Ulrik Fahnøe

Mouse models of acute and chronic hepacivirus infection

New York City rats provide a gift to virologists Despite the development of curative drugs for hepatitis C virus (HCV) infection, global eradication of HCV will likely require a prophylactic vaccine. Progress toward a vaccine has been impeded by the absence of mouse models suitable for studying the immune response to HCV. Billerbeck et al. found that a HCV-related virus isolated from New York City rats produces an infection in laboratory mice that shares several immunological features with human infections (see the Perspective by Klenerman and Barnes). Their initial analyses of the infected mice revealed that acute clearance of the virus was dependent on T cells but not on natural killer cells. Science, this issue p. 204; see also p. 129 A mouse model may provide mechanistic insights into the immune response to hepatitis C virus infection. An estimated 71 million people worldwide are infected with hepatitis C virus (HCV). The lack of small-animal models has impeded studies of antiviral immune mechanisms. Here we show that an HCV-related hepacivirus discovered in Norway rats can establish high-titer hepatotropic infections in laboratory mice with immunological features resembling those seen in human viral hepatitis. Whereas immune-compromised mice developed persistent infection, immune-competent mice cleared the virus within 3 to 5 weeks. Acute clearance was T cell dependent and associated with liver injury. Transient depletion of CD4+ T cells before infection resulted in chronic infection, characterized by high levels of intrahepatic regulatory T cells and expression of inhibitory molecules on intrahepatic CD8+ T cells. Natural killer cells controlled early infection but were not essential for viral clearance. This model may provide mechanistic insights into hepatic antiviral immunity, a prerequisite for the development of HCV vaccines.

A fast and robust method for full genome sequencing of Porcine Reproductive and Respiratory Syndrome Virus (PRRSV) Type 1 and Type 2

PRRSV is a positive-sense RNA virus with a high degree of genetic variability among isolates. For diagnostic sensitivity and vaccine design it is essential to monitor PRRSV genetic diversity. However, to date only a few full genome sequences of PRRSV isolates have been made publicly available. In the present study, fast and robust methods for long range RT-PCR amplification and subsequent next generation sequencing (NGS) were developed and validated on nine Type 1 and nine Type 2 PRRSV viruses. The methods generated robust and reliable sequences both on primary material and cell culture adapted viruses and the protocols performed well on all three NGS platforms tested (Roche 454 FLX, Illumina HiSeq2000, and Ion Torrent PGM™ Sequencer). These methods will greatly facilitate the generation of more full genome PRRSV sequences globally.

Analysis of classical swine fever virus RNA replication determinants using replicons

Self-replicating RNAs (replicons), with or without reporter gene sequences, derived from the genome of the Paderborn strain of classical swine fever virus (CSFV) have been produced. The full-length viral cDNA, propagated within a bacterial artificial chromosome, was modified by targeted recombination within Escherichia coli. RNA transcripts were produced in vitro and introduced into cells by electroporation. The translation and replication of the replicon RNAs could be followed by the accumulation of luciferase (from Renilla reniformis or Gaussia princeps) protein expression (where appropriate), as well as by detection of CSFV NS3 protein production within the cells. Inclusion of the viral E2 coding region within the replicon was advantageous for replication efficiency. Production of chimeric RNAs, substituting the NS2 and NS3 coding regions (as a unit) from the Paderborn strain with the equivalent sequences from the highly virulent Koslov strain or the vaccine strain Riems, blocked replication. However, replacing the Paderborn NS5B coding sequence with the RNA polymerase coding sequence from the Koslov strain greatly enhanced expression of the reporter protein from the replicon. In contrast, replacement with the Riems NS5B sequence significantly impaired replication efficiency. Thus, these replicons provide a system for determining specific regions of the CSFV genome required for genome replication without the constraints of maintaining infectivity.

Determinants of the VP1/2A junction cleavage by the 3C protease in foot-and-mouth disease virus-infected cells

The foot-and-mouth disease virus (FMDV) capsid precursor, P1-2A, is cleaved by FMDV 3C protease to yield VP0, VP3, VP1 and 2A. Cleavage of the VP1/2A junction is the slowest. Serotype O FMDVs with uncleaved VP1-2A (having a K210E substitution in VP1; at position P2 in cleavage site) have been described previously and acquired a second site substitution (VP1 E83K) during virus rescue. Furthermore, introduction of the VP1 E83K substitution alone generated a second site change at the VP1/2A junction (2A L2P, position P2′ in cleavage site). These virus adaptations have now been analysed using next-generation sequencing to determine sub-consensus level changes in the virus; this revealed other variants within the E83K mutant virus population that changed residue VP1 K210. The construction of serotype A viruses with a blocked VP1/2A cleavage site (containing K210E) has now been achieved. A collection of alternative amino acid substitutions was made at this site, and the properties of the mutant viruses were determined. Only the presence of a positively charged residue at position P2 in the cleavage site permitted efficient cleavage of the VP1/2A junction, consistent with analyses of diverse FMDV genome sequences. Interestingly, in contrast to the serotype O virus results, no second site mutations occurred within the VP1 coding region of serotype A viruses with the blocked VP1/2A cleavage site. However, some of these viruses acquired changes in the 2C protein that is involved in enterovirus morphogenesis. These results have implications for the testing of potential antiviral agents targeting the FMDV 3C protease.

Efficient generation of recombinant RNA viruses using targeted recombination-mediated mutagenesis of bacterial artificial chromosomes containing full-length cDNA

BackgroundInfectious cDNA clones are a prerequisite for directed genetic manipulation of RNA viruses. Here, a strategy to facilitate manipulation and rescue of classical swine fever viruses (CSFVs) from full-length cDNAs present within bacterial artificial chromosomes (BACs) is described. This strategy allows manipulation of viral cDNA by targeted recombination-mediated mutagenesis within bacteria.ResultsA new CSFV-BAC (pBeloR26) derived from the Riems vaccine strain has been constructed and subsequently modified in the E2 coding sequence, using the targeted recombination strategy to enable rescue of chimeric pestiviruses (vR26_E2gif and vR26_TAV) with potential as new marker vaccine candidates. Sequencing of the BACs revealed a high genetic stability during passages within bacteria. The complete genome sequences of rescued viruses, after extensive passages in mammalian cells showed that modifications in the E2 protein coding sequence were stably maintained. A single amino acid substitution (D3431G) in the RNA dependent RNA polymerase was observed in the rescued viruses vR26_E2gif and vR26, which was reversion to the parental Riems sequence.ConclusionsThese results show that targeted recombination-mediated mutagenesis provides a powerful tool for expediting the construction of novel RNA genomes and should be applicable to the manipulation of other RNA viruses.

Creation of Functional Viruses from Non-Functional cDNA Clones Obtained from an RNA Virus Population by the Use of Ancestral Reconstruction.

RNA viruses have the highest known mutation rates. Consequently it is likely that a high proportion of individual RNA virus genomes, isolated from an infected host, will contain lethal mutations and be non-functional. This is problematic if the aim is to clone and investigate high-fitness, functional cDNAs and may also pose problems for sequence-based analysis of viral evolution. To address these challenges we have performed a study of the evolution of classical swine fever virus (CSFV) using deep sequencing and analysis of 84 full-length cDNA clones, each representing individual genomes from a moderately virulent isolate. In addition to here being used as a model for RNA viruses generally, CSFV has high socioeconomic importance and remains a threat to animal welfare and pig production. We find that the majority of the investigated genomes are non-functional and only 12% produced infectious RNA transcripts. Full length sequencing of cDNA clones and deep sequencing of the parental population identified substitutions important for the observed phenotypes. The investigated cDNA clones were furthermore used as the basis for inferring the sequence of functional viruses. Since each unique clone must necessarily be the descendant of a functional ancestor, we hypothesized that it should be possible to produce functional clones by reconstructing ancestral sequences. To test this we used phylogenetic methods to infer two ancestral sequences, which were then reconstructed as cDNA clones. Viruses rescued from the reconstructed cDNAs were tested in cell culture and pigs. Both reconstructed ancestral genomes proved functional, and displayed distinct phenotypes in vitro and in vivo. We suggest that reconstruction of ancestral viruses is a useful tool for experimental and computational investigations of virulence and viral evolution. Importantly, ancestral reconstruction can be done even on the basis of a set of sequences that all correspond to non-functional variants.

Spd2 assists Spd1 in the modulation of ribonucleotide reductase architecture but does not regulate deoxynucleotide pools

ABSTRACT In yeasts, small intrinsically disordered proteins (IDPs) modulate ribonucleotide reductase (RNR) activity to ensure an optimal supply of dNTPs for DNA synthesis. The Schizosaccharomyces pombe Spd1 protein can directly inhibit the large RNR subunit (R1), import the small subunit (R2) into the nucleus and induce an architectural change in the R1–R2 holocomplex. Here, we report the characterization of Spd2, a protein with sequence similarity to Spd1. We show that Spd2 is a CRL4Cdt2-controlled IDP that functions together with Spd1 in the DNA damage response and in modulation of RNR architecture. However, Spd2 does not regulate dNTP pools and R2 nuclear import. Furthermore, deletion of spd2 only weakly suppresses the Rad3ATR checkpoint dependency of CRL4Cdt2 mutants. However, when we raised intracellular dNTP pools by inactivation of RNR feedback inhibition, deletion of spd2 could suppress the checkpoint dependency of CRL4Cdt2 mutant cells to the same extent as deletion of spd1. Collectively, these observations suggest that Spd1 on its own regulates dNTP pools, whereas in combination with Spd2 it modulates RNR architecture and sensitizes cells to DNA damage.

Sequence adaptations during growth of rescued classical swine fever viruses in cell culture and within infected pigs.

Classical swine fever virus (CSFV) causes an economically important disease of swine. Four different viruses were rescued from full-length cloned cDNAs derived from the Paderborn strain of CSFV. Three of these viruses had been modified by mutagenesis (with 7 or 8 nt changes) within stem 2 of the subdomain IIIf of the internal ribosome entry site (IRES) that directs the initiation of protein synthesis. Rescued viruses were inoculated into pigs. The rescued vPader10 virus, without modifications in the IRES, induced clinical disease in pigs that was very similar to that observed previously with the parental field strain and transmission to in-contact pigs occurred. Two sequence reversions, in the NS2 and NS5B coding regions, became dominant within the virus populations in these infected pigs. Rescued viruses, with mutant IRES elements, did not induce disease and only very limited circulation of viral RNA could be detected. However, the animals inoculated with these mutant viruses seroconverted against CSFV. Thus, these mutant viruses were highly attenuated in vivo. All 4 rescued viruses were also passaged up to 20 times in cell culture. Using full genome sequencing, the same two adaptations within each of four independent virus populations were observed that restored the coding sequence to that of the parental field strain. These adaptations occurred with different kinetics. The combination of reverse genetics and in depth, full genome sequencing provides a powerful approach to analyse virus adaptation and to identify key determinants of viral replication efficiency in cells and within host animals.

Ribavirin-induced mutagenesis across the complete open reading frame of hepatitis C virus genotypes 1a and 3a

Ribavirin (RBV) has been used for the last 20 years to treat patients with chronic hepatitis C virus (HCV) infection. This pluripotent drug is believed to induce mutagenesis in HCV RNA. However, for cell-cultured HCV (HCVcc) this phenomenon has only been investigated in genotype 2a recombinants. Here we studied the mutations that developed in HCVcc of genotypes 1a and 3a treated with RBV or ribavirin triphosphate (RBV-TP) compared to non-treated controls. Analysis was performed on the amplified full-length open reading frame (ORF) of recovered viruses following next-generation sequencing and clonal analyses. Compared to non-treated controls, the spread of TNcc(1a) and DBN3acc(3a) HCVcc was delayed by RBV and RBV-TP at concentrations of 40 µM or higher. The delay in HCVcc spread was associated with increased new single-nucleotide polymorphisms (SNP). Significantly higher numbers of new SNP were observed in TNcc(1a) viruses treated with RBV or RBV-TP compared to matched non-treated controls. RBV or RBV-TP treatment led to significantly increased proportions of new G-to-A and C-to-U SNP compared to non-treated TNcc(1a). Clonal analyses confirmed a significantly increased mutation rate in RBV-treated TNcc(1a). Synonymous pairwise distances increased in both viruses across the complete ORF under RBV and RBV-TP treatment compared to controls. Consensus-shifts in single samples of RBV- or RBV-TP-treated TNcc(1a) viruses occurred in proteins E1, p7, NS3 and NS4B. No non-synonymous consensus changes were observed in DBN3acc(3a). This study supports a biased G-to-A and C-to-U mutagenic effect of RBV and RBV-TP throughout the entire ORF of HCV genotypes 1a and 3a.

Deoxynucleoside salvage in fission yeast allows rescue of ribonucleotide reductase deficiency but not Spd1-Mediated inhibition of replication

In fission yeast, the small, intrinsically disordered protein S-phase delaying protein 1 (Spd1) blocks DNA replication and causes checkpoint activation at least in part, by inhibiting the enzyme ribonucleotide reductase, which is responsible for the synthesis of DNA building blocks. The CRL4Cdt2 E3 ubiquitin ligase mediates degradation of Spd1 and the related protein Spd2 at S phase of the cell cycle. We have generated a conditional allele of CRL4Cdt2, by expressing the highly unstable substrate-recruiting protein Cdt2 from a repressible promoter. Unlike Spd1, Spd2 does not regulate deoxynucleotide triphosphate (dNTP) pools; yet we find that Spd1 and Spd2 together inhibit DNA replication upon Cdt2 depletion. To directly test whether this block of replication was solely due to insufficient dNTP levels, we established a deoxy-nucleotide salvage pathway in fission yeast by expressing the human equilibrative nucleoside transporter 1 (hENT1) and the Drosophila deoxynucleoside kinase. We present evidence that this salvage pathway is functional, as 2 µM of deoxynucleosides in the culture medium is able to rescue the growth of two different temperature-sensitive alleles controlling ribonucleotide reductase. However, salvage completely failed to rescue S phase delay, checkpoint activation, and damage sensitivity, which was caused by CRL4Cdt2 inactivation, suggesting that Spd1—in addition to repressing dNTP synthesis—together with Spd2, can inhibit other replication functions. We propose that this inhibition works at the point of the replication clamp proliferating cell nuclear antigen, a co-factor for DNA replication.