Optimization of S. aureus dCas9 and CRISPRi Elements for a Single Adeno-Associated Virus that Targets an Endogenous Gene.

The power of CRISPRi to decrease targeted gene expression for clinical applications has been inhibited by delivery challenges. Existing constructs are too large to fit within the ∼4.7 kb packaging size limitation of adeno-associated virus (AAV), the only FDA approved viral vector for clinical use. Therefore, we optimized CRISPRi components to generate a single AAV vector that contains all functional elements and effectively knocks down expression of an endogenous gene in vivo. First, we increased nuclear targeting of Staphylococcus aureus deactivated Cas9 (SadCas9) 4-fold by using a helical linker and the c-Myc nuclear localization signal. Second, we identified an amino-terminal Krüppel associated box (KRAB) construct as the most effective in decreasing expression of target genes in vitro. Third, we optimized promoters for guide RNA and evaluated mini-promoters for expression of KRAB-SadCas9 in liver cells. Our final construct decreased protein convertase subtilisin/kexin type 9 (Pcsk9) mRNA and secreted protein 5-fold in vitro. The corresponding AAV2/8 vector was localized in nuclei of liver cells and decreased Pcsk9 mRNA and serum protein levels by 30% in vivo. This single AAV approach provides a potential clinically translatable method for decreasing targeted gene transcription by CRISPRi in vivo.

Phenotypes of primary retinal macroglia: Implications for purification and culture conditions.

Many neurodegenerations, including those of the visual system, have complex etiologies that include roles for both neurons and glia. In the retina there is evidence that retinal astrocytes play an important role in neurodegeneration. There are several approaches for isolating and growing primary retinal astrocytes, however, they often lead to different results. In this study, we examined the influence of culture conditions on phenotypic maturation of primary, purified retinal glia. We compared retinal astrocytes and Müller glia purified by immunomagnetic separation, as differentiation between these astrocyte subtypes is critical and immuno-based methods are the standard practice of purification. We found that while time in culture impacts the health and phenotype of both astrocytes and Müller glia, the phenotypic maturation of retinal astrocytes was most impacted by serum factors. These factors appeared to actively regulate intermediate filament phenotypes in a manner consistent with the induction of astrocyte-mesenchymal transition (AMT). This propensity for retinal astrocytes to shift along an AMT continuum should be considered when interpreting resulting data. Our goal is that this study will help standardize the field so that studies are replicable, comparable, and as accurate as possible for subsequent interpretation of findings.

Systems Biology-Based Investigation of Cellular Antiviral Drug Targets Identified by Gene-Trap Insertional Mutagenesis.

Viruses require host cellular factors for successful replication. A comprehensive systems-level investigation of the virus-host interactome is critical for understanding the roles of host factors with the end goal of discovering new druggable antiviral targets. Gene-trap insertional mutagenesis is a high-throughput forward genetics approach to randomly disrupt (trap) host genes and discover host genes that are essential for viral replication, but not for host cell survival. In this study, we used libraries of randomly mutagenized cells to discover cellular genes that are essential for the replication of 10 distinct cytotoxic mammalian viruses, 1 gram-negative bacterium, and 5 toxins. We herein reported 712 candidate cellular genes, characterizing distinct topological network and evolutionary signatures, and occupying central hubs in the human interactome. Cell cycle phase-specific network analysis showed that host cell cycle programs played critical roles during viral replication (e.g. MYC and TAF4 regulating G0/1 phase). Moreover, the viral perturbation of host cellular networks reflected disease etiology in that host genes (e.g. CTCF, RHOA, and CDKN1B) identified were frequently essential and significantly associated with Mendelian and orphan diseases, or somatic mutations in cancer. Computational drug repositioning framework via incorporating drug-gene signatures from the Connectivity Map into the virus-host interactome identified 110 putative druggable antiviral targets and prioritized several existing drugs (e.g. ajmaline) that may be potential for antiviral indication (e.g. anti-Ebola). In summary, this work provides a powerful methodology with a tight integration of gene-trap insertional mutagenesis testing and systems biology to identify new antiviral targets and drugs for the development of broadly acting and targeted clinical antiviral therapeutics.

Identification of an epithelial cell receptor responsible for Clostridium difficile TcdB-induced cytotoxicity.

Clostridium difficile is the leading cause of hospital-acquired diarrhea in the United States. The two main virulence factors of C. difficile are the large toxins, TcdA and TcdB, which enter colonic epithelial cells and cause fluid secretion, inflammation, and cell death. Using a gene-trap insertional mutagenesis screen, we identified poliovirus receptor-like 3 (PVRL3) as a cellular factor necessary for TcdB-mediated cytotoxicity. Disruption of PVRL3 expression by gene-trap mutagenesis, shRNA, or CRISPR/Cas9 mutagenesis resulted in resistance of cells to TcdB. Complementation of the gene-trap or CRISPR mutants with PVRL3 resulted in restoration of TcdB-mediated cell death. Purified PVRL3 ectodomain bound to TcdB by pull-down. Pretreatment of cells with a monoclonal antibody against PVRL3 or prebinding TcdB to PVRL3 ectodomain also inhibited cytotoxicity in cell culture. The receptor is highly expressed on the surface epithelium of the human colon and was observed to colocalize with TcdB in both an explant model and in tissue from a patient with pseudomembranous colitis. These data suggest PVRL3 is a physiologically relevant binding partner that can serve as a target for the prevention of TcdB-induced cytotoxicity in C. difficile infection.

A role for H/ACA and C/D small nucleolar RNAs in viral replication.

We have employed gene-trap insertional mutagenesis to identify candidate genes whose disruption confer phenotypic resistance to lytic infection, in independent studies using 12 distinct viruses and several different cell lines. Analysis of >2,000 virus-resistant clones revealed >1,000 candidate host genes, approximately 20 % of which were disrupted in clones surviving separate infections with 2-6 viruses. Interestingly, there were 83 instances in which the insertional mutagenesis vector disrupted transcripts encoding H/ACA-class and C/D-class small nucleolar RNAs (SNORAs and SNORDs, respectively). Of these, 79 SNORAs and SNORDs reside within introns of 29 genes (predominantly protein-coding), while 4 appear to be independent transcription units. siRNA studies targeting candidate SNORA/Ds provided independent confirmation of their roles in infection when tested against cowpox virus, Dengue Fever virus, influenza A virus, human rhinovirus 16, herpes simplex virus 2, or respiratory syncytial virus. Significantly, eight of the nine SNORA/Ds targeted with siRNAs enhanced cellular resistance to multiple viruses suggesting widespread involvement of SNORA/Ds in virus-host interactions and/or virus-induced cell death.

The safety, pharmacokinetics, and efficacy of intraocular celecoxib.

PURPOSE: To determine safety, pharmacokinetics, and anti-inflammatory effects of intraocular celecoxib. METHODS: The right eye of animals was injected with 1.5, 3, or 6 mg celecoxib prepared in dimethyl sulfoxide (DMSO). Left eyes served as controls and received 0.1 mL DMSO. Electroretinograms (ERG) were obtained at baseline and at 1, 4, and 12 weeks, and eyes were enucleated afterward for histopathologic analysis. For pharmacokinetics, 3 mg celecoxib was injected, and vitreous and retina/choroid drug levels were then analyzed at specific time points. For efficacy, 1 µg lipopolysaccharide was injected to induce inflammation; the right eye was then injected with 3 mg celecoxib (six eyes) or 2 mg triamcinolone acetonide (six eyes) and the left eye with saline. Twenty-four hours later, aqueous fluid was removed, and total leukocyte concentration and prostaglandin E2 (PGE2) concentration were determined. RESULTS: Histologic and ERG studies demonstrated no signs of retinal or optic nerve toxicity. After a single 3-mg injection, vitreous (0.06 µg/mL) and retina/choroid (132.31 µg/g) celecoxib concentrations at 8 weeks exceeded median inhibitory concentration. Treatment with celecoxib and triamcinolone significantly reduced total leukocyte count by 40% (P = 0.02) and 31% (P = 0.01), respectively. Reduction in PGE2 levels paralleled reduction in leukocyte counts (P < 0.05). There was no increase in intraocular pressure, but cataract formation was observed at higher concentrations. CONCLUSIONS: Intraocular injection of celecoxib appeared to be nontoxic and demonstrated excellent penetration into the retina/choroid and sustained drug levels out to 8 weeks. Celecoxib demonstrated potent anti-inflammatory effects, but there was an association with cataract formation at higher doses.

Reduction of interleukin 8 and platelet-derived growth factor levels by topical ketorolac, 0.45%, in patients with diabetic retinopathy.

IMPORTANCE: Inhibition of inflammatory cytokines may have therapeutic effects in diabetic retinopathy (DR). OBJECTIVE: To compare aqueous and vitreous levels of 17 inflammatory cytokines in patients treated preoperatively with topical ketorolac tromethamine, 0.45%, or placebo before pars plana vitrectomy for complications related to proliferative DR (PDR). DESIGN, SETTING, AND PARTICIPANTS: A prospective, randomized, placebo-controlled, patient-masked interventional study performed in a university academic hospital included 20 eyes from 20 patients undergoing pars plana vitrectomy for complications of PDR. INTERVENTIONS: Eyes were randomized to ketorolac tromethamine, 0.45% (Acuvail), or placebo 4 times daily for 3 days before pars plana vitrectomy. Undiluted aqueous and vitreous samples were taken at the time of surgery and immediately frozen at -80°C. MAIN OUTCOMES AND MEASURES: Aqueous and vitreous levels of prostaglandin E2 and 16 other inflammatory cytokines implicated in the pathogenesis of DR. RESULTS: Prostaglandin E2, platelet-derived growth factor (PDGF) AA, eotaxin, vascular endothelial growth factor, interferon γ-inducible protein of 10 kDa, monocyte chemoattractant protein 1, growth-related oncogene, interleukin 6, interleukin 8 (IL-8), and tumor necrosis factor were detectable in the aqueous and vitreous of at least half of the eyes, and these cytokines were analyzed further. Aqueous levels were lower in the ketorolac group for all cytokines detected, but only the difference in IL-8 was statistically significant (52% reduction; P = .04). Levels of IL-8 (41% reduction; P = .002) and PDGF-AA (21% reduction; P = .009) were significantly lower in the vitreous of patients treated with ketorolac. CONCLUSIONS AND RELEVANCE: Topical ketorolac tromethamine, 0.45%, significantly lowered aqueous IL-8 levels and vitreous IL-8 and PDGF-AA levels in this series of eyes, suggesting that it may cause meaningful inhibition of inflammatory cytokines implicated in the pathogenesis of DR. TRIAL REGISTRATION: clinicaltrials.gov Identifier: NCT01609881.

Antibacterial properties of 2% lidocaine and reduced rate of endophthalmitis after intravitreal injection.

PURPOSE: To determine whether the application of subconjunctival 2% lidocaine/0.1% methylparaben for anesthesia may reduce rates of endophthalmitis after intravitreal (IVT) injection. METHODS: We performed in vitro experiments to determine the antibacterial properties of 2% lidocaine/0.1% methylparaben (lidocaine) against causative organisms of endophthalmitis. Isolates of Staphylococcus aureus, Staphylococcus epidermidis, and Streptococcus viridans from patients with endophthalmitis were incubated with or without lidocaine. Aliquots (100 µL) were plated on Mueller-Hinton (S. aureus and S. epidermidis) or blood agar plates (S. viridans) at 0, 10, 30, 120, and 240 minutes, and colonies were counted after 24 hours. A retrospective review of 15,042 IVT injections was performed from January 2004 to February 2011 to determine the rate of endophthalmitis with or without application of subconjunctival lidocaine for anesthesia. RESULTS: Lidocaine demonstrated rapid bactericidal effects against all 3 organisms. After 10 minutes of exposure, there was approximately a 90% (P < 0.01), 95% (P < 0.001), and 92% (P < 0.001) reduction in colony forming units when compared with time 0 for S. aureus, S. epidermidis, and S. viridans, respectively. Complete elimination of colony forming units occurred at subsequent time points for each organism in contrast to logarithmic increase for control plates. There were a total of 0 cases of endophthalmitis of 6,853 IVT injections performed with subconjunctival lidocaine and 8 cases of endophthalmitis of 8,189 (0.1%) IVT injections performed with other methods of anesthesia (P = 0.03). CONCLUSION: Application of subconjunctival 2% lidocaine/0.1% methylparaben for anesthesia may reduce the incidence of endophthalmitis after IVT injection.

Role of connexin 43 in Helicobacter pylori VacA-induced cell death.

Helicobacter pylori colonizes the human stomach and confers an increased risk for the development of peptic ulceration, noncardia gastric adenocarcinoma, and gastric lymphoma. A secreted H. pylori toxin, VacA, can cause multiple alterations in gastric epithelial cells, including cell death. In this study, we sought to identify host cell factors that are required for VacA-induced cell death. To do this, we analyzed gene trap and short hairpin RNA (shRNA) libraries in AZ-521 human gastric epithelial cells and selected for VacA-resistant clones. Among the VacA-resistant clones, we identified multiple gene trap library clones and an shRNA library clone with disrupted expression of connexin 43 (Cx43) (also known as gap junction protein alpha 1 [GJA1]). Further experiments with Cx43-specific shRNAs confirmed that a reduction in Cx43 expression results in resistance to VacA-induced cell death. Immunofluorescence microscopy experiments indicated that VacA did not colocalize with Cx43. We detected production of the Cx43 protein in AZ-521 cells but not in AGS, HeLa, or RK-13 cells, and correspondingly, AZ-521 cells were the most susceptible to VacA-induced cell death. When Cx43 was expressed in HeLa cells, the cells became more susceptible to VacA. These results indicate that Cx43 is a host cell constituent that contributes to VacA-induced cell death and that variation among cell types in susceptibility to VacA-induced cell death is attributable at least in part to cell type-specific differences in Cx43 production.

Reduction of vitreous prostaglandin E2 levels after topical administration of ketorolac 0.45%.

IMPORTANCE: Inhibition of proinflammatory prostaglandins in the retina may have therapeutic effects for retinal disease. OBJECTIVE: To determine vitreous levels of ketorolac and prostaglandin E2 (PGE2) in eyes treated with topical ketorolac tromethamine 0.45% (Acuvail). DESIGN, SETTING, AND PARTICIPANTS: A prospective comparative interventional study, performed in a university academic hospital, included 24 eyes in 22 consecutive patients undergoing pars plana vitrectomy. INTERVENTION: Application of topical ketorolac 0.45%, 4 times daily, for 3 days before pars plana vitrectomy in the first 12 consecutive eyes. The next 12 eyes were untreated and served as controls. Undiluted vitreous samples were obtained at the time of surgery and immediately frozen at -80 °C. MAIN OUTCOMES AND MEASURES: Vitreous ketorolac and PGE2 levels. RESULTS: Seven of the 12 eyes (58%) had ketorolac levels above the lower limit of quantitation. All 7 were in pseudophakic eyes, and 4 of the 5 below this limit were phakic (P = .01). The mean ketorolac level in the 7 eyes was 7.55 ng/mL (range, 5.0-14.9 ng/mL). The mean (SD) PGE2 levels were 13.8 (3.8) pg/mL in control eyes and 11.7 (4.4) pg/mL in ketorolac-treated eyes (P = .04). Treatment with ketorolac resulted in a 15% reduction in PGE2 levels. When only pseudophakic eyes were analyzed, mean (SD) PGE2 levels were 14.1 (4.1) pg/mL in control eyes and 11.6 (4.5) pg/mL in ketorolac-treated eyes (P < .05). CONCLUSIONS AND RELEVANCE: Topical ketorolac 0.45% can obtain a vitreous level that exceeds its median inhibitory concentration and can significantly decrease vitreous PGE2 levels. Vitreous levels of ketorolac were significantly higher in pseudophakic eyes than in phakic eyes. The results of this study suggest that topically administered ketorolac 0.45% may allow meaningful inhibition of prostaglandins in the retina. TRIAL REGISTRATION: clinicaltrials.gov Identifier: NCT01609881.

Oligomerization of Clostridium perfringens epsilon toxin is dependent upon caveolins 1 and 2.

Evidence from multiple studies suggests that Clostridium perfringens ε-toxin is a pore-forming toxin, assembling into oligomeric complexes in the plasma membrane of sensitive cells. In a previous study, we used gene-trap mutagenesis to identify mammalian factors contributing to toxin activity, including caveolin-2 (CAV2). In this study, we demonstrate the importance of caveolin-2 and its interaction partner, caveolin-1 (CAV1), in ε-toxin-induced cytotoxicity. Using CAV2-specific shRNA in a toxin-sensitive human kidney cell line, ACHN, we confirmed that cells deficient in CAV2 exhibit increased resistance to ε-toxin. Similarly, using CAV1-specific shRNA, we demonstrate that cells deficient in CAV1 also exhibit increased resistance to the toxin. Immunoprecipitation of CAV1 and CAV2 from ε-toxin-treated ACHN cells demonstrated interaction of both CAV1 and -2 with the toxin. Furthermore, blue-native PAGE indicated that the toxin and caveolins were components of a 670 kDa protein complex. Although ε-toxin binding was only slightly perturbed in caveolin-deficient cells, oligomerization of the toxin was dramatically reduced in both CAV1- and CAV2-deficient cells. These results indicate that CAV1 and -2 potentiate ε-toxin induced cytotoxicity by promoting toxin oligomerization - an event which is requisite for pore formation and, by extension, cell death.

Increased prostaglandin E2 (PGE2) levels in proliferative diabetic retinopathy, and correlation with VEGF and inflammatory cytokines.

PURPOSE: We determined vitreous levels of prostaglandin E2 (PGE(2)), VEGF, and 15 other cytokines in diabetic and nondiabetic patients undergoing vitrectomy. METHODS: Of 26 eyes of 26 patients enrolled consecutively, 13 eyes underwent vitrectomy for complications related to proliferative diabetic retinopathy, and the other 13 for epiretinal membrane, macular hole, vitreous opacities, or dislocated intraocular lens. Undiluted vitreous samples were taken at the time of surgery and frozen immediately at -80°C, and later analyzed for PGE(2), VEGF, and 15 other cytokines. RESULTS: PGE(2) levels were 53% higher in diabetic eyes. Mean ± standard deviation PGE(2) levels were 25.11 ± 11 pg/mL and 16.40 ± 7 pg/mL in diabetic and nondiabetic eyes, respectively (P < 0.03). Mean ± standard deviation VEGF levels were 2225 ± 3798 pg/mL and 66 ± 185 pg/mL in diabetic and nondiabetic eyes, respectively (P < 0.001). Other cytokines, including eotaxin-1, growth related oncogene (GRO), interleukin (IL)-6, IL-8, interferon-γ-inducible protein of 10 kDa (IP-10), monocyte chemoattractant protein-1 (MCP-1), tumor necrosis factor alpha (TNF-α), and platelet-derived growth factor-AA, also were elevated significantly in diabetic eyes. A significant correlation was seen between PGE(2) levels and IP-10 and VEGF (P = 0.04). CONCLUSIONS: PGE(2) levels are significantly higher in the vitreous of patients with complications from proliferative diabetic retinopathy, and correlate with IP-10 and VEGF. The results of our study suggest that PGE(2) may have a pathogenic role in diabetic retinopathy and implicates a potential therapeutic role for nonsteroidal anti-inflammatory drugs. (ClinicalTrials.gov number, NCT01609881.).

Identification of cellular proteins required for replication of human immunodeficiency virus type 1.

Cellular proteins are essential for human immunodeficiency virus type 1 (HIV-1) replication and may serve as viable new targets for treating infection. Using gene trap insertional mutagenesis, a high-throughput approach based on random inactivation of cellular genes, candidate genes were found that limit virus replication when mutated. Disrupted genes (N=87) conferring resistance to lytic infection with several viruses were queried for an affect on HIV-1 replication by utilizing small interfering RNA (siRNA) screens in TZM-bl cells. Several genes regulating diverse pathways were found to be required for HIV-1 replication, including DHX8, DNAJA1, GTF2E1, GTF2E2, HAP1, KALRN, UBA3, UBE2E3, and VMP1. Candidate genes were independently tested in primary human macrophages, toxicity assays, and/or Tat-dependent ß-galactosidase reporter assays. Bioinformatics analyses indicated that several host factors present in this study participate in canonical pathways and functional processes implicated in prior genome-wide studies. However, the genes presented in this study did not share identity with those found previously. Novel antiviral targets identified in this study should open new avenues for mechanistic investigation.

Inhibition of influenza A virus replication by antagonism of a PI3K-AKT-mTOR pathway member identified by gene-trap insertional mutagenesis.

BACKGROUND: Host genes serving potential roles in virus replication may be exploited as novel antiviral targets. METHODS: Small interfering RNA (siRNA)-mediated knockdown of host gene expression was used to validate candidate genes in screens against six unrelated viruses, most importantly influenza. A mouse model of influenza A virus infection was used to evaluate the efficacy of a candidate FDA-approved drug identified in the screening effort. RESULTS: Several genes in the PI3K-AKT-mTOR pathway were found to support broad-spectrum viral replication in vitro by RNA interference. This led to the discovery that everolimus, an mTOR inhibitor, showed in vitro antiviral activity against cowpox, dengue type 2, influenza A, rhino- and respiratory syncytial viruses. In a lethal mouse infection model of influenza A (H1N1 and H5N1) virus infection, everolimus treatment (1 mg/kg/day) significantly delayed death but could not prevent mortality. Fourteen days of treatment was more beneficial in delaying the time to death than treatment for seven days. Pathological findings in everolimus-treated mice showed reduced lung haemorrhage and lung weights in response to infection. CONCLUSIONS: These results provide proof of concept that cellular targets can be identified by gene knockout methods, and highlight the importance of the PI3K-AKT-mTOR pathway in supporting viral infections.

A functional role for ADAM10 in human immunodeficiency virus type-1 replication.

BACKGROUND: Gene trap insertional mutagenesis was used as a high-throughput approach to discover cellular genes participating in viral infection by screening libraries of cells selected for survival from lytic infection with a variety of viruses. Cells harboring a disrupted ADAM10 (A Disintegrin and Metalloprotease 10) allele survived reovirus infection, and subsequently ADAM10 was shown by RNA interference to be important for replication of HIV-1. RESULTS: Silencing ADAM10 expression with small interfering RNA (siRNA) 48 hours before infection significantly inhibited HIV-1 replication in primary human monocyte-derived macrophages and in CD4⁺ cell lines. In agreement, ADAM10 over-expression significantly increased HIV-1 replication. ADAM10 down-regulation did not inhibit viral reverse transcription, indicating that viral entry and uncoating are also independent of ADAM10 expression. Integration of HIV-1 cDNA was reduced in ADAM10 down-regulated cells; however, concomitant 2-LTR circle formation was not detected, suggesting that HIV-1 does not enter the nucleus. Further, ADAM10 silencing inhibited downstream reporter gene expression and viral protein translation. Interestingly, we found that while the metalloprotease domain of ADAM10 is not required for HIV-1 replication, ADAM15 and γ-secretase (which proteolytically release the extracellular and intracellular domains of ADAM10 from the plasma membrane, respectively) do support productive infection. CONCLUSIONS: We propose that ADAM10 facilitates replication at the level of nuclear trafficking. Collectively, our data support a model whereby ADAM10 is cleaved by ADAM15 and γ-secretase and that the ADAM10 intracellular domain directly facilitates HIV-1 nuclear trafficking. Thus, ADAM10 represents a novel cellular target class for development of antiretroviral drugs.

Gene-trap mutagenesis identifies mammalian genes contributing to intoxication by Clostridium perfringens ε-toxin.

The Clostridium perfringens ε-toxin is an extremely potent toxin associated with lethal toxemias in domesticated ruminants and may be toxic to humans. Intoxication results in fluid accumulation in various tissues, most notably in the brain and kidneys. Previous studies suggest that the toxin is a pore-forming toxin, leading to dysregulated ion homeostasis and ultimately cell death. However, mammalian host factors that likely contribute to ε-toxin-induced cytotoxicity are poorly understood. A library of insertional mutant Madin Darby canine kidney (MDCK) cells, which are highly susceptible to the lethal affects of ε-toxin, was used to select clones of cells resistant to ε-toxin-induced cytotoxicity. The genes mutated in 9 surviving resistant cell clones were identified. We focused additional experiments on one of the identified genes as a means of validating the experimental approach. Gene expression microarray analysis revealed that one of the identified genes, hepatitis A virus cellular receptor 1 (HAVCR1, KIM-1, TIM1), is more abundantly expressed in human kidney cell lines than it is expressed in human cells known to be resistant to ε-toxin. One human kidney cell line, ACHN, was found to be sensitive to the toxin and expresses a larger isoform of the HAVCR1 protein than the HAVCR1 protein expressed by other, toxin-resistant human kidney cell lines. RNA interference studies in MDCK and in ACHN cells confirmed that HAVCR1 contributes to ε-toxin-induced cytotoxicity. Additionally, ε-toxin was shown to bind to HAVCR1 in vitro. The results of this study indicate that HAVCR1 and the other genes identified through the use of gene-trap mutagenesis and RNA interference strategies represent important targets for investigation of the process by which ε-toxin induces cell death and new targets for potential therapeutic intervention.

Rab9 GTPase is required for replication of human immunodeficiency virus type 1, filoviruses, and measles virus.

Rab proteins and their effectors facilitate vesicular transport by tethering donor vesicles to their respective target membranes. By using gene trap insertional mutagenesis, we identified Rab9, which mediates late-endosome-to-trans-Golgi-network trafficking, among several candidate host genes whose disruption allowed the survival of Marburg virus-infected cells, suggesting that Rab9 is utilized in Marburg replication. Although Rab9 has not been implicated in human immunodeficiency virus (HIV) replication, previous reports suggested that the late endosome is an initiation site for HIV assembly and that TIP47-dependent trafficking out of the late endosome to the trans-Golgi network facilitates the sorting of HIV Env into virions budding at the plasma membrane. We examined the role of Rab9 in the life cycles of HIV and several unrelated viruses, using small interfering RNA (siRNA) to silence Rab9 expression before viral infection. Silencing Rab9 expression dramatically inhibited HIV replication, as did silencing the host genes encoding TIP47, p40, and PIKfyve, which also facilitate late-endosome-to-trans-Golgi vesicular transport. In addition, silencing studies revealed that HIV replication was dependent on the expression of Rab11A, which mediates trans-Golgi-to-plasma-membrane transport, and that increased HIV Gag was sequestered in a CD63+ endocytic compartment in a cell line stably expressing Rab9 siRNA. Replication of the enveloped Ebola, Marburg, and measles viruses was inhibited with Rab9 siRNA, although the non-enveloped reovirus was insensitive to Rab9 silencing. These results suggest that Rab9 is an important cellular target for inhibiting diverse viruses and help to define a late-endosome-to-plasma-membrane vesicular transport pathway important in viral assembly.

Discovery of mammalian genes that participate in virus infection.

BACKGROUND: Viruses are obligate intracellular parasites that rely upon the host cell for different steps in their life cycles. The characterization of cellular genes required for virus infection and/or cell killing will be essential for understanding viral life cycles, and may provide cellular targets for new antiviral therapies. RESULTS: Candidate genes required for lytic reovirus infection were identified by tagged sequence mutagenesis, a process that permits rapid identification of genes disrupted by gene entrapment. One hundred fifty-one reovirus resistant clones were selected from cell libraries containing 2 x 105 independently disrupted genes, of which 111 contained mutations in previously characterized genes and functionally anonymous transcription units. Collectively, the genes associated with reovirus resistance differed from genes targeted by random gene entrapment in that known mutational hot spots were under represented, and a number of mutations appeared to cluster around specific cellular processes, including: IGF-II expression/signalling, vesicular transport/cytoskeletal trafficking and apoptosis. Notably, several of the genes have been directly implicated in the replication of reovirus and other viruses at different steps in the viral lifecycle. CONCLUSIONS: Tagged sequence mutagenesis provides a rapid, genome-wide strategy to identify candidate cellular genes required for virus infection. The candidate genes provide a starting point for mechanistic studies of cellular processes that participate in the virus lifecycle and may provide targets for novel anti-viral therapies.

Effects of transforming growth factor-alpha (TGF-alpha) in vitro and in vivo on reovirus replication.

We have utilized growth factors in in vitro and in vivo systems to examine the role of cellular proliferation in reovirus replication. In vitro, proliferating RIE-1 cells can be infected with whole reovirus virions, but are relatively resistant to infection once confluent (Go arrest). It has been shown that TGF-alpha, which signals through the EGF-receptor (EGF-R), is capable of dramatically increasing the number of RIE-1 cells entering the S-phase in the presence of additional serum factors. Stimulation of the EGF-R without serum results in minimal increases in cells entering the S-phase with a restriction in reovirus replication. Therefore, other factors in serum are essential for fully permissive infection. In vivo, we used metallothionein (MT) promoter/enhancer-TGF-alpha transgenic mice to study the effect of cytokine activation on reovirus type 1 infection. Virus replication decreased following oral infection in these transgenic mice at 1 month of age, concordant with increased mucin production. Titers of reovirus obtained from the livers of 1 year old transgenic mice were approximately 10-fold higher than titers obtained in control mice. Taken together, these data indicate that while growth factor activation ultimately leads to an increase in virus infectivity, other factors may be necessary for reovirus replication.

Mutations in the IGF-II pathway that confer resistance to lytic reovirus infection.

BACKGROUND: Viruses are obligate intracellular parasites and rely upon the host cell for different steps in their life cycles. The characterization of cellular genes required for virus infection and/or cell killing will be essential for understanding viral life cycles, and may provide cellular targets for new antiviral therapies. RESULTS: A gene entrapment approach was used to identify candidate cellular genes that affect reovirus infection or virus induced cell lysis. Four of the 111 genes disrupted in clones selected for resistance to infection by reovirus type 1 involved the insulin growth factor-2 (IGF-II) pathway, including: the mannose-6-phosphate/IGF2 receptor (Igf2r), a protease associated with insulin growth factor binding protein 5 (Prss11), and the CTCF transcriptional regulator (Ctcf). The disruption of Ctcf, which encodes a repressor of Igf2, was associated with enhanced Igf2 gene expression. Plasmids expressing either the IGF-II pro-hormone or IGF-II without the carboxy terminal extension (E)-peptide sequence independently conferred high levels of cellular resistance to reovirus infection. Forced IGF-II expression results in a block in virus disassembly. In addition, Ctcf disruption and forced Igf2 expression both enabled cells to proliferate in soft agar, a phenotype associated with malignant growth in vivo. CONCLUSION: These results indicate that IGF-II, and by inference other components of the IGF-II signalling pathway, can confer resistance to lytic reovirus infection. This report represents the first use of gene entrapment to identify host factors affecting virus infection. Concomitant transformation observed in some virus resistant cells illustrates a potential mechanism of carcinogenesis associated with chronic virus infection.