected cells were split on day 3 after infection and used on day 5 after infection for preparation of RNA samples for hybridization. 2.4. Preparation of cRNA for hybridization Total RNA was isolated from mock-infected and SV-infected S2 cells (3–4 × 107 cells) at 5 dpi, using Trizol reagent (Ambion, Austin, TX). Integrity of total RNA was tested by obtaining absorbance A260 and A260 /A280 ratio for each sample. Samples showing A260 /A280 ratio value >1.7 were used for mRNA isolation. The presence of rRNA bands in the total RNA sample in 1% agarose gel electrophoresis was also examined to determine the integrity of each sample. Messenger RNA was isolated from total RNA using a poly (A) isolation kit (Ambion, Austin, TX). Spike controls coding different genes expressing the enzymes involved in metabolism of different amino acids—Lys Phe, Thr, Trp, Dap of Bacillus subtilis (obtained from ATTC 87482, 87483, 87484, 87485, and 87486) were added to the total RNA before isolation of mRNA to check for any loss of RNA during subsequent isolation, labeling and hybridization steps of the protocol. Genechips used for hybridization had the probes corresponding to these bacterial genes. “Absent” call for any of these genes during preliminary analysis (see below) in MAS 5.0 (Affymetrix MicroArray Suite 5.0) indicated loss of RNA and the dataset showing “Absent” call for these bacterial genes was rejected. mRNA was processed according to the Affymetrix genechip protocol provided by the manufacturer. Briefly, cDNA was obtained by reverse transcription of the mRNA using Superscript II Reverse Transcriptase (Invitrogen, Carlsbad, CA) and was transcribed in

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vitro using biotinylated rNTPs using Enzo Bioarray kit (Affymetrix, Santa Clara, CA) to obtain 15 ␮g biotinylated cRNA. cRNA representing all the polyadenylated RNA of S2 cells was fragmented to obtain 400–600 nt fragments. These target cRNA fragments were hybridized to gene-specific probes on Affymetrix drosophila genechips (Drosogenome1 genechips, Affymetrix, Santa Clara, CA). The arrays used contained probesets, i.e., a set of probes designed to target a specific gene according to information obtained from more than 13,500 drosophila genes, mRNA, predicted Open Reading Frame (Drosogenome1) information and Expressed Sequence Tags [200–500 bp sequence from either 5 or 3 end of cDNA (obtained from RNA using oligo-dT primers) representing a specific gene]. B2 Oligonucleotide (Affymetrix, Santa Clara, CA) was added to target cRNA sample to serve as a positive control for hybridization. Hybridization was done at 45 ◦ C for 16 h in a hybridization oven (Affymetrix, Santa Clara, CA) with rotation at 60 rpm. Staining and washing were done using fluidics station 400 (Affymetrix, Santa Clara, CA). Protocols specified by manufacturers were used and the image was scanned using an Agilent scanner (Affymetrix, Santa Clara, CA). Signal intensity data in terms of Perfect Match probeset–Mis Match probeset was obtained in MicroArray Suite (MAS) 5.0 2.5. Preliminary examination of the data using MicroArray Suite (MAS) 5.0 Spike controls (see above) that were added to total RNA samples served as controls to identify any degradation of sample during processing of RNA for hybridization. Samples giving “Present” call for spike controls and house keeping genes (GADPH, Actin, Eukaryotic initiation factor Eif-4a), signal intensity ratios from probesets designed to target the 3 end and 5 end of the house keeping genes (3 /5 ratios) were considered to assess quality of the dataset. Datasets showing 3 /5 ratios of <3 for all the house keeping genes were considered for analysis. Datasets showing “Present” call for rRNA were rejected. Array intensity signal data from independent, biological replicates of 3 mock and 2 samples from 5 dpi that met these criteria were considered for analyses steps described below. 2.6. Statistical analysis using Genespring 7.2 Intensity signal values from 3 biological replicates of mockinfected and 2 biological replicates of infected (5 dpi) samples obtained from MAS (MicroArray Suite) 5.0 were analyzed using Genespring 7.2. (PerfectMatch Intensity–Mismatch Intensity) values corresponding to each probeset on the array were considered for analysis. Probesets with intensity values of less than 10 were omitted from normalization and further analyses. Statistical analysis was done using the Cross gene error model (this uses measurement of variation within a sample and sample-to-sample variation) in Genespring 7.2. Sample-to-sample variability of gene expression measurements between two experimental conditions, standard error (represents precision of the mean of gene expression measurement in condition with respect to true condition mean) and t-test p-values were obtained using the cross gene error model. “Per chip” normalization was done using the distribution of signal intensity values of all the genes (i.e., probeset IDs) in a sample. Each intensity measurement was divided by the 50th percentile of all (probesets with intensity values >10 in MAS) measurements in that sample. “Per gene” normalization was done using specific control samples in which the intensity signal of each gene was divided by the average signal of the identical gene in control samples. Each measurement corresponding to a gene in a sample was divided by the median of its measurement in all the samples to yield a scaled value ∼1 and these values were log-transformed (log2 ) to represent the fold change of expression

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of each gene. These fold changes were listed and are discussed in this manuscript. Line graphs were colored (Fig. 1B) based on how far the gene is over or under the expressed level relative to the normalized expression level of 1, in terms of the standard error of measurement (ranging from +3 to −3). Differentially expressed genes were identified using t-test (one-way ANOVA) not assuming equal variances between samples. Genes showing 1.5–2-fold or greater change in expression levels with t-test p-values below 0.05 were filtered. Genes showing 1.5–2 or greater change (decrease or increase) in these scaled values from mock replicas (3 independent biological samples) to 5 dpi sample replicas (2 independent biological samples) were hand-picked and reported here. For example, genes that gave a normalized intensity value below 1 (0.3–0.9) in the mock samples and 1.4 or above in the 5 dpi samples were hand-picked and labeled as changing 1.5–2-fold. Since the infected sample was chosen from 5 dpi samples, 1.5-fold or more change in gene expression was considered significant. Gene ontologies and relevant information pertaining to the probesets selected by the analyses steps described above were obtained from NetAffx analysis center at Affymetrix website. 2.7. Dot blots of mRNA from SV-infected S2 and U4.4 cells using TEPII specific probes RNA was isolated from 6 × 106 drosophila S2 cells or mosquito U4.4 cells, using Trizol reagent (Ambion, Austin, TX). Messenger RNA was isolated from total RNA from mock-infected, 4.5 hpi or 5 dpi samples, using poly (A) isolation kit (Ambion, Austin, TX). RNA from an equal number of cells (6 × 106 ) in each treatment condition was blotted onto positively charged Genescreen membrane (Perkin Elmer, Boston, MA) using a dot blotter (Schleicher-Schuell, Florham Park, NJ). Each sample was diluted 1.7-fold, with a total of 8 dilutions. Hybridization was done using a probe made from TEPII e (an alternately spliced form e) cDNA clone. For preparation of the probe, 100 ng of linearized DNA (in 20 ␮l H2 O), NEB Buffer 2 (1× buffer: 50 mM NaCl, 10 mM Tris–HCl, 10 mM 10 mM MgCl2 , 1 mM dithiothreitol) (New England Biolabs, Ipswich, MA) 0.5 ␮l of 0.1 M DTT was boiled for 5 min and quickly cooled on ice. After cooling, 0.5 ␮l of 50 mg/ml bovine serum albumin (BSA), 2.5 ␮l random oligomers (New England Biolabs, Ipswich, MA), 0.5 ␮l each of 20 mM dTTP, dCTP, dGTP, 5 ␮l of ␣-P32 dATP (Perkin Elmer, Boston, MA) and 2 ␮l of Klenow enzyme (New England Biolabs, Ipswich, MA) were added to the denaturing reaction (final reaction volume 50 ␮l) and incubated for 4 h at 37 ◦ C. The probe was purified using a Sephadex G25 (Sigma, St. Louis, MO) column in 1× TE buffer (10 mM Tris–HCl, 1 mM EDTA), before use. Hybridization was done at 42 ◦ C for 16–20 h in 5× SSPE diluted from a 20× buffer stock (1× SSPE: 0.15 M NaCl, 10 mM NaH2 PO4 –H2 O and 1 mM EDTA–Na2 pH 7.4), 50% deionized formamide, 5× Denhardt’s solution (0.05% polyvinylpyrrolidone, 0.05% BSA, 0.05% Ficoll 400), 1% SDS, 10% dextran sulphate (mol. wt. 500,000). Hybridizations were done in 10 ml of hybridization buffer. Prior to hybridization the membrane was prehybridized for 4 h at 42 ◦ C in hybridization buffer containing 50 ␮l of 100 ␮g/ml denatured salmon sperm DNA. The membrane was washed twice in 200 ml of 2× SSPE at RT. High stringency washes were done at 65 ◦ C for 1 h 30 min twice in buffer containing 2× SSPE and 2%SDS. Low-stringency washes were done twice in 200 ml 0.1× SSPE at RT, for 30 min. Autoradiograms were obtained by exposing the membrane to Kodak BioMax XAR film (Source One, Jacksonville, FL) for 72 h. For each mock/infected sample most diluted sample containing a radioactive signal on the blot was considered for quantification. Intensity values corresponding to each sample were measured as integrated density values (IDV) in adobe photoshop CS3. Dilution factor (1.7) was considered to obtain the relative intensity values in different samples as—IDV × folddilution (2) × dilution factor (1.7).

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Fig. 1. Findings of the array experiments to compare transcriptional profiles of mock-infected S2 cells and S2 cells infected with SV, at 5 dpi. (A) A table showing number of genes increased or decreased in expression at different significance levels. (B) Are shown the t-test results using Genespring 7.2. Samples 1–3 are mock infected and 4 and 5 are infected samples at 5 dpi. Genes showing less than 1.5-fold difference in expression were filtered from t-test results (881 genes) and shown here. Color of the line (ranging from yellow to blue) indicates degrees of significance of differential expression of a particular gene as measured by standard error (described in Section 2). (C) Are shown different functional classes of genes involved in establishment of SV persistence in insect cells. Given are the numbers of genes coding for factors involved in different cellular functions. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of the article.)

2.8. Western Blot of nsPs with anti-nsP1 and anti-nsP2 antibodies 1 × 107 S2 cells were infected with SVHR at 1000 moi. For superinfections, these infected cells were infected again at 5 dpi, at 1000 moi. Cell lysates were prepared from S2 cells that were mockinfected, 1.5, 4 h after infection, at 5 dpi after initial infection and at 1.5 or 4 h after superinfection on 5 dpi. Mock infections were done with 1× PBS containing 3% FBS. Cell pellets were obtained by centrifugation at 1200 rpm, in a desktop centrifuge and lysed in 400 ␮l ice-cold TNT lysis buffer [0.02 M Tris pH 7.5, 0.5% NP40, 150 mM NaCl containing 0.2 mM phenylmethylsulfonyl fluoride (PMSF)]. Lysates were incubated at RT for 5 min and centrifuged at 10 K rpm for 10 min. The supernatants were used in Western blots using anti-nsP2 antibody. For blots using anti-nsP1, samples were obtained from mock-infected S2 cells, 1.5 h after infection or superinfection and processed in a similar fashion, to that described above. For Western blot analysis, 20 ␮l of the supernatant (from above) was mixed with 5 ␮l of 5× sample buffer (62.5 mM Tris, pH 6.8, 50% glycerol, 5% SDS, 0.05% bromo phenol blue, 10% ␤mercaptoethanol) and run on a 10.8% SDS-PAGE gel and transferred onto a PVDF membrane (Sequiblot 0.2 ␮m membrane, Bio-rad, Philadelphia, PA). The blots were incubated in blocking solution (10%; w/v non-fat dry milk, 1%; v/v goat serum, made with 1× PBS) for 1 h, incubated in incubation solution (0.05% Tween-20, 10%; w/v non-fat dry milk, 1% goat serum, made with 1× PBS) with 10 ␮l anti-nsP1 or anti-nsP2 antibody for 1 h, washed 3 times in washing solution (0.05%; v/v Tween-20, 1%; v/v goat serum, made with 1× PBS) for 5 min each time and then incubated with goat 125 [I] anti-

rabbit IgG (Perkin Elmer, Boston, MA) for 1 h. All incubations were done at RT. The blots were washed 3 times in washing solution, air-dried and exposed to Kodak XAR film (Source One, Jacksonville, FL) for 3–4 days. Signal intensities corresponding to ns polyprotein were obtained as Integrated Density Values using adobe photoshop CS3. 2.9. Immunoprecipitation to detect levels of SV nonstructural polyproteins under different conditions of infection 7 × 106 S2 or U4.4 cells were infected at 1000 moi with SVHR and superinfected on 5 dpi, at 1000 moi. Samples were prepared from cells that were mock-infected, 4 hpi, 4 hpi after superinfection on 5 dpi, and at 5 dpi after the initial infection. Lysates were prepared on ice, using 150 ␮l of TNT lysis buffer (10 mM Tris pH 7.4, 1% NP-40, 150 mM, NaCl, 0.2 mM PMSF) at 4 ◦ C and the samples were incubated for 5 min at RT. The lysates were incubated for 20–24 h, at 4 ◦ C, with Protein A sepharose beads (Invitrogen, Carlsbad, CA). The lysates were then incubated with 2 ␮l of primary antibody (anti-nsP1 or anti-nsP2), for 36 h, at 4 ◦ C. The proteins bound to antibodies were separated by incubating the lysates with a fresh batch of Protein A sepharose beads for 2 h, at 4 ◦ C. The proteins bound to Sepharose beads were collected by spinning the beads + lysate at 10 K rpm in a tabletop centrifuge. For running gels, the beads were boiled with 2× loading buffer (25 mM Tris, pH 6.8, 20% glycerol, 2% SDS, 0.02% bromo phenol blue, 4% ␤-mercaptoethanol) for 3–5 min and spun for 2 min at maximum speed in a tabletop centrifuge. Proteins separated by SDS-PAGE were quantified by obtaining integrated

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Table 1 Components of Notch pathway that are differentially expressed in drosophila S2 cells infected with SV, at 5 dpi. Gene ID

Gene product

Function

CG11988

Neur

CG3779 CG7555

Numb Nedd4

CG10653 CG1725

Hook Dlg1

CG5692 CG13281

Raps Cas

CG8532

Lqf

CG7467

Osa

CG5203

CHIP

Ubiquitination of ligand of Notch, Delta Interaction with Notch Ubiquitination of Notch and endocytosis Endocytosis of Delta Tumor suppressor interacting with raps Partner of Inscuteable Nuclear export receptor protein Required in signal donor/receptor cell Chromatin remodeling to repress ac/sc Required for maximal enhancer activity

Fold change increase 2

p-Value

>0.05

Identities with A. gambiae proteins

A. gambiae Gene ID

50%

ENSANGP00000007854

Identities with A. aegypti proteins

A. aegypti Gene ID

2 2

0.02 0.038

61% 62%

ENSANGP00000006150 ENSANGP00000025653

1.5 2.5-3.5

0.02 0.027

53% 77%

ENSANGP00000016709 ENSANGP00000012487

69% 64%

EAT33528 EAT39461(ranBP)

0.0003 0.0006

65% 63%

ENSANGP00000019406 ENSANGP00000018477

66%

EAT39510

36%

ENSANGP00000002840

62%

EAT36891(Lim BP)

47%

ENSANGP00000008445

60%

ENSANGP00000020684

>3 1.5-2 1.5-2 1.5-2 1.5-2

>0.05 0.026 >0.05

density values for ns polyprotein bands, using adobe photoshop CS3. 3. Results Transcriptional profiles of the drosophila genome were obtained from mock-infected and SV-infected S2 cells at 5 dpi, using Affymetrix genechip array technique and the results of the analysis are presented in Fig. 1. Fig. 1A shows the number of up or down regulated genes, at different p-values, obtained by analysis using Genespring 7.2 (as described in Section 2). Transcription profiles of the differentially regulated genes are also shown (Fig. 1B). t-Test results showed 881 genes to be differentially expressed (p-value <0.05) at 5 dpi in S2 cells upon SV infection. 210 genes were up-regulated and 68 genes were down-regulated at a p-value <0.05. Our results identified cytoskeletal and membrane trafficking components (18), immune-related components (9), transcription factors (8), factors affecting the cell cycle or cell division (9), Ras (6) and Notch pathway components (10) to be potentially involved in establishment of SV persistence in insect cells (Fig. 1C). Lists of 210 up-regulated and 68 down-regulated genes with corresponding pvalues can be found in the supplementary material. The 5 dpi time point was chosen based on previous data that showed that S2 cells produce virus at levels characteristic of persistent infection (100fold reduction in virus levels) by day 5 post-infection (Mudiganti et al., 2006). The number of genes changing in expression, at different significance levels, is given in Fig. 1A. Factors that could play a role in the reduction in growth rates (Karpf et al., 1997a), in formation and functioning of intracellular vesicles in mosquito cells (Gliedman et al., 1975) and drosophila cells (Mudiganti et al., 2006) upon SV infection, are discussed in the following paragraphs. 3.1. Notch pathway may be involved in establishment of SV persistence in insect cells Table 1 shows up-regulated genes that encode factors that mediate regulatory aspects of cellular functions involving evolutionarily conserved Notch pathway (Artavanis-Tsakonas et al., 1999; Kopan and Ilagan, 2009; Mumm and Kopan, 2000). Notch pathway plays a role in either inhibition or induction of apoptosis depending on the cellular contexts in different tissues of drosophila (Baron et al., 2002; Krejci et al., 2009; Lundell et al., 2003). Notch signaling is activated by intramembranous cleavage of the Notch receptor upon binding of the ligand (Delta in drosophila) from an adjacent

cell (Mumm and Kopan, 2000). The intracellular domain of Notch receptor travels to nucleus and associates with specific DNA binding proteins to activate transcription of target genes (Krejci et al., 2009). Adjacent cells need to be in physical contact with each other for Notch signaling to be activated (Kopan and Ilagan, 2009). S2 cells used for SV infection in the present study showed a high affinity for clumping making activation of Notch, a possibility. Notch pathway components and functions of the gene products discussed in this manuscript (see Section 4) were studied in detail in neuroepithelial cells (Cayouette and Raff, 2002) or malphigian tubule (Orgogozo et al., 2002; Wan et al., 2000) imaginal discs of drosophila eye, wing (Parks et al., 2000) and oocytes of drosophila. S2 cells were used for some of the experiments in these studies. Up-regulation of significant number of Notch components (Table 1) in light of the fact that cell growth rate changes are observed in invertebrate cell cultures persistently infected with SV (Karpf et al., 1997a; Mudiganti et al., 2006), prompts us to suggest activation of Notch pathway in invertebrate cells infected with SV, at 5 dpi. SV-infected insect cells do not undergo apoptosis as do the vertebrate cells (Karpf et al., 1997a) and cellular functions mediated by Notch signaling might contribute to this inhibition of apoptosis. Drosophila Notch, Delta and other gene products identified here have significant similarities with the mosquito A. aegypti and A. gambiae proteins (Table 1). It is interesting to note that gamma herpesviruses imitate or manipulate the Notch pathway to establish lifelong viral persistence in mammalian cells (Curry et al., 2005, 2007; Hayward, 2004, Hayward et al., 2006; Lan et al., 2005). S2 and U4.4 cells used in this study maintained SV persistence for as long as the cell cultures are maintained. 3.2. Cytoskeleton and intracellular trafficking-related factors up-regulated at 5 dpi Table 2 lists factors involved in the intracellular transport and vesicle formation that potentially mediate the intracellular changes observed in SV-infected insect cells (Miller and Brown, 1992; Mudiganti et al., 2006). Strikingly many of the genes up-regulated in this group are involved in vesicle formation, ER-Golgi transport or exocytosis; the secretion-like activities needed to accumulate and release SV virions from insect cells (Miller and Brown, 1992; Mudiganti et al., 2006). We speculate that csp (Table 2), which is studied as synaptic vesicle protein (Heckmann et al., 1997) interacts with the product of syx13 (Table 2) (Nie et al., 1999) to regulate release of the virus. Gene products of mammalian unc-13 (Table 2) interact with Syntaxins (Madison et al., 2005). Hence, it is possi-

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Table 2 Factors involved in different cellular functions up-regulated in drosophila S2 cells infected with SV, at 5 dpi. Gene ID

Gene name

Function

Fold change

Cell cycle/cell division CG8203 CG1081 CG5692 CG8374 CG3443 CG5553 CG1403 CG14066 CG7749 CG5650

cdk5 rheb rapsynoid dalmatian pecanex DNAprim sep1 larp fat2 pp1-87B

Synaptic vesicle secretion, cell cycle G1/S transition, GTPase activity Mitotic spindle orientation Nervous system development Nervous system dev and differentiation DNA primase, successful S phase Cytokinesis and cell cycle La-related protein involved in autophagic cell death Negative regulation of cell growth Essential for mitosis

2 1.5–2 2–3 2–3 2–3 >3 1.5–2 1.5–2 1.5–2 1.5–2

Transcription CG6711 CG4195 CG11992 CG2679 CG8625 CG8815 CG3660 CG6222 CG3458 CG1070

TafII150 l(3)73Ah relish gol iswi sin3A kis su(s) top3 alhambra

DNA methylation, G2/M transition Zinc ion binding, ubiquitination NF-kB factor, Response to bacteria Mesoderm formation, ubiquitination Chromatin assembly, disassembly Histone deacetylation, transcriptional repression Chromatin remodeling Transcriptional repressor Topoisomerase activity DNA binding transcription factor

2–3 2–3 1.5–2 1.5–2 1.5–2 1.5 1.5–2 1.5–2 1.5–2 2

Ubiquitination CG4195 CG2679 CG1782

l(3)73Ah gol uba1

Zinc ion binding, Transcription factor Transcription regulator Ubiquitin activating enzyme

2–3 1.5–2 1.5–2

Vescicle transport/fusion/cytoskeleton CG5722 CG1651 CG5105 CG11278 CG8156 CG3320 CG3269 CG8287 CG7838 CG9012 CG8705 CG7210 CG8266 CG6395 CG10653 CG1483 CG3637 CG18734 CG2999

NPC1 ankyrin plap syntaxin13 arf51F rab1 rab2 rab8 bub1 chc peanut kel sec31 csp hook map205 cortactin furin 2 unc-13

Cholesterol transport, hedgehog receptor Actin binding, receptor binding Fusome Excocytosis, synaptic vescicle ER to Golgi transport Endocytosis Endocytosis, protein transport Endocytosis, protein transport Ser/Thr Kinase, Exit from mitosis Clathrin heavy chain Cytokinesis, vesicle docking, targeting Actin binding in oocyte ring canal ER and golgi component, exocytosis Synaptic vesicle exocytosis Microtubule binding, endocytosis Microtubule binding Actin binding, defense response Pro-protein processing Synaptic vescicle, exocytosis

1.5–2 2–3 >3 >3 1.5–2 1.5–2 1.5–2 1.5–2 1.5–2 1.5–2 1.5–2 2 2 1.5–2 1.5–2 1.5–2 1.5–2 1.5–2 1.5–2

Signaling CG6721 CG8174 CG12019 CG8107 CG14217 CG6235 CG10072 CG18485 CG12819

gap1 SRPK cdc37 calpB tao-1 twins sugarless stumps sle

Neg. regulation of Ras, EGF pathway Ser-Thr Kinase, Spliceosome Chaperone, cell cycle Ca binding, Calmodulin binding Influences FGF pathway Regulation of Wnt signaling Important for Wnt & FGF signaling Effector downstream of FGF receptor Influences Ras signaling

2–3 6 >3 1.5–2 1.5–2 1.5–2 1.5–2 1.5–2 1.5–2

ble that products of syx13 (Hirling et al., 2000; Collins et al., 2002) and unc-13 may function independently or together during vesicle fusion events to ultimately release the virus from the infected cell. Functions of increased levels of rab1and rab8 gene products (Table 2) here may be essential for fusion of vesicles carrying SV structural proteins from ER to Golgi, similar to function of these gene products during transport of Vesicular Stomatitis Virus glycoproteins in mammalian cells (Peter et al., 1994; Pind et al., 1994; Tisdale et al., 1992). NPC1 (Nieman-Pick Disease 1) (Table 2) also increased in expression in the midgut of A. aegypti upon SV infection, particularly on 4 dpi (Sanders et al., 2005), suggesting a specific function for this gene product during establishment of viral persistence.

3.3. SV-induced immune processes in insect cells NF-kB family transcription factor Relish (rel) is increased in expression at 5 dpi in SV infected S2 cells (Table 2). In a recent study rel −/− drosophila mutant flies showed increased levels of viral RNA compared to WT flies, upon infection with SV (Avadhanula et al., 2009). TEP II, a member of the family of Thiol-ester containing proteins that were shown to have immune-related functions in invertebrates (Armstrong, 2006; Blandin and Levashina, 2004) is increased in expression at 5 dpi (see following paragraphs) in SV-infected S2 cells (Table 3). In previous studies, infection of drosophila flies (Lagueux et al., 2000) and S2 cells (Kallio et al., 2005) with bacteria lead to increase in levels of TEP II. TEPII belongs to the

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Table 3 Immune-related genes up-regulated in drosophila S2 cells infected with SV, at 5 dpi. Gene ID Defense response CG7668 CG18503 CG32557 CG8896 CG6395 CG15573 CG14919 CG6895 CG8874 Complement-related CG7052 CG7586 CG2198

Gene name

Function

Fold change

– svr – 18w csp femcoat allatostatin-C GNBP1 Fps85D

Predicted receptor, defense response Melanization Hemolymph coagulation Embryonic morphogenesis, receptor activity Synaptic vescicle, exocytosis, chaperone Insect chorion formation Stress Response Pattern recognition receptor of Toll pathway JAK-STAT, Predicted defense response,

2.5 >3 >3 1.5–2 1.5–2 1.5–2 1.5–2 1.5–2

TEP II Mcr amalgam

Wide-spectrum protease inhibitor, opsonization Opsonization Immunoglobulin superfamily, secreted

3–4 1.5–2 1.5

super family of proteins containing members like human complement and ␣-macroglobulin proteins (AMCOMs) (Armstrong, 2006). A previous study showed that TEP II is important for phagocytosis of Escherichia coli bacteria by S2 cells (Stroschein-Stevenson et al., 2006). 3.4. TEP II transcript levels increase upon SV infection At 5 dpi, reduced levels of SV, indicative of a persistent infection, are seen in infected drosophila S2 and mosquito U4.4 cells (Karpf et al., 1997a; Mudiganti et al., 2006). Our array analysis identified increased levels expression of complement-related gene product TEPII, at 5 dpi (Table 2), in S2 cells. A DNA probe made from drosophila TEP II e cDNA clone was used to probe for TEP II homologues in drosophila S2 cells and mosquito U4.4 cells using dot blot technique. Messenger RNA was obtained from mock-infected cells and virus infected cells at 4.5 hpi, 5 dpi, and 4.5 hpi after superinfection on 5 dpi (Fig. 2). TEP II expression levels in superinfected samples were examined at the same time point that was tested for inhibition of viral protease activity upon superinfection on 5 dpi (see below, Figs. 3 and 4). Dot blots showed that TEP II expression levels increased by ∼3-fold in both S2 and U4.4 cells, at 5 dpi

(Fig. 2A and C). These levels increased further by ∼20% upon superinfection, at 4.5 hpi after superinfection on 5 dpi (Fig. 2C) in S2 cells. This increase in TEP II levels after superinfection was not significant in U4.4 cells (Fig. 2C). The levels at 4.5 hpi (acute phase of infection) were similar to the levels of TEP II in mock-infected samples (Fig. 2A), indicating that its production is unaffected in the early stages of infection. Though there was a high background in the blots using mRNA samples from mosquito U4.4 cells, the levels of TEP II related sequences appeared to increase at 5 dpi (Fig. 2B and C). In a separate study TEPI, another member of TEP family of gene products, increased in expression at 6 hpi after bacterial infection in drosophila (Kallio et al., 2005; Lagueux et al., 2000), Anopheles gambiae and mosquito cell lines (Levashina et al., 2001). In this study, TEP II levels were not increased at 4.5 hpi after SV infection (Fig. 2A), suggesting that up-regulation of TEP II is not an immediate response of the cell to SV infection and that this protein may have a specific function during establishment of the persistent infection. TEP II gene product is involved in immune-related activities and belongs to family of proteins containing mammalian complement and protease inhibitors (Lagueux et al., 2000). This raised the possibility that viral protease activity may be affected in SV-

Fig. 2. Up-regulation of TEP II gene product in invertebrate cells infected with SV. (A) Dot blot showing increased levels of TEP II mRNA levels at 5 dpi and 4.5 h after superinfection of drosophila S2 cells with SV. Positive control, TEPIIe Probe; Mock, mock-infection; 4.5 hpi, 4.5 h after infection with SV; 5 dpi, 5 dpi after infection with SV; 4.5 hpi on 5 dpi, 4.5 h after superinfection on 5 dpi. (B) Dot blot showing increased levels of TEP II mRNA levels at 5 dpi and 4.5 h after superinfection of mosquito U4.4 cells with SV. Mock, mock infection; 4.5 hpi, 4.5 h after infection with SV, 5 dpi, 5 dpi after infection with SV, 4.5 hpi on 5 dpi, 4.5 h after superinfection on 5 dpi. (C) The graph obtained plotting radioactive signal intensities corresponding to the levels of TEP II expression in S2 and U4.4 cells at various times after infection with SV, as indicated on X axis. Signal intensities were quantified using adobe photoshop CS3.

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Fig. 3. Accumulation of ns polyproteins in drosophila S2 cells at 5 dpi, upon SV infection. Western blots of drosophila S2 cells with SV. (A) The accumulation of nsP123 at 5 dpi and upon superinfection of Drosophila S2 cells with SV, using anti nsP2 Ab. SV superinfections were done on day 5 post-infection. Lane 1, molecular weight markers; lane 2, mock infected cells; lane 3, 1.5 h after infection, lane 4, 1.5 h after superinfection on 5 dpi; lane 5, 4 h after infection; lane 6, 4 h after superinfection on 5 dpi; and lane 7, 5 dpi. Solid arrow (–→) shows nsP123 accumulated in 5 dpi and superinfected samples that are infected with SV on 5 dpi. Broken arrow (– →) indicates cross-reacting host protein. (B) A Western blot using anti-nsP1 antibody showing accumulation of nsP123 at 5 dpi and upon superinfection of drosophila S2 cells with SV. Lane 1, molecular weight markers; lane 2, mock infection; lane 3, 1.5 h after infection; and lane 4, 1.5 h after superinfection. nsP123 accumulated in superinfected samples is shown by solid arrow (–→). (C) The graph obtained by plotting relative amounts of polyprotein at different time points after infection, as indicated on X axis. Signal intensities corresponding to ns polyprotein are quantified using adobe photoshop CS3 and plotted here.

infected insect cells. Because one of the early functions of the SV nonstructural proteins is to autocatalytically process the precursor polyprotein, we assessed levels of the ns polyproteins during establishment of persistence using IP and Western blot techniques. 3.5. Analysis of SV nonstructural polyproteins by Western blots of persistently infected insect cells Sindbis viral replicase-transcriptase complexes are regulated to preferentially synthesize different viral RNA species in the infected cell (see introduction). This is achieved by specific ns polyproteins, cleavage intermediates or final cleavage products (Lemm et al., 1994, 1998; Shirako and Strauss, 1994) that form the viral replication complex. Inhibition of protease activity of the nsPs during persistence was examined by determining the levels of ns polyprotein precursors after superinfecting the SV-infected S2 cells at 5 dpi. Superinfections were done at 5 dpi to examine the levels of viral ns polyproteins when more viral RNA is introduced into cells that contained the viral and host factors expressed after the initial round of viral infection. Western blots using anti-nsP2 (Fig. 3A ) and anti-nsP1 (Fig. 3B) antibodies showed higher levels of nsP123 in S2 cells at 5 dpi, compared to the levels at 4.5 hpi that used identical moi’s (Fig. 3A–C) for infection. These levels further increased by ∼25% upon superinfection on 5 dpi (Fig. 3A and C). A host protein of ∼70 kDa was seen to cross-react with the antinsP2 antibody in many of the samples, as this can also be seen in mock-infected samples (Fig. 3A, indicated by broken arrow). We did not find any 120 kDa or 250 kDa host protein cross-reacting with the anti-nsP1 or anti-nsP2 antibodies in the invertebrate cell lines, in contrast to the observations seen in vertebrate cells (Barton et al., 1991; Hardy and Strauss, 1989).

3.6. Analysis of SV nonstructural polyproteins in persistently infected insect cells using immunoprecipitation technique We used IP technique (i) to confirm the results of Western blots that used samples from drosophila S2 cells and (ii) to examine inhibition of the cleavage of nsP123 in mosquito U4.4 cells. U4.4 cell samples were immunoprecipitated using either anti-nsP1 (or) anti-nsP2 antibody (Fig. 4A and B) and S2 cell supernantants were immunoprecipitated using anti-nsP1 antibody (Fig. 4D). Even though the protein band corresponding to nsP123 was faint (shown by broken arrow in Fig. 4A and B), the levels of ns polyprotein increased at 5 dpi (shown by broken arrow in Fig. 4A and B), compared to the amounts at 4 hpi (Fig. 4A–C). These levels further increased upon superinfection, on 5 dpi (Fig. 4C). The level of nsP123 (∼200 kDa protein shown by solid arrow) in the superinfected U4.4 cells was higher (Fig. 4A and B) compared to the negligible amounts of polyprotein in the samples infected at the same moi and harvested at 4hpi (no signal—Fig. 4A and B). The levels of ns polyprotein doubled at 4.5 h after superinfection compared to levels on 5 dpi (Fig. 4C). Similar results were obtained when IPs were done using samples from S2 cells—i.e., nsP123 (Fig. 4D, indicated by solid arrow) was not seen at 4 hpi compared to the amounts of nsP123 at 5 dpi and at 4 hpi after superinfection on 5 dpi. Quantification of IPs from S2 cells did not show further increase in ns polyprotein upon superinfection, compared to the amounts at 5 dpi. This may be due to saturation of viral replication sites in this cell line during the initial round of infection. IPs further confirmed the results obtained in Western blot analysis, i.e., the superinfected samples showed higher levels of nsP123 compared to the negligible amounts at 4 hpi (Fig. 4). These results

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Fig. 4. Accumulation of viral ns polyprotein in insect cells at 5 dpi, upon infection with SV. IPs of SV-infected mosquito U4.4 cells and drosophila S2 cells. (A) The accumulation of nsP123 using anti nsP1 Ab at 5 dpi and upon superinfection of mosquito U4.4 cells with SV. SV superinfections were done on 5 dpi. Lane 1, mock-infection; lane 2, 5 dpi; lane 3, 4 h after infection; lane 4, 4 h after superinfection on 5 dpi. (B) Immunoprecipitations using anti-nsP2 Ab showing accumulation of nsP123 at 5 dpi and upon superinfection of Drosophila S2 cells with SV. Lane 1, mock-infection; lane 2, 5 dpi; lane 3, 4 h after infection; lane 4, 4 h after superinfection on 5 dpi; and lane 5, molecular weight markers. nsP123 is indicated by solid arrow (–→). Faint band corresponding to nsP123 in 5 dpi samples is indicated by broken arrow (- - →). (C) The graph obtained by plotting the amounts of ns polyprotein inU4.4 cells at different time points after SV infection, as indicated on X axis. (D) The results of IPs of SV-infected S2 cells using anti-nsP1Ab. Accumulation of nsP123 at 5 dpi and upon superinfection on 5 dpi, with SV, is shown. Lane 1, molecular weight markers; lane 2, mock-infection, 1.5 h after SV infection, lane 3, 1.5 h after superinfection on 5 dpi; lane 4, 4 h after infection, 4 h after superinfection on 5 dpi; lane 5, 5 dpi. nsP123 is indicated by solid arrow (–→). ∼70 kDa host protein cross-reacting with anti nsP1 antibody is indicated by broken arrow.

suggest that the polyprotein nsP123 is cleaved by 4.5 hpi (no signal at 4 hpi, in Figs. 3 and 4) and accumulates by day 5 after infection resulting in detection of the nsP123 in the 5 dpi sample. This could be a result of increased rate of synthesis of polyprotein compared to its degradation by proteosomes OR due to inhibition of autoproteolysis. The latter possibility seems to be the case as introducing more viral RNA by superinfection at day 5 post-infection, lead to accumulation of even higher amounts of nsP123 (Figs. 3C and 4C). These data suggest that two factors are playing a role in establishment of SV viral persistence—(i) ns polyprotein accumulates in SV-infected cells as cells establish viral persistence. (ii) When cells are reinfected with SV, the ns polyprotein produced during replication of superinfecting virus accumulates in cells. 4. Discussion Using drosophila S2 cell cultures for array analysis, the present study identified gene expression changes in response to alphavirus infection at the cellular level, omitting the cross talk between different tissues in whole organism. This may account for differences in specific gene products identified in the present study, compared to previous in vivo studies using SV (Sanders et al., 2005) or o’nyong-nyong virus (ONNV) (Sim et al., 2005), an alphavirus closely related to SV. For example, heat shock protein cognate 70B (hsp70B) that may function in maintaining mosquito homeostasis was up-regulated in A. gambiae after infection with ONNV (Sim et

al., 2007) and it is not identified in the present study. Notch pathway components are identified as up-regulated in this study and not in the previous in vivo study (Sanders et al., 2005). It is interesting to note that membrane trafficking components were up-regulated in previous in vivo study (Sanders et al., 2005) and the present study. At 5 dpi, the virus levels were reduced by 100-fold in whole mosquitoes (Bowers et al., 1995), suggesting the biological significance of gene expression changes at this time point. In a recent study U4.4 cells infected with SFV, an alphavirus closely related to SV, showed reduced levels of gene expression during acute phase of infection (Fragkoudis et al., 2008). We identified increased levels of Notch components and cytoskeletal factors in our array experiments at 5 dpi, suggesting that specific genes important for virus–vector coexistence are activated at this time point. Increase in levels of TEPII at 5 dpi, but not at 4.5 hpi, also reflects the fact that this gene product would not be identified at time points during acute infection, but, is up-regulated at later time points. Notch-related activities of the numb (Table 1) gene product have been reported to affect cellular fates of daughter cells during mitosis in different drosophila cell lineages (Cayouette and Raff, 2002; Frise et al., 1996; Orgogozo et al., 2002; Wan et al., 2000). Products of the genes raps and dlg1 (Table 1) have been shown to aid in asymmetric distribution of Numb (Bellaiche et al., 2001; Carmena et al., 1998). Gene products of nedd4 and hook (Table 1) were shown to regulate endocytosis of the Notch receptor (Sakata et al., 2004) and Delta (Parks et al., 2000) respectively. cas (Table 1), which encodes a pro-

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tein necessary for nuclear transport of Notch (Tekotte et al., 2002) is also increased in expression in SV-infected S2 cells. Activation of Notch results in the expression of Achaete/Scute (Ac/Sc) proneural gene suppressors (Artavanis-Tsakonas et al., 1999, Bardin et al., 2004). Up-regulation of gene osa (Table 1), which codes for a negative regulator of Ac/Sc (Heitzler et al., 2003) adds to the evidence that Notch signaling may be involved in establishment of SV persistence in insect cells. JAK-STAT and Toll pathways are two important antimicrobial pathways in insects (Hoffmann, 2003). Evidence is accumulating to show that these are important for virus-vector coexistence—JAKSTAT pathway was shown to function in response to viral infection in drosophila (Dostert et al., 2005). In a previous study (Fragkoudis et al., 2008) activation of pathway involving STAT (upon bacterial infection of U4.4 cells) reduced SFV viral RNA levels up to 40–60% in U4.4 cells, presenting a possibility of STAT-related pathways being involved in antiviral activities against alphavirus infections (Fragkoudis et al., 2008). JAK-STAT pathway was also shown to be involved in mosquito antiviral defense against Dengue virus (Family: Flaviviridae) that contains a positive-sense RNA genome, similar to SV. Silencing of JAK-STAT pathway receptor in A. aegypti mosquitoes lead to increased susceptibility to Dengue infection (Souza-Neto et al., 2009). TEPI, closely related to TEPII was shown to be involved in JAK-STAT and/or Toll pathways. Toll and Hopscotch (drosophila homologue of mammalian Jak) gain-of-function mutant flies showed constitutive expression of TEPI (Lagueux et al., 2000). Complement-like activity of TEPI has been shown to determine the vectorial capacity of A. gambiae to carry malaria parasite (Blandin et al., 2004). Hence, up-regulation of TEP II at 5 dpi may indicate a immune-related role for TEP II in establishment of SV persistence. The single time-point selected for the expression analysis described here proved to be useful as we could identify factors relevant to previous observations in the insect cells infected or persistently infected with SV. For example, the Notch pathway could play a role in growth rate changes and cell survival, upon SV infection. The number of cytoskeletal factors that showed increased levels of expression was more numerous compared to any other functional class of gene products, reflecting the cytoskeletal changes seen in insect cells infected with SV, including appearance of intracellular vesicles (Miller and Brown, 1992; Mudiganti et al., 2006). Though not a natural host, the S2 cells provided a suitable system to study insect response to alphavirus infection. Extensive drosophila gene product information, i.e., gene ontologies, clones for specific candidate genes and information from gene silencing experiments involving various signaling pathways, certainly gave us more information to understand the functions of different factors up-regulated during establishment of alphavirus persistence.

4.1. TEPII expression correlates with inhibition of protease activity of viral ns polyproteins As TEPII gene product that codes for a protease inhibitor was identified to be up-regulated at 5 dpi, the inhibition of viral ns polyprotein cleavage in SV-infected drosophila S2 and mosquito U4.4 cells was probed using two different techniques. An increase in the polyprotein species nsP123 was seen in both cell lines using both western blot and IP techniques. The data presented shows that inhibition of processing of nsP123 correlated with an increase in TEPII shown by dot blots. This correlates with reduction in virus levels at 5 dpi (Mudiganti et al., 2006). While these data do not show the mechanism of inhibition of protease activity, they show accumulation of ns polyprotein to be involved in establishment of viral persistence.

Replicases formed by nsP123 and nsP4 synthesized (minus)RNA with high efficiency and (plus)-RNA with very low efficiency (Shirako and Strauss, 1994). Identification of nsP123 in S2 and U4.4 cells at 5 dpi suggests continuous (minus)-strand synthesis and very low levels of (plus)-strand synthesis during SV persistence. These decreased levels of (plus)-RNA must be responsible for reduction in virus levels seen at 5 dpi (see Section 1) in S2 and U4.4 cells (Mudiganti et al., 2006). Taking the observations (i) inhibition of cleavage of the ns polyprotein, (ii) TEPII up-regulation and (iii) reduction in virus levels at 5 dpi, together, we propose a model for the mechanism of SV persistence in invertebrates. In this model TEPII binds and traps the viral polyprotein to inhibit its activity, similar to the functioning of other members of the super family, such as mammalian ␣-macroglobulin (Arakawa et al., 1989). Continuous synthesis of nsP1234 and nsP123 was required to maintain continuous synthesis of minus-strands in mammalian cells infected with SV or SFV replicons that establish persistent infections (Frolov et al., 1999; Sawicki et al., 2006). The data presented here show that a similar mechanism is used in maintenance of SV persistence in the invertebrate host. Acknowledgements Authors would like to thank Elena Levashina (EMBL, Heidelberg, Germany) for providing TEP II e clone, Jim and Ellen Strauss for providing anti-nsP1 and anti-nsP2 antibodies. This research was supported by the Foundation for Research, Carson City NV. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.virusres.2010.02.016. References Arakawa, H., Nishigai, M., Ikai, A., 1989. alpha 2-macroglobulin traps a proteinase in the midregion of its arms. An immunoelectron microscopic study. J. Biol. Chem. 264 (4), 2350–2356. Armstrong, P.B., 2006. Proteases and protease inhibitors: a balance of activities in host–pathogen interaction. Immunobiology 211 (4), 263–281. Artavanis-Tsakonas, S., Rand, M.D., Lake, R.J., 1999. Notch signaling: cell fate control and signal integration in development. Science 284 (5415), 770–776. Avadhanula, V., Weasner, B.P., Hardy, G.G., Kumar, J.P., Hardy, R.W., 2009. A novel system for the launch of alphavirus RNA synthesis reveals a role for the Imd pathway in arthropod antiviral response. PLoS Pathogen. 5 (9), e1000582. Bardin, A.J., Le Borgne, R., Schweisguth, F., 2004. Asymmetric localization and function of cell-fate determinants: a fly’s view. Curr. Opin. Neurobiol. 14 (1), 6–14. Baron, M., Aslam, H., Flasza, M., Fostier, M., Higgs, J.E., Mazaleyrat, S.L., Wilkin, M.B., 2002. Multiple levels of Notch signal regulation (review). Mol. Membr. Biol. 19 (1), 27–38. Barton, D.J., Sawicki, S.G., Sawicki, D.L., 1991. Solubilization and immunoprecipitation of alphavirus replication complexes. J. Virol. 65 (3), 1496–1506. Bellaiche, Y., Radovic, A., Woods, D.F., Hough, C.D., Parmentier, M.L., O’Kane, C.J., Bryant, P.J., Schweisguth, F., 2001. The Partner of Inscuteable/Discs-large complex is required to establish planar polarity during asymmetric cell division in Drosophila. Cell 106 (3), 355–366. Blandin, S., Levashina, E.A., 2004. Thioester-containing proteins and insect immunity. Mol. Immunol. 40 (12), 903–908. Blandin, S., Shiao, S.H., Moita, L.F., Janse, C.J., Waters, A.P., Kafatos, F.C., Levashina, E.A., 2004. Complement-like protein TEP1 is a determinant of vectorial capacity in the malaria vector Anopheles gambiae. Cell 116 (5), 661–670. Bowers, D.F., Abell, B.A., Brown, D.T., 1995. Replication and tissue tropism of the alphavirus Sindbis in the mosquito Aedes albopictus. Virology 212 (1), 1–12. Bowers, D.F., Coleman, C.G., Brown, D.T., 2003. Sindbis virus-associated pathology in Aedes albopictus (Diptera: Culicidae). J. Med. Entomol. 40 (5), 698–705. Bras-Herreng, F., 1973. Changes in the properties of Sindbis virus during passage experiments in “Drosophila” (author’s transl.). Ann. Microbiol. (Paris) 124A (4), 507–533. Bras-Herreng, F., 1975. Multiplication of sindbis virus in Drosophila cells cultivated in vitro (author’s transl.). Arch. Virol. 48 (2), 121–129. Bras-Herreng, F., 1976. Adaptation of a Sindbis virus population to “Drosophila melanogaster” (author’s transl.). Ann. Microbiol. (Paris) 127B (4), 541–565. Brown, D.T., 1980. The assembly of Alphaviruses. In: Schlesingers, R.W. (Ed.), The Togaviruses. Academic Press, NY, pp. 473–501.

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