A promoterless AAV6.2FF-based lung gene editing platform for the correction of surfactant protein B deficiency.

Surfactant protein B (SP-B) deficiency is a rare genetic disease that causes fatal respiratory failure within the first year of life. Currently, the only corrective treatment is lung transplantation. Here, we co-transduced the murine lung with adeno-associated virus 6.2FF (AAV6.2FF) vectors encoding a SaCas9-guide RNA nuclease or donor template to mediate insertion of promoterless reporter genes or the (murine) Sftpb gene in frame with the endogenous surfactant protein C (SP-C) gene, without disrupting SP-C expression. Intranasal administration of 3 × 1011 vg donor template and 1 × 1011 vg nuclease consistently edited approximately 6% of lung epithelial cells. Frequency of gene insertion increased in a dose-dependent manner, reaching 20%-25% editing efficiency with the highest donor template and nuclease doses tested. We next evaluated whether this promoterless gene editing platform could extend survival in the conditional SP-B knockout mouse model. Administration of 1 × 1012 vg SP-B-donor template and 5 × 1011 vg nuclease significantly extended median survival (p = 0.0034) from 5 days in the untreated off doxycycline group to 16 days in the donor AAV and nuclease group, with one gene-edited mouse living 243 days off doxycycline. This AAV6.2FF-based gene editing platform has the potential to correct SP-B deficiency, as well as other disorders of alveolar type II cells.

Oral delivery of a dsRNA-Phytoglycogen nanoparticle complex enhances both local and systemic innate immune responses in rainbow trout.

Salmonids are one of the most farmed fish species worldwide. These aquatic vertebrates rely heavily on their innate immune responses as the first line of defense to defend themselves against invading pathogens. Although commercial vaccines are available against some viral and bacterial pathogens affecting salmonids, their protective efficacy varies. Using a prophylactic inducer of local and systemic innate immune responses to limit infection could have significant implications in salmonid aquaculture. A potent inducer of innate immune responses in fish is double-stranded RNA (dsRNA), a molecule that all viruses make during their replicative cycle. Polyinosinic: polycytidylic acid (polyI:C) is a synthetic dsRNA commonly used to induce type I interferons (IFNs), interferon stimulated genes (ISGs) as well as an antiviral state in vertebrate species. Based on in vitro data it was hypothesized that both local and systemic innate immune responses, in salmonids, would be enhanced by orally delivering high molecular weight polyI:C (HMW polyI:C) using cationic phytoglycogen nanoparticles (NPs) as a delivery method. The present study investigates this hypothesis using two feed delivery methods. In the first in vivo study, to ensure an equal distribution of dose, individual rainbow trout (Oncorhynchus mykiss) were orally gavaged with feed moistened with a solution containing HMW-NP (polyI:C complexed with cationic phytoglycogen nanoparticles) or HMW polyI:C alone. In a second in vivo experiment, to better mimic a more realistic feeding scenario, rainbow trout were fed feed pellets to which HMW, or HMW-NP was added. The expression of IFN1 and ISGs (vig-3, Mx1) were quantified using real-time PCR in the intestine (local response) and head kidney (systemic response). The results of these studies indicate that HMW-NP induced a higher level of IFN1 and ISG expression in the intestine and head kidney compared to the HMW fed fish. The results of this study could lead to new advances in therapeutics for the aquaculture industry by utilizing the innate immune response against invading pathogens using an orally delivered stimulant.

Truncation of the enzootic nasal tumor virus envelope protein cytoplasmic tail increases Env-mediated fusion and infectivity.

Enzootic nasal tumor virus (ENTV) and Jaagsiekte sheep retrovirus (JSRV) are highly related ovine betaretroviruses that induce nasal and lung tumours in small ruminants, respectively. While the ENTV and JSRV envelope (Env) glycoproteins mediate virus entry using the same cellular receptor, the glycosylphosphatidylinositol-linked protein hyaluronoglucosaminidase, ENTV Env pseudovirions mediate entry into cells from a much more restricted range of species than do JSRV Env pseudovirions. Unlike JSRV Env, ENTV Env does not induce cell fusion at pH 5.0 or above, but rather requires a much lower pH (4.0-4.5) for fusion to occur. The cytoplasmic tail of retroviral envelope proteins is a key modulator of envelope-mediated fusion and pseudotype efficiency, especially in the context of virions composed of heterologous Gag proteins. Here we report that progressive truncation of the ENTV Env cytoplasmic tail improves transduction efficiency of pseudotyped retroviral vectors and that complete truncation of the ENTV Env cytoplasmic tail increases transduction efficiency to wild-type JSRV Env levels by increasing fusogenicity without affecting sensitivity to inhibition by lysosomotropic agents, subcellular localization or efficiency of inclusion into virions. Truncation of the cytoplasmic domain of ENTV Env resulted in a significant advantage in viral entry into all cell types tested, including foetal ovine lung and nasal cells. Taken together, we demonstrate that the cytoplasmic tail modulates the fusion activity of the ENTV Env protein and that truncation of this region enhances Eenv-mediated entry into target cells.

AAV vector distribution in the mouse respiratory tract following four different methods of administration.

BACKGROUND: Targeted delivery of gene therapy vectors to the mouse respiratory tract is often performed via intranasal or intratracheal administration; however, there can be a great deal of variability between these methods, which could potentially influence experimental results. Improving the accuracy and precision of lung delivery will not only reduce the number of animals required to detect statistically significant differences, but may reduce the variability of studies from different laboratories. RESULTS: Here we evaluated three different methods of adeno-associated virus (AAV) vector administration to the respiratory tract in mice (intranasal, intubation, and intratracheal injection) and discuss the advantages, challenges, and shortcomings of each. We also present a modified-intranasal delivery technique that is superior to passive administration of vector into the nares of anesthetized supine animals. Transgene expression was consistently visible in the nasal cavity, trachea, and proximal to middle aspect of all lung lobes for all four methods, whereas transgene expression was consistently observed in the most distal aspect of lung lobes only with the intubation and intratracheal injection techniques. AAV vector genome copy numbers in the lung were approximately four-fold lower in mice that received vector via intranasal administration in comparison to the other three methods of vector delivery. The modified intranasal, intubation and intratracheal injection methods of vector administration did not yield statistical differences in AAV vector genome copy numbers in the lung. With regard to reproducibility of vector distribution within and between animals, the modified-intranasal technique was superior. CONCLUSION: Our results show that mode of AAV vector administration to the murine respiratory tract should be selected based on desired target site and skill of the researcher, and that appropriate technique selection may greatly influence experimental outcomes.

Nuclear Translocation Sequence and Region in Autographa californica Multiple Nucleopolyhedrovirus ME53 That Are Important for Optimal Baculovirus Production.

UNLABELLED: Autographa californicamultiple nucleopolyhedrovirus (AcMNPV) is in the familyBaculoviridae, genusAlphabaculovirus AcMNPVme53is a highly conserved immediate early gene in all lepidopteran baculoviruses that have been sequenced and is transcribed up to late times postinfection. Althoughme53is not essential for viral DNA synthesis, infectious budded virus (BV) production is greatly attenuated when it is deleted. ME53 associates with the nucleocapsid on both budded virus and occlusion-derived virus, but not with the virus envelope. ME53 colocalizes in plasma membrane foci with the envelope glycoprotein GP64 in a GP64-dependent manner. ME53 localizes in the cytoplasm early postinfection, and despite the lack of a reported nuclear localization signal (NLS), ME53 translocates to the nucleus at late times postinfection. To map determinants of ME53 that facilitate its nuclear translocation, recombinant AcMNPV bacmids containing a series of ME53 truncations, internal deletions, and peptides fused with hemagglutinin (HA) or green fluorescent protein (GFP) tags were constructed. Intracellular-localization studies identified residues within amino acids 109 to 137 at the N terminus of ME53 that acted as the nuclear translocation sequence (NTS), facilitating its nuclear transport at late times postinfection. The first 100 N-terminal amino acids and the last 50 C-terminal amino acids of ME53 are dispensable for high levels of budded virus production. The region within amino acids 101 to 398, which also contains the NTS, is critical for optimal levels of budded virus production. IMPORTANCE: Baculovirusme53is a conserved immediate early gene found in all sequenced lepidopteran alpha- and betabaculoviruses. We first identified residues within amino acids 109 to 137 at the N terminus that act as the ME53 nuclear translocation sequence (NTS) to facilitate its nuclear translocation and defined an internal region within amino acids 101 to 398, which includes the NTS, as being necessary for optimal budded virus production. Altogether, these results indicate a previously unidentified nuclear role that ME53 plays in virus replication.

Selection and characterization of Autographa californica multiple nucleopolyhedrovirus DNA polymerase mutations.

Autographa californica multiple nucleopolyhedrovirus (AcMNPV) DNA polymerase (DNApol) is essential for viral DNA replication. AcMNPV mutants resistant to aphidicolin, a selective inhibitor of viral DNA replication, and abacavir, an efficacious nucleoside analogue with inhibitory activity against reverse transcriptase, were selected by the serial passage of the parental AcMNPV in the presence of increasing concentrations of aphidicolin or abacavir. These drug-resistant mutants had either a single (C543R) (aphidicolin) or a double (C543R and S611T) (abacavir) point mutation within conserved regions II and III. To confirm the role of these point mutations in AcMNPV DNA polymerase, a dnapol knockout virus was first generated, and several repair viruses were constructed by transposing the dnapol wild-type gene or ones containing a single or double point mutation into the polyhedrin locus of the dnapol knockout bacmid. The single C543R or double C543R/S611T mutation showed increased resistance to both aphidicolin and abacavir and, even in the absence of drug, decreased levels of virus and viral DNA replication compared to the wild-type repair virus. Surprisingly, the dnapol mutant repair viruses led to the generation of occlusion-derived viruses with mostly single and only a few multiple nucleocapsids in the ring zone and within polyhedra. Thus, these point mutations in AcMNPV DNA polymerase increased drug resistance, slightly compromised virus and viral DNA replication, and influenced the viral morphogenesis of occlusion-derived virus.

Immediate-early protein ME53 forms foci and colocalizes with GP64 and the major capsid protein VP39 at the cell membranes of Autographa californica multiple nucleopolyhedrovirus-infected cells.

me53 is an immediate-early/late gene found in all lepidopteran baculoviruses sequenced to date. Deletion of me53 results in a greater-than-1,000-fold reduction in budded-virus production in tissue culture (J. de Jong, B. M. Arif, D. A. Theilmann, and P. J. Krell, J. Virol. 83:7440-7448, 2009). We investigated the localization of ME53 using an ME53 construct fused to green fluorescent protein (GFP). ME53:GFP adopted a primarily cytoplasmic distribution at early times postinfection and a primarily nuclear distribution at late times postinfection. Additionally, at late times ME53:GFP formed distinct foci at the cell periphery. These foci colocalized with the major envelope fusion protein GP64 and frequently with VP39 capsid protein, suggesting that these cell membrane regions may represent viral budding sites. Deletion of vp39 did not influence the distribution of ME53:GFP; however, deletion of gp64 abolished ME53:GFP foci at the cell periphery, implying an association between ME53 and GP64. Despite the association of ME53 and GP64, ME53 fractionated with the nucleocapsid only after budded-virus fractionation. Together these findings suggest that ME53 may be providing a scaffold that bridges the viral envelope and nucleocapsid.

Changes to fine structure, size and mechanical modulus of phytoglycogen nanoparticles subjected to high-shear extrusion.

This study aims to enhance the understanding of the structure of maize phytoglycogen nanoparticles, and the effect of shear scission on their architecture, radius, stiffness, and deformability. Compared to amylopectin, phytoglycogen had a lower A:B chain ratio, a lower number of chains per B chain, and a much higher number of Afingerprint chains. Phytoglycogen (Mw = 28.0 × 106 g/mol) was subjected to high-shear extrusion with varying Specific Mechanical Energies (SMEs) using different screw speeds, showing a maximum stable molecular weight Mw of ∼9.31 × 106 g/mol and a particle radius R reduction of 36 %, with a corresponding 20 % increase in the average mass density. Atomic force microscopy force spectroscopy revealed that nanoparticles extruded at the lowest SME (122 Wh/kg) exhibited a 20 % increase in Young's modulus. Higher SME values (up to 488 Wh/kg) resulted in an overall decrease in stiffness without further significant reductions in radius.

Using a Prime-Boost Vaccination Strategy That Proved Effective for High Resolution Epitope Mapping to Characterize the Elusive Immunogenicity of Survivin.

Survivin is a member of the inhibitor of apoptosis family of proteins and has been reported to be highly expressed in a variety of cancer types, making it a high priority target for cancer vaccination. We previously described a heterologous prime-boost strategy using a replication-deficient adenovirus, followed by an oncolytic rhabdovirus that generates unprecedented antigen-specific T cell responses. We engineered each vector to express a mutated version of full-length murine survivin. We first sought to uncover the complete epitope map for survivin-specific T cell responses in C57BL/6 and BALB/c mice by flow cytometry. However, no T cell responses were detected by intracellular cytokine staining after re-stimulation of T cells. Survivin has been found to be expressed by activated T cells, which could theoretically cause T cell-mediated killing of activated T cells, known as fratricide. We were unable to recapitulate this phenomenon in experiments. Interestingly, the inactivated survivin construct has been previously shown to directly kill tumor cells in vitro. However, there was no evidence in our models of induction of death in antigen-presenting cells due to treatment with a survivin-expressing vector. Using the same recombinant virus-vectored prime-boost strategy targeting the poorly immunogenic enhanced green fluorescent protein proved to be a highly sensitive method for mapping T cell epitopes, particularly in the context of identifying novel epitopes recognized by CD4+ T cells. Overall, these results suggested there may be unusually robust tolerance to survivin in commonly used mouse strains that cannot be broken, even when using a particularly potent vaccination platform. However, the vaccination method shows great promise as a strategy for identifying novel and subdominant T cell epitopes.

Autographa californica multiple nucleopolyhedrovirus me53 (ac140) is a nonessential gene required for efficient budded-virus production.

me53 is a highly conserved baculovirus gene found in all lepidopteran baculoviruses that have been fully sequenced to date. The putative ME53 protein contains a zinc finger domain and has been previously described as a major early transcript. We generated an me53-null bacmid (AcDeltame53GFP), as well as a repair virus (AcRepME53:HA-GFP) carrying me53 with a C-terminal hemagglutinin (HA) tag, under the control of its native early and late promoter elements. Sf9 and BTI-Tn-5b1 cells transfected with AcDeltame53GFP resulted in a 3-log reduction in budded-virus (BV) production compared to both the parental Autographa californica multiple nucleopolyhedrosis virus and the repair bacmids, demonstrating that although me53 is not essential for replication, replication is compromised in its absence. Our data also suggest that me53 does not affect DNA replication. Cell fractionation showed that ME53 is found in both the nucleus and the cytoplasm as early as 6 h postinfection. Deletion of the early transcriptional start site resulted in a 10- to 360-fold reduction of BV yield; however, deletion of the late promoter (ATAAG) resulted in a 160- to 1,000-fold reduction, suggesting that, in the context of BV production, ME53 is required both early and late in the infection cycle. Additional Western blot analysis of purified virions from the repair virus revealed that ME53:HA is associated with both BV and occlusion-derived virions. Together, these results indicate that me53, although not essential for viral replication, is required for efficient BV production.

Phytoglycogen Nanoparticle Delivery System for Inorganic Selenium Reduces Cytotoxicity without Impairing Selenium Bioavailability.

PURPOSE: Selenium is an essential trace element that supports animal health through the antioxidant defense system by protecting cells from oxidative-related damage. Using inorganic selenium species, such as sodium selenite (Na Sel), as a food supplement is cost-effective; however, its limitation as a nutritional supplement is its cytotoxicity. One strategy to mitigate this problem is by delivering inorganic selenium using a nanoparticle delivery system (SeNP). METHODS: Rainbow trout intestinal epithelial cells, bovine turbinate cells and bovine intestinal myofibroblasts were treated with soluble Na Sel or SeNPs. Two SeNP formulations were tested; SeNP-Ionic where inorganic selenium was ionically bound to cationic phytoglycogen (PhG) NPs, and SeNP-Covalent, where inorganic selenium was covalently bound to PhG NPs. Selenium-induced cytotoxicity along with selenium bioavailability were measured. RESULTS: SeNPs (SeNP-Ionic or SeNP-Covalent) substantially reduced cytotoxicity in all cell types examined compared to similar doses of soluble inorganic selenium. The SeNP formulations did not affect selenium bioavailability, as selenium-induced glutathione peroxidase (GPx) activity and GPx1 transcript levels were similarly elevated whether cells were treated with soluble Na Sel or SeNPs. This was the case for all three cell types tested. CONCLUSION: Nanoparticle-assisted inorganic selenium delivery, which demonstrated equal bioavailability without causing deleterious cytotoxic side effects, has potential applications for safely supplementing animal diets with inorganic selenium at what are usually toxic doses.

Optimized Pre-Clinical Grade Production of Two Novel Lentiviral Vector Pseudotypes for Lung Gene Delivery.

Lung gene therapy requires efficient transduction of slow-replicating epithelia and stable expression of delivered transgenes in the respiratory tract. Lentiviral (LV) vectors have the ideal coding, expression, and transducing capacity required for gene therapy. A modified envelope glycoprotein from the Jaagsiekte Sheep Retrovirus, termed Jenv, is well suited for LV-mediated lung gene therapy due to its inherent lung tropism. Here, two novel Jenv-pseudotyped LVs that effectively transduce lung tissue and yield titers similar to the gold standard, vesicular stomatitis virus glycoprotein (VSVg)-pseudotyped LVs, were generated. As the concentration efficiency of LVs was found to depend on envelope pseudotype, a large-scale production method tailored for Jenv-pseudotyped LVs was developed and the most appropriate method of concentration was determined. In contrast to VSVg and Ebola virus glycoprotein-pseudotyped LVs, ultracentrifugation through a sucrose cushion drastically reduced the yield of Jenv LVs, whereas polyethylene glycol precipitation and tangential flow filtration (TFF) proved to be more suitable methods for concentrating Jenv LVs. Importantly, pressure during TFF was found to be crucial for increasing LV recovery. Finally, a unique mouse model was developed to test the suitability of these novel Jenv-pseudotyped LVs for use in lung gene therapy applications.

Enhancing innate antiviral immune responses in rainbow trout by double stranded RNA delivered with cationic phytoglycogen nanoparticles.

Innate immunity is induced when pathogen-associated molecular patterns (PAMPs) bind host pattern recognition receptors (PRRs). Polyinosinic:polycytidylic acid [poly(I:C)] is a synthetic analogue of viral dsRNA that acts as a PAMP, inducing type I interferons (IFNs) in vertebrates. In the present study, the immunostimulatory effects of high molecular weight (HMW) poly(I:C) in rainbow trout cells were measured when bound to a cationic phytoglycogen nanoparticle (Nano-HMW). The physical characteristics of the nanoparticle itself, when bound to different lengths of dsRNA and when cell associated was evaluated. Optimal concentration and timing for innate immune stimulation was measured using the RTG-P1 reporter cell line. The immunostimulatory effects of HMW poly (I:C) was compared to Nano-HMW in vitro using the RTgutGC cell line cultured in a conventional monolayer or a transwell culture system. The ability of an activated intestinal epithelium to transmit an antiviral signal to macrophages was evaluated using a co-culture of RTgutGC cells and RTSll (a monocyte/macrophage cell). In all culture conditions, Nano-HMW was a more effective inducer of IFN-related antiviral immune responses compared to HMW poly (I:C) alone. This study introduces the use of cationic phytoglycogen nanoparticles as a novel delivery system for immunomodulatory molecules to enhance immune responses in aquatic vertebrates.

Use of Precision-Cut Lung Slices as an Ex Vivo Tool for Evaluating Viruses and Viral Vectors for Gene and Oncolytic Therapy.

Organotypic slice cultures recapitulate many features of an intact organ, including cellular architecture, microenvironment, and polarity, making them an ideal tool for the ex vivo study of viruses and viral vectors. Here, we describe a procedure for generating precision-cut ovine and murine tissue slices from agarose-perfused normal and murine melanoma tumor-bearing lungs. Furthermore, we demonstrate that these precision-cut lung slices can be maintained up to 1 month and can be used for a range of applications, which include characterizing the tissue tropism of viruses that cannot be propagated in cell monolayers, evaluating the transducing properties of gene therapy vectors, and, finally, investigating the tumor specificity of oncolytic viruses. Our results suggest that ex vivo lung slices are an ideal platform for studying the tissue specificity and cancer cell selectivity of gene therapy vectors and oncolytic viruses prior to in vivo studies, providing justification for pre-clinical work.

Rapid generation of fowl adenovirus 9 vectors.

Fowl adenoviruses (FAdV) have the largest genomes of any fully sequenced adenovirus genome, and are widely considered as excellent platforms for vaccine development and gene therapy. As such, there is a strong need for stream-lined protocols/strategies for the generation of recombinant adenovirus genomes. Current genome engineering strategies rely upon plasmid based homologous recombination in Escherichia coli BJ5183. This process is time-consuming, involves multiple cloning steps, and low efficiency recombination. This report describes a novel system for the more rapid generation of recombinant fowl adenovirus genomes using the lambda Red recombinase system in E. coli DH10B. In this strategy, PCR based amplicons with around 50 nt long homologous arms, a unique SwaI site and a chloramphenicol resistance gene fragment (CAT cassette), are introduced into the FAdV-9 genome in a highly efficient and site-specific manner. To demonstrate the efficacy of this system we generated FAdV-9 ORF2, and FAdV-9 ORF11 deleted, CAT marked and unmarked FAdV-9 infectious clones (FAdmids), and replaced either ORF2 or ORF11, with an EGFP expression cassette or replaced ORF2 with an EGFP coding sequence via the unique SwaI sites, in approximately one month. All recombinant FAdmids expressed EGFP and were fully infectious in CH-SAH cells.

Genetic characterization of small ruminant lentiviruses circulating in naturally infected sheep and goats in Ontario, Canada.

Maedi-visna virus (MVV) and caprine arthritis encephalitis virus (CAEV) are related members of a group of small ruminant lentiviruses (SRLVs) that infect sheep and goats. SRLVs are endemic in many countries, including Canada. However, very little is known about the genetic characteristics of Canadian SRLVs, particularly in the province of Ontario. Given the importance of surveillance and eradication programs for the control of SRLVs, it is imperative that the diagnostic tests used to identify infected animals are sensitive to local strains of SRLVs. The aim of this work was to characterize SRLV strains circulating in Ontario and to evaluate the variability of the immunodominant regions of the Gag protein. In this study, the nearly complete gag sequence of 164 SRLVs, from 130 naturally infected sheep and 32 naturally infected goats from Ontario, was sequenced. Animals belonged to distantly located single and mixed species (sheep and goats) farms. Ovine lentiviruses from the same farm tended to cluster more closely together than did caprine lentiviruses from the same farm. Sequence analysis revealed a higher degree of heterogeneity among the caprine lentivirus sequences with an average inter-farm pairwise DNA distance of 10% and only 5% in the ovine lentivirus group. Interestingly, amplification of SRLVs from ELISA positive sheep was successful in 81% of cases, whereas amplification of SRLV proviral DNA was only possible in 55% of the ELISA positive goat samples; suggesting that a significant portion of caprine lentiviruses circulating in Ontario possess heterogeneity at the primer binding sites used in this study. Sequences of sheep and goat SRLVs from Ontario were assembled into phylogenetic trees with other known SRLVs and were found to belong to sequence groups A2 and B1, respectively, as defined by Shah et al. (2004a). A novel caprine lentivirus with a pairwise genetic difference of 15.6-25.4% relative to other group B subtypes was identified. Thus we suggest the designation of a novel subtype, B4, within the caprine lentivirus-like cluster. Lastly, we demonstrate evidence of recombination between ovine lentiviruses. These results emphasize the broad genetic diversity of SRLV strains circulating in the province of Ontario and show that the gag region is suitable for phylogenetic studies and may be applied to monitor SRLV eradication programs.

Analysis of the Choristoneura fumiferana nucleopolyhedrovirus genome.

The double-stranded DNA genome of Choristoneura fumiferana nucleopolyhedrovirus (CfMNPV) was sequenced and analysed in the context of other group I nucleopolyhedroviruses (NPVs). The genome consists of 129,593 bp with a G + C content of 50.1 mol%. A total of 146 open reading frames (ORFs) of greater than 150 bp, and with no or minimal overlap were identified. In addition, five homologous regions were identified containing 7-10 repeats of a 36 bp imperfect palindromic core. Comparison with other completely sequenced baculovirus genomes revealed that 139 of the CfMNPV ORFs have homologues in at least one other baculovirus and seven ORFs are unique to CfMNPV. Of the 117 CfMNPV ORFs common to all group I NPVs, 12 are exclusive to group I NPVs. Overall, CfMNPV is most similar to Orgyia pseudotsugata MNPV based on gene content, arrangement and overall amino acid identity. Unlike other group I baculoviruses, however, CfMNPV encodes a viral enhancing factor (vef) and has two copies of p26.

Choristoneura fumiferana nucleopolyhedrovirus encodes a functional 3'-5' exonuclease.

The Choristoneura fumiferana nucleopolyhedrovirus (CfMNPV) encodes an ORF homologous to type III 3'-5' exonucleases. The CfMNPV v-trex ORF was cloned into the Bac-to-Bac baculovirus expression-vector system, expressed in insect Sf21 cells with an N-terminal His tag and purified to homogeneity by using Ni-NTA affinity chromatography. Biochemical characterization of the purified V-TREX confirmed that this viral protein is a functional 3'-5' exonuclease that cleaves oligonucleotides from the 3' end in a stepwise, distributive manner, suggesting a role in proofreading during viral DNA replication and DNA repair. Enhanced degradation of a 5'-digoxigenin- or 5'-(32)P-labelled oligo(dT)(30) substrate was observed at increasing incubation times or increased amounts of V-TREX. The 3'-excision activity of V-TREX was maximal at alkaline pH (9.5) in the presence of 5 mM MgCl(2), 2 mM dithiothreitol and 0.1 mg BSA ml(-1).