Processing of the 3C/D Region of the Deformed Wing Virus (DWV).

The deformed wing virus (DWV) belongs to the genus Iflavirus and the family Iflaviridae within the order Picornavirales. It is an important pathogen of the Western honey bee, Apis mellifera, causing major losses among honey bee colonies in association with the ectoparasitic mite Varroa destructor. Although DWV is one of the best-studied insect viruses, the mechanisms of viral replication and polyprotein processing have been poorly studied in the past. We investigated the processing of the protease-polymerase region at the C-terminus of the polyprotein in more detail using recombinant expression, novel serological reagents, and virus clone mutagenesis. Edman degradation of purified maturated polypeptides uncovered the C- and N-termini of the mature 3C-like (3CL) protease and RNA-dependent RNA polymerase (3DL, RdRp), respectively. Autocatalytic processing of the recombinant DWV 3CL protease occurred at P1 Q2118 and P1' G2119 (KPQ/GST) as well as P1 Q2393 and P1' S2394 (HAQ/SPS) cleavage sites. New monoclonal antibodies (Mab) detected the mature 3CL protease with an apparent molecular mass of 32 kDa, mature 3DL with an apparent molecular mass of 55 kDa as well as a dominant 3CDL precursor of 90 kDa in DWV infected honey bee pupae. The observed pattern corresponds well to data obtained via recombinant expression and N-terminal sequencing. Finally, we were able to show that 3CL protease activity and availability of the specific protease cleavage sites are essential for viral replication, protein synthesis, and establishment of infection using our molecular clone of DWV-A.

DNAJC14-Independent Replication of the Atypical Porcine Pestivirus.

Atypical porcine pestiviruses (APPV; Pestivirus K) are a recently discovered, very divergent species of the genus Pestivirus within the family Flaviviridae. The presence of APPV in piglet-producing farms is associated with the occurrence of so-called "shaking piglets," suffering from mild to severe congenital tremor type A-II. Previous studies showed that the cellular protein DNAJC14 is an essential cofactor of the NS2 autoprotease of all classical pestiviruses. Consequently, genetically engineered DNAJC14 knockout cell lines were resistant to all tested noncytopathogenic (non-cp) pestiviruses. Surprisingly, we found that the non-cp APPV can replicate in these cells in the absence of DNAJC14, suggesting a divergent mechanism of polyprotein processing. A complete laboratory system for the study of APPV was established to learn more about the replication of this unusual virus. The inactivation of the APPV NS2 autoprotease using reverse genetics resulted in nonreplicative genomes. To further investigate whether a regulation of the NS2-3 cleavage is also existing in APPV, we constructed synthetic viral genomes with deletions and duplications leading to the NS2 independent release of mature NS3. As observed with other pestiviruses, the increase of mature NS3 resulted in elevated viral RNA replication levels and increased protein expression. Our data suggest that APPV exhibit a divergent mechanism for the regulation of the NS2 autoprotease activity most likely utilizing a different cellular protein for the adjustment of replication levels. IMPORTANCE DNAJC14 is an essential cofactor of the pestiviral NS2 autoprotease, limiting replication to tolerable levels as a prerequisite for the noncytopathogenic biotype of pestiviruses. Surprisingly, we found that the atypical porcine pestivirus (APPV) is able to replicate in the absence of DNAJC14. We further investigated the NS2-3 processing of APPV using a molecular clone, monoclonal antibodies, and DNAJC14 knockout cells. We identified two potential active site residues of the NS2 autoprotease and could demonstrate that the release of NS3 by the NS2 autoprotease is essential for APPV replication. Defective interfering genomes and viral genomes with duplicated NS3 sequences that produce mature NS3 independent of the NS2 autoprotease activity showed increased replication and antigen expression. It seems likely that an alternative cellular cofactor controls NS2-3 cleavage and thus replication of APPV. The replication-optimized synthetic APPV genomes might be suitable live vaccine candidates, whose establishment and testing warrant further research.

Characterization of a Cytopathogenic Reporter CSFV.

Cytopathogenic (cp) pestiviruses frequently emerge in cattle that are persistently infected with the bovine viral diarrhea virus (BVDV) as a consequence of RNA recombination and mutation. They induce apoptosis in infected tissue cultures, are highly attenuated in the immunocompetent host, and unable to establish persistent infections after diaplacental infections. Cp strains of BVDV have been used as naturally attenuated live vaccines and for species-specific plaque reduction tests for the indirect serological detection of BVDV. Here, we present a genetically engineered cp strain of the classical swine fever virus (CSFV). Cytopathogenicity of the strain was induced by the insertion of ubiquitin embedded in a large NS3 to NS4B duplication. The CSFV RNA genome was stabilized by the inactivation of the NS2 autoprotease, hindering the deletion of the insertion and the reversion to a wild-type genome. Additional insertion of a mCherry gene at the 5'-end of the E2 gene allowed fluorescence-verified plaque reduction assays for CSFV, thus providing a novel, cost-efficient diagnostic tool. This genetically stabilized cp CSFV strain could be further used as a basis for potential new modified live vaccines. Taken together, we applied reverse genetics to rationally fixate a typical cp NS3 duplication in a CSFV genome.

Organization of the Structural Protein Region of La Jolla Virus Isolated from the Invasive Pest Insect Drosophila suzukii.

Drosophila suzukii (Ds) is an invasive pest insect that infests ripening fruit, causing severe economic losses. Control measures based on chemical pesticides are inefficient and undesirable, so biological alternatives have been considered, including native Ds viruses. We previously isolated a strain of La Jolla virus (LJV-Ds-OS20) from Ds in Germany as a candidate biopesticide. Here we characterized the new strain in detail, focusing on the processing of its capsid proteins. We tested LJV growth during Ds development to optimize virus production, and established a laboratory production system using adult flies. This system was suitable for the preparation of virions for detailed analysis. The LJV-Ds-OS20 isolate was cloned by limiting dilution and the complete nucleotide sequence was determined as a basis for protein analysis. The terminal segments of the virus genome were completed by RACE-PCR. LJV virions were also purified by CsCl gradient centrifugation and analyzed by SDS-PAGE and electron microscopy. The capsid proteins of purified LJV virions were resolved by two-dimensional SDS-PAGE for N-terminal sequencing and peptide mass fingerprinting. The N-terminal sequences of VP1 and VP2, together with MS data representing several capsid proteins, allowed us to develop a model for the organization of the LJV structural protein region. This may facilitate the development of new viral strains as biopesticides.

Fluorophore labelled BVDV: a novel tool for the analysis of infection dynamics.

Genetic labelling of viruses with a fluorophore allows to study their life cycle in real time, without the need for fixation or staining techniques. Within the family Flaviviridae, options for genetic labelling of non-structural proteins exist. Yet, no system to genetically label structural proteins has been put forward to date. Taking advantage of a previously described site within the structural protein E2, a fluorophore was introduced into a cytopathogenic (cpe) BVDV-1 virus (BVDVE2_fluo). This insertion was well tolerated, resulting in a 2-fold drop in titer compared to the parental virus, and remained stably integrated into the genome for more than 10 passages. The fluorophore E2 fusion protein was readily detectable in purified virus particles by Western blot and fluorescence microscopy and the particle integrity and morphology was confirmed by cryo electron microscopy. The same integration site could also be used to label the related Classical swine fever virus. Also, BVDVE2_fluo particles bound to fluorophore labelled CD46 expressing cells could be resolved in fluorescence microscopy. This underlines the applicability of BVDVE2_fluo as a tool to study the dynamics of the whole life cycle of BVDV in real time.

The core protein of a pestivirus protects the incoming virus against IFN-induced effectors.

A multitude of viral factors - either inhibiting the induction of the IFN-system or its effectors - have been described to date. However, little is known about the role of structural components of the incoming virus particle in protecting against IFN-induced antiviral factors during or immediately after entry. In this study, we take advantage of the previously reported property of Classical swine fever virus (family Flaviviridae, genus Pestivirus) to tolerate a deletion of the core protein if a compensatory mutation is present in the NS3-helicase-domain (Vp447∆c). In contrast to the parental virus (Vp447), which causes a hemorrhagic-fever-like disease in pigs, Vp447∆c is avirulent in vivo. In comparison to Vp447, growth of Vp447∆c in primary porcine cells and IFN-treated porcine cell lines was reduced >20-fold. Also, primary porcine endothelial cells and IFN-pretreated porcine cell lines were 8-24 times less susceptible to Vp447∆c. This reduction of susceptibility could be partially reversed by loading Vp447∆c particles with different levels of core protein. In contrast, expression of core protein in the recipient cell did not have any beneficial effect. Therefore, a protective effect of core protein in the incoming virus particle against the products of IFN-stimulated genes could be demonstrated.

Construction and Rescue of a Molecular Clone of Deformed Wing Virus (DWV).

European honey bees are highly important in crop pollination, increasing the value of global agricultural production by billions of dollars. Current knowledge about virulence and pathogenicity of Deformed wing virus (DWV), a major factor in honey bee colony mortality, is limited. With this study, we close the gap between field research and laboratory investigations by establishing a complete in vitro model for DWV pathogenesis. Infectious DWV was rescued from a molecular clone of a DWV-A genome that induces DWV symptoms such as crippled wings and discoloration. The expression of DWV proteins, production of infectious virus progeny, and DWV host cell tropism could be confirmed using newly generated anti-DWV monoclonal antibodies. The recombinant RNA fulfills Koch's postulates circumventing the need of virus isolation and propagation of pure virus cultures. In conclusion, we describe the development and application of a reverse genetics system for the study of DWV pathogenesis.

Influenza A Virus Infection in Pigs Attracts Multifunctional and Cross-Reactive T Cells to the Lung.

UNLABELLED: Pigs are natural hosts for influenza A viruses and play a critical role in influenza epidemiology. However, little is known about their influenza-evoked T-cell response. We performed a thorough analysis of both the local and systemic T-cell response in influenza virus-infected pigs, addressing kinetics and phenotype as well as multifunctionality (gamma interferon [IFN-γ], tumor necrosis factor alpha [TNF-α], and interleukin-2 [IL-2]) and cross-reactivity. A total of 31 pigs were intratracheally infected with an H1N2 swine influenza A virus (FLUAVsw) and consecutively euthanized. Lungs, tracheobronchial lymph nodes, and blood were sampled during the first 15 days postinfection (p.i.) and at 6 weeks p.i. Ex vivo flow cytometry of lung lymphocytes revealed an increase in proliferating (Ki-67(+)) CD8(+) T cells with an early effector phenotype (perforin(+) CD27(+)) at day 6 p.i. Low frequencies of influenza virus-specific IFN-γ-producing CD4(+) and CD8(+) T cells could be detected in the lung as early as 4 days p.i. On consecutive days, influenza virus-specific CD4(+) and CD8(+) T cells produced mainly IFN-γ and/or TNF-α, reaching peak frequencies around day 9 p.i., which were up to 30-fold higher in the lung than in tracheobronchial lymph nodes or blood. At 6 weeks p.i., CD4(+) and CD8(+) memory T cells had accumulated in lung tissue. These cells showed diverse cytokine profiles and in vitro reactivity against heterologous influenza virus strains, all of which supports their potential to combat heterologous influenza virus infections in pigs. IMPORTANCE: Pigs not only are a suitable large-animal model for human influenza virus infection and vaccine development but also play a central role in the emergence of new pandemic strains. Although promising candidate universal vaccines are tested in pigs and local T cells are the major correlate of heterologous control, detailed and targeted analyses of T-cell responses at the site of infection are scarce. With the present study, we provide the first detailed characterization of magnitude, kinetics, and phenotype of specific T cells recruited to the lungs of influenza virus-infected pigs, and we could demonstrate multifunctionality, cross-reactivity, and memory formation of these cells. This, and ensuing work in the pig, will strengthen the position of this species as a large-animal model for human influenza virus infection and will immediately benefit vaccine development for improved control of influenza virus infections in pigs.

Single amino acid substitution (G42E) in the receptor binding domain of mouse mammary tumour virus envelope protein facilitates infection of non-murine cells in a transferrin receptor 1-independent manner.

BACKGROUND: Mouse mammary tumour virus (MMTV) is a betaretrovirus that infects rodent cells and uses mouse tranferrin receptor 1 (TfR1) for cell entry. Several MMTV strains have been shown to productively infect, in addition to murine cells, various heterologous cell lines including those of human origin, albeit less efficiently than murine cells. Furthermore, there have been reports that the continued passage of MMTV in heterologous cell lines gives rise to novel variants that are able to infect naive non-murine cells with higher efficiency than the parental virus. RESULTS: We show that MMTV(C3H), like other MMTV strains, that had undergone a number of replication cycles in non-murine cells displayed an increased replication kinetic, as compared to parental virus, when applied on naive human cells. Sequence analysis of several replication kinetic variants and the parental virus, together with calculation of the ratio of non-synonymous to synonymous mutations at individual codons, revealed that several regions within the viral genome were under strong positive selection pressure during viral replication in human cells. The mutation responsible, at least in part, for the phenotypic change was subsequently mapped to the segment of env encoding the receptor binding site (F40HGFR44). Introduction of the identified mutation, leading to single amino acid substitution (G42E), into egfp-containing recombinant MMTV virions enhanced their ability to bind to and infect human cells. Interestingly, neither the replication kinetic mutant nor the parental virus required human TfR1 for infection. Knock-out of TFR1 gene from the human genome did not decrease the susceptibility of Hs578T cells to virus infection. Furthermore, the expression of human TfR1, in contrast to mouse TfR1, did not enhance the susceptibility of MMTV-resistant Chinese hamster ovary cells. Thus, human TfR1 is dispensable for infection and another cell surface molecule mediates the MMTV entry into human cells. CONCLUSION: Taken together, our data explain the mechanism enabling MMTV to form 'host-range variants' in non-murine cells that has been known for a long time, the basis of which remained obscure. Our findings may expand our understanding of how viruses gain capability to cross species-specific barriers to infect new hosts.

Autocatalytic cleavage within classical swine fever virus NS3 leads to a functional separation of protease and helicase.

Classical swine fever virus (CSFV) is a positive-stranded RNA virus belonging to the genus Pestivirus within the Flaviviridae family. Pivotal for processing of a large portion of the viral polyprotein is a serine protease activity within nonstructural protein 3 (NS3) that also harbors helicase and NTPase activities essential for RNA replication. In CSFV-infected cells, NS3 appears as two forms, a fully processed NS3 of 80 kDa and the precursor molecule NS2-3 of 120 kDa. Here we report the identification and mapping of additional autocatalytic intramolecular cleavages. One cleavable peptide bond occurs between Leu1781 and Met1782, giving rise to a helicase subunit of 55 kDa and, depending on the substrate, a NS2-3 fragment of 78 kDa (NS2-3p) or a NS3 protease subunit of 26 kDa (NS3p). In trans-cleavage assays using NS4-5 as a substrate, NS3p acts as a fully functional protease that is able to process the polyprotein. NS3p comprises the minimal essential protease, as deletion of Leu1781 results in inactivation. A second intramolecular cleavage was mapped to the Leu1748/Lys1749 peptide bond that yields a proteolytically inactive NS3 fragment. Deletion of either of the cleavage site residues resulted in a loss of RNA infectivity, indicating the functional importance of amino acid identity at the respective positions. Our data suggest that internal cleavage within the NS3 moiety is a common process that further extends the functional repertoires of the multifunctional NS2-3 or NS3 and represents another level of the complex polyprotein processing of Flaviviridae.

The core protein of classical Swine Fever virus is dispensable for virus propagation in vitro.

Core protein of Flaviviridae is regarded as essential factor for nucleocapsid formation. Yet, core protein is not encoded by all isolates (GBV- A and GBV- C). Pestiviruses are a genus within the family Flaviviridae that affect cloven-hoofed animals, causing economically important diseases like classical swine fever (CSF) and bovine viral diarrhea (BVD). Recent findings describe the ability of NS3 of classical swine fever virus (CSFV) to compensate for disabling size increase of core protein (Riedel et al., 2010). NS3 is a nonstructural protein possessing protease, helicase and NTPase activity and a key player in virus replication. A role of NS3 in particle morphogenesis has also been described for other members of the Flaviviridae (Patkar et al., 2008; Ma et al., 2008). These findings raise questions about the necessity and function of core protein and the role of NS3 in particle assembly. A reverse genetic system for CSFV was employed to generate poorly growing CSFVs by modification of the core gene. After passaging, rescued viruses had acquired single amino acid substitutions (SAAS) within NS3 helicase subdomain 3. Upon introduction of these SAAS in a nonviable CSFV with deletion of almost the entire core gene (Vp447(Δc)), virus could be rescued. Further characterization of this virus with regard to its physical properties, morphology and behavior in cell culture did not reveal major differences between wildtype (Vp447) and Vp447(Δc). Upon infection of the natural host, Vp447(Δc) was attenuated. Hence we conclude that core protein is not essential for particle assembly of a core-encoding member of the Flaviviridae, but important for its virulence. This raises questions about capsid structure and necessity, the role of NS3 in particle assembly and the function of core protein in general.

High-level secretion of recombinant monomeric murine and human single-chain Fv antibodies from Drosophila S2 cells.

Single-chain variable fragment (scFvs) antibodies are small polypeptides (∼26 kD) containing the heavy (V(H)) and light (V(L)) immunoglobulin domains of a parent antibody connected by a flexible linker. In addition to being frequently used in diagnostics and therapy for an increasing number of human diseases, scFvs are important tools for structural biology as crystallization chaperones. Although scFvs can be expressed in many different organisms, the expression level of an scFv strongly depends on its particular amino acid sequence. We report here a system allowing for easy and efficient cloning of (i) scFvs selected by phage display and (ii) individual heavy and light chain sequences from hybridoma cDNA into expression plasmids engineered for secretion of the recombinant fragment produced in Drosophila S2 cells. We validated the method by producing five scFvs derived from human and murine parent antibodies directed against various antigens. The production yields varied between 5 and 12 mg monomeric scFv per liter of supernatant, indicating a relative independence on the individual sequences. The recombinant scFvs bound their cognate antigen with high affinity, comparable with the parent antibodies. The suitability of the produced recombinant fragments for structural studies was demonstrated by crystallization and structure determination of one of the produced scFvs, derived from a broadly neutralizing antibody against the major glycoprotein E2 of the hepatitis C virus. Structural comparison with the Protein Data Bank revealed the typical spatial organization of V(H) and V(L) domains, further validating the here-reported expression system.

Biosynthesis of classical swine fever virus nonstructural proteins.

Proteolytic processing of polyproteins is considered a crucial step in the life cycle of most positive-strand RNA viruses. An enhancement of NS2-3 processing has been described as a major difference between the noncytopathogenic (non-CP) and the cytopathogenic (CP) biotypes of pestiviruses. The effects of accelerated versus delayed NS2-3 processing on the maturation of the other nonstructural proteins (NSP) have never been compared. In this study, we analyzed the proteolytic processing of NSP in Classical swine fever virus (CSFV). Key to the investigation was a panel of newly developed monoclonal antibodies (MAbs) that facilitated monitoring of all nonstructural proteins involved in virus replication (NS2, NS3, NS4A, NS5A, and NS5B). Applying these MAbs in Western blotting and radioimmunoprecipitation allowed an unambiguous identification of the mature proteins and precursors in non-CP CSFV-infected cells. Furthermore, the kinetics of processing were determined by pulse-chase analyses for non-CP CSFV, CP CSFV, and a CP CSFV replicon. A slow but constant processing of NS4A/B-5A/B occurred in non-CP CSFV-infected cells, leading to balanced low-level concentrations of mature NSP. In contrast, the turnover of the polyprotein precursors was three times faster in CP CSFV-infected cells and in cells transfected with a CP CSFV replicon, causing a substantial increase of mature NSP concentrations. We conclude that a delayed processing not only of NS3 but further of all NSP represents a hallmark of regulation in non-CP pestiviruses.

Characterization of essential domains and plasticity of the classical Swine Fever virus Core protein.

Pestiviruses are pathogens of cloven-hoofed animals, belonging to the Flaviviridae. The pestiviral particle consists of a lipid membrane containing the three envelope glycoproteins Erns, E1, and E2 and a nucleocapsid of unknown symmetry, which is composed of the Core protein and the viral positive-sense RNA genome. The positively charged pestiviral Core protein consists of 86 to 89 amino acids. To analyze the organization of essential domains, N- and C-terminal truncations, as well as internal deletions, were introduced into the Core coding sequence in the context of an infectious cDNA clone of classical swine fever virus strain Alfort. Amino acids 179 to 180, 194 to 198, and 208 to 212 proved to be of special importance for the generation of progeny virus. The results of transcomplementation of a series of C-terminally truncated Core molecules indicate the importance of Ala255 at the C terminus. The plasticity of Core protein was examined by the construction of concatemeric arrays of Core coding regions and the insertion of up to three yellow fluorescent protein (YFP) genes between two Core genes. Even a Core fusion protein with more than 10-fold-increased molecular mass was integrated into the viral particle and supported the production of infectious progeny virus. The unexpected plasticity of Core protein brings into question the formation of a regular icosahedric particle and supports the idea of a histone-like protein-RNA interaction. All viruses with a duplicated Core gene were unstable and reverted to the wild-type sequence. Interestingly, a nonviable YFP-Core construct was rescued by a mutation within the C-terminal domain of the nonstructural protein NS3.