Cristina C. P. Celma

Development of Reverse Genetics Systems for Bluetongue Virus: Recovery of Infectious Virus from Synthetic RNA Transcripts

ABSTRACT Bluetongue virus (BTV), an insect-vectored emerging pathogen of both wild ruminants and livestock, has had a severe economic impact in agriculture in many parts of the world. The investigation of BTV replication and pathogenesis has been hampered by the lack of a reverse genetics system. Recovery of infectious BTV is possible by the transfection of permissive cells with the complete set of 10 purified viral mRNAs derived in vitro from transcribing cores (M. Boyce and P. Roy, J. Virol. 81:2179-2186, 2007). Here, we report that in vitro synthesized T7 transcripts, derived from cDNA clones, can be introduced into the genome of BTV using a mixture of T7 transcripts and core-derived mRNAs. The replacement of genome segment 10 and the simultaneous replacement of segments 2 and 5 encoding the two immunologically important outer capsid proteins, VP2 and VP5, are described. Further, we demonstrate the recovery of infectious BTV entirely from T7 transcripts, proving that synthetic transcripts synthesized in the presence of cap analogue can functionally substitute for viral transcripts at all stages of the BTV replication cycle. The generation of BTV with a fully defined genome permits the recovery of mutations in a defined genetic background. The ability to generate specific mutants provides a new tool to investigate the BTV replication cycle as well as permitting the generation of designer vaccine strains, which are greatly needed in many countries.

A Viral Nonstructural Protein Regulates Bluetongue Virus Trafficking and Release

ABSTRACT Bluetongue virus (BTV), a nonenveloped insect-borne virus, is released from infected cells by multiple pathways. Unlike other nonenveloped viruses, in addition to cell lysis the newly synthesized virus particles also appear to use a unique “budding” process. The nonstructural protein NS3, the only membrane protein encoded by BTV in infected cells, has been implicated in this process, since it appears to interact not only with the outermost viral capsid protein VP2 but also with a component of the cellular ESCRT pathway. However, to date it had not been possible to obtain direct evidence for the involvement of NS3 in BTV morphogenesis due to the lack of a genetic system that would allow introducing the targeted mutation in NS3 gene. In this study, we have used the recently developed T7 transcript-based reverse genetics system for BTV to introduce mutations in the sequence of NS3 into the viral genome and have investigated the effect of these mutations in the context of a replicating virus. While certain NS3 mutations exhibited drastic effects on newly synthesized virus release, others had less pronounced effects. In particular, mutations of two residues in the Tsg101 binding motif, the putative L domain of NS3, altered normal virus egress patterns and left nascent particles tethered to the cellular membrane, apparently arrested in the process of budding. In cells infected with a mutant virus that was incapable of an NS3-VP2 interaction, no budding particles were visualized. These data suggest that NS3 may act like the membrane protein of enveloped viruses and is responsible for intracellular trafficking and budding of virus particles. NS3 is thus a bridge between the maturing virion particles and cellular proteins during virus egress.

Generation of Replication-Defective Virus-Based Vaccines That Confer Full Protection in Sheep against Virulent Bluetongue Virus Challenge

ABSTRACT The reverse genetics technology for bluetongue virus (BTV) has been used in combination with complementing cell lines to recover defective BTV-1 mutants. To generate a potential disabled infectious single cycle (DISC) vaccine strain, we used a reverse genetics system to rescue defective virus strains with large deletions in an essential BTV gene that encodes the VP6 protein (segment S9) of the internal core. Four VP6-deficient BTV-1 mutants were generated by using a complementing cell line that provided the VP6 protein in trans. Characterization of the growth properties of mutant viruses showed that each mutant has the necessary characteristics for a potential vaccine strain: (i) viral protein expression in noncomplementing mammalian cells, (ii) no infectious virus generated in noncomplementing cells, and (iii) efficient replication in the complementing VP6 cell line. Further, a defective BTV-8 strain was made by reassorting the two RNA segments that encode the two outer capsid proteins (VP2 and VP5) of a highly pathogenic BTV-8 with the remaining eight RNA segments of one of the BTV-1 DISC viruses. The protective capabilities of BTV-1 and BTV-8 DISC viruses were assessed in sheep by challenge with specific virulent strains using several assay systems. The data obtained from these studies demonstrated that the DISC viruses are highly protective and could offer a promising alternative to the currently available attenuated and killed virus vaccines and are also compliant as DIVA (differentiating infected from vaccinated animals) vaccines.

Bluetongue virus non-structural protein 1 is a positive regulator of viral protein synthesis

BackgroundBluetongue virus (BTV) is a double-stranded RNA (dsRNA) virus of the Reoviridae family, which encodes its genes in ten linear dsRNA segments. BTV mRNAs are synthesised by the viral RNA-dependent RNA polymerase (RdRp) as exact plus sense copies of the genome segments. Infection of mammalian cells with BTV rapidly replaces cellular protein synthesis with viral protein synthesis, but the regulation of viral gene expression in the Orbivirus genus has not been investigated.ResultsUsing an mRNA reporter system based on genome segment 10 of BTV fused with GFP we identify the protein characteristic of this genus, non-structural protein 1 (NS1) as sufficient to upregulate translation. The wider applicability of this phenomenon among the viral genes is demonstrated using the untranslated regions (UTRs) of BTV genome segments flanking the quantifiable Renilla luciferase ORF in chimeric mRNAs. The UTRs of viral mRNAs are shown to be determinants of the amount of protein synthesised, with the pre-expression of NS1 increasing the quantity in each case. The increased expression induced by pre-expression of NS1 is confirmed in virus infected cells by generating a replicating virus which expresses the reporter fused with genome segment 10, using reverse genetics. Moreover, NS1-mediated upregulation of expression is restricted to mRNAs which lack the cellular 3′ poly(A) sequence identifying the 3′ end as a necessary determinant in specifically increasing the translation of viral mRNA in the presence of cellular mRNA.ConclusionsNS1 is identified as a positive regulator of viral protein synthesis. We propose a model of translational regulation where NS1 upregulates the synthesis of viral proteins, including itself, and creates a positive feedback loop of NS1 expression, which rapidly increases the expression of all the viral proteins. The efficient translation of viral reporter mRNAs among cellular mRNAs can account for the observed replacement of cellular protein synthesis with viral protein synthesis during infection.

Interaction of Calpactin Light Chain (S100A10/p11) and a Viral NS Protein Is Essential for Intracellular Trafficking of Nonenveloped Bluetongue Virus

ABSTRACT Bluetongue virus (BTV), a member of the Reoviridae family, is an insect-borne animal pathogen. Virus release from infected cells is predominantly by cell lysis, but some BTV particles are also released from the plasma membrane. The nonstructural protein NS3 has been implicated in this process. Using alternate initiator methionine residues, NS3 is expressed as a full-length protein and as a truncated variant that lacks the initial 13 residues, which, by yeast-two hybrid analyses, have been shown to interact with a cellular trafficking protein S100A10/p11. To understand the physiological significance of this interaction in virus-infected cells, we have used reverse genetics to investigate the roles of NS3 and NS3A in virus replication and localization in both mammalian and insect vector-derived cells. A virus expressing NS3 but not NS3A was able to propagate in and release from mammalian cells efficiently. However, growth of a mutant virus expressing only NS3A was severely attenuated, although protein expression, replication, double-stranded RNA (dsRNA) synthesis, and particle assembly in the cytoplasm were observed. Two of three single-amino-acid substitutions in the N-terminal 13 residues of NS3 showed phenotypically similar effects. Pulldown assay and confocal microscopy demonstrated a lack of interaction between NS3 and S100A10/p11 in mutants with poor replication. The role of NS3/NS3A was also assessed in insect cells where virus grew, albeit with a reduced titer. Notably, however, while wild-type particles were found within cytoplasmic vesicles in insect cells, mutant viruses were scattered throughout the cytoplasm and not confined to vesicles. These results provide support for a role for the extreme amino terminus of NS3 in the late stages of virus growth in mammalian cells, plausibly in egress. However, both NS3 and NS3A were required for efficient BTV growth in insect cells.

A reverse genetics system of African horse sickness virus reveals existence of primary replication

African horse sickness virus (AHSV), a member of the orbivirus genus of the family Reoviridae, is an insect‐vectored pathogen of horses of concern to the equine industry. Studies on AHSV replication and pathogenesis have been hampered by the lack of reverse genetics allowing targeted mutation of viral genomes. We demonstrate that AHSV single‐stranded RNA synthesized in vitro (core transcripts) is infectious and that there are distinct primary and secondary stages of the replication cycle. Transfection with a mixture of core transcripts from two different serotypes or a mixture of core transcripts and a T7 derived transcript resulted in the recovery of reassortant viruses. Recovery of infectious ASHV from nucleic acid will benefit investigation of the virus and the generation of attenuated vaccines.

Rapid Generation of Replication-Deficient Monovalent and Multivalent Vaccines for Bluetongue Virus: Protection against Virulent Virus Challenge in Cattle and Sheep

ABSTRACT Since 1998, 9 of the 26 serotypes of bluetongue virus (BTV) have spread throughout Europe, and serotype 8 has suddenly emerged in northern Europe, causing considerable economic losses, direct (mortality and morbidity) but also indirect, due to restriction in animal movements. Therefore, many new types of vaccines, particularly subunit vaccines, with improved safety and efficacy for a broad range of BTV serotypes are currently being developed by different laboratories. Here we exploited a reverse genetics-based replication-deficient BTV serotype 1 (BTV-1) (disabled infectious single cycle [DISC]) strain to generate a series of DISC vaccine strains. Cattle and sheep were vaccinated with these viruses either singly or in cocktail form as a multivalent vaccine candidate. All vaccinated animals were seroconverted and developed neutralizing antibody responses to their respective serotypes. After challenge with the virulent strains at 21 days postvaccination, vaccinated animals showed neither any clinical reaction nor viremia. Further, there was no interference with protection with a multivalent preparation of six distinct DISC viruses. These data indicate that a very-rapid-response vaccine could be developed based on which serotypes are circulating in the population at the time of an outbreak.

Positive and Negative Modulation of Virus Infectivity and Envelope Glycoprotein Incorporation into Virions by Amino Acid Substitutions at the N Terminus of the Simian Immunodeficiency Virus Matrix Protein

ABSTRACT The matrix (MA) protein of the simian immunodeficiency viruses (SIVs) is encoded by the amino-terminal region of the Gag precursor and is the component of the viral capsid that lines the inner surface of the virus envelope. Previously, we identified domains in the SIV MA that are involved in the transport of Gag to the plasma membrane and in particle assembly. In this study, we characterized the role in the SIV life cycle of highly conserved residues within the SIV MA region spanning the two N-terminal α-helices H1 and H2. Our analyses identified two classes of MA mutants: (i) viruses encoding amino acid substitutions within α-helices H1 or H2 that were defective in envelope (Env) glycoprotein incorporation and exhibited impaired infectivity and (ii) viruses harboring mutations in the β-turn connecting helices H1 and H2 that were more infectious than the wild-type virus and displayed an enhanced ability to incorporate the Env glycoprotein. Remarkably, among the latter group of MA mutants, the R22L/G24L double amino acid substitution increased virus infectivity eightfold relative to the wild-type virus in single-cycle infectivity assays, an effect that correlated with a similar increase in Env incorporation. Furthermore, the R22L/G24L MA mutation partially or fully complemented single-point MA mutations that severely impair or block Env incorporation and virus infectivity. Our finding that the incorporation of the Env glycoprotein into virions can be upregulated by specific mutations within the SIV MA amino terminus strongly supports the notion that the SIV MA domain mediates Gag-Env association during particle formation.

Multigene expression of protein complexes by iterative modification of genomic Bacmid DNA

BackgroundMany cellular multi-protein complexes are naturally present in cells at low abundance. Baculovirus expression offers one approach to produce milligram quantities of correctly folded and processed eukaryotic protein complexes. However, current strategies suffer from the need to produce large transfer vectors, and the use of repeated promoter sequences in baculovirus, which itself produces proteins that promote homologous recombination. One possible solution to these problems is to construct baculovirus genomes that express each protein in a complex from a separate locus within the viral DNA. However current methods for selecting such recombinant genomes are too inefficient to routinely modify the virus in this way.ResultsThis paper reports a method which combines the lambda red and bacteriophage P1 Cre-recombinase systems to efficiently generate baculoviruses in which protein complexes are expressed from multiple, single-locus insertions of foreign genes. This method is based on an 88 fold improvement in the selection of recombinant viruses generated by red recombination techniques through use of a bipartite selection cassette. Using this system, seven new genetic loci were identified in the AcMNPV genome suitable for the high level expression of recombinant proteins. These loci were used to allow the recovery two recombinant virus-like particles with potential biotechnological applications (influenza A virus HA/M1 particles and bluetongue virus VP2/VP3/VP5/VP7 particles) and the mammalian chaperone and cancer drug target CCT (16 subunits formed from 8 proteins).Conclusion1. Use of bipartite selections can significantly improve selection of modified bacterial artificial chromosomes carrying baculovirus DNA. Furthermore this approach is sufficiently robust to allow routine modification of the virus genome. 2. In addition to the commonly used p10 and polyhedrin loci, the ctx, egt, 39k, orf51, gp37, iap2 and odv-e56 loci in AcMNPV are all suitable for the high level expression of heterologous genes. 3. Two protein, four protein and eight protein complexes including virus-like particles and cellular chaperone complexes can be produced using the new approach.

Mutational analysis of the feline immunodeficiency virus matrix protein

To study the process of feline immunodeficiency virus (FIV) assembly, we examined the suitability of the vaccinia vector system to reproduce FIV particle formation. To this end, we constructed a recombinant vaccinia virus carrying the FIV gag gene. Biochemical and electron microscopy analyses of cells infected with this recombinant virus showed that the FIV Gag polyprotein self-assembled into lentivirus-like particles that were released into the culture medium. As a first step in the identification of molecular determinants in FIV Gag that are involved in virus assembly, we performed a site-directed mutagenesis analysis of the N-terminal matrix (MA) domain of the FIV Gag precursor. To this end, a series of amino acid substitutions and small in-frame deletions were introduced into the FIV MA and the mutated FIV gag gene constructs were expressed by means of the vaccinia system. Characterization of the assembly phenotype of these FIV Gag mutants led to the identification of amino acidic regions within the MA domain that are necessary for efficient transport of the Gag precursor to the plasma membrane and particle assembly. Our results reveal the role that the FIV MA plays in virus morphogenesis and contribute to the understanding of the assembly process in non-primate lentiviruses.