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

Fahnøe U, Pedersen AG, Dräger C, Orton RJ, Blome S, Höper D, Beer M, Rasmussen TB - PLoS ONE (2015)

Bottom Line: To test this we used phylogenetic methods to infer two ancestral sequences, which were then reconstructed as cDNA clones.Both reconstructed ancestral genomes proved functional, and displayed distinct phenotypes in vitro and in vivo.Importantly, ancestral reconstruction can be done even on the basis of a set of sequences that all correspond to non-functional variants.

View Article: PubMed Central - PubMed

Affiliation: DTU National Veterinary Institute, Technical University of Denmark, Lindholm, Kalvehave, Denmark; Center for Biological Sequence Analysis, DTU Systems Biology, Technical University of Denmark, Kgs. Lyngby, Denmark.

ABSTRACT
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.

No MeSH data available.


Related in: MedlinePlus

Replication kinetics of both reconstructed ancestral nodes.Replication of viruses in PK-15 cells was measured using RT-qPCR relative to 2 hour measurement at 8 and 12 hours after infection. Means ± s.d. are shown for biological replicates (n = 3). The replication rates of the two constructs was significantly different at both 8h (p = 0.003, t-test) and 12h (p = 0.008).
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pone.0140912.g005: Replication kinetics of both reconstructed ancestral nodes.Replication of viruses in PK-15 cells was measured using RT-qPCR relative to 2 hour measurement at 8 and 12 hours after infection. Means ± s.d. are shown for biological replicates (n = 3). The replication rates of the two constructs was significantly different at both 8h (p = 0.003, t-test) and 12h (p = 0.008).

Mentions: Based on the computationally inferred ancestral sequences, we produced constructs corresponding to the black and red internal nodes in Fig 3A, with the purpose of testing their functionality in vitro and in vivo. These constructs were created using site-directed mutagenesis, starting from one of the infectious clones and removing mutations step by step to produce the desired cDNA clones. Specifically, the clone from which we started had 2 missense and one silent mutation compared to the inferred sequence of the black internal node. We first constructed a cDNA clone corresponding to the ancestral sequence at the black diamond shaped node (here termed “Ros”; Fig 3B). This inferred ancestral sequence is identical to the consensus sequence of the viral population. RNA transcripts derived from Ros proved infectious in PK-15 cells, with growth curves showing that the virus rescued from Ros (termed “vRos”) proliferates at least as well as the virus rescued from RNA transcripts obtained from the parental cDNA (vRos_cDNA) in cell culture (Fig 4). Starting from the Ros cDNA clone the ancestral sequence at the red node was subsequently constructed using two additional steps of site-directed mutagenesis. This sequence, named “Ros_S1359N_A2668T”, had the two missense SNVs (S1359N in NS2 and A2668T in NS4B), but not the silent change in NS5B (T11992C; Fig 3B). Each step added one missense mutation and transcripts containing each of the individual changes (Ros_S1359N and Ros_A2668T) were found to be infectious in PK-15 cells (data not shown). The final construct Ros_S1359N_A2668T also proved to be infectious in cell culture. As both ancestral reconstructions led to infectious viruses, we further tested their replication efficiency in cell culture. PK-15 cells were infected with the same infectious dose; RNA was extracted at 2, 8 and 12 hours, and the level of CSFV genomes then measured by RT-qPCR. This analysis showed that the ancestor at the red node, vRos_S1359N_A2668T, replicated significantly faster than the ancestor at the black node, vRos (Fig 5). This was seen at both 8 (t-test, p = 0.003) and 12 hours post infection (t-test, p = 0.008).


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

Fahnøe U, Pedersen AG, Dräger C, Orton RJ, Blome S, Höper D, Beer M, Rasmussen TB - PLoS ONE (2015)

Replication kinetics of both reconstructed ancestral nodes.Replication of viruses in PK-15 cells was measured using RT-qPCR relative to 2 hour measurement at 8 and 12 hours after infection. Means ± s.d. are shown for biological replicates (n = 3). The replication rates of the two constructs was significantly different at both 8h (p = 0.003, t-test) and 12h (p = 0.008).
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4613144&req=5

pone.0140912.g005: Replication kinetics of both reconstructed ancestral nodes.Replication of viruses in PK-15 cells was measured using RT-qPCR relative to 2 hour measurement at 8 and 12 hours after infection. Means ± s.d. are shown for biological replicates (n = 3). The replication rates of the two constructs was significantly different at both 8h (p = 0.003, t-test) and 12h (p = 0.008).
Mentions: Based on the computationally inferred ancestral sequences, we produced constructs corresponding to the black and red internal nodes in Fig 3A, with the purpose of testing their functionality in vitro and in vivo. These constructs were created using site-directed mutagenesis, starting from one of the infectious clones and removing mutations step by step to produce the desired cDNA clones. Specifically, the clone from which we started had 2 missense and one silent mutation compared to the inferred sequence of the black internal node. We first constructed a cDNA clone corresponding to the ancestral sequence at the black diamond shaped node (here termed “Ros”; Fig 3B). This inferred ancestral sequence is identical to the consensus sequence of the viral population. RNA transcripts derived from Ros proved infectious in PK-15 cells, with growth curves showing that the virus rescued from Ros (termed “vRos”) proliferates at least as well as the virus rescued from RNA transcripts obtained from the parental cDNA (vRos_cDNA) in cell culture (Fig 4). Starting from the Ros cDNA clone the ancestral sequence at the red node was subsequently constructed using two additional steps of site-directed mutagenesis. This sequence, named “Ros_S1359N_A2668T”, had the two missense SNVs (S1359N in NS2 and A2668T in NS4B), but not the silent change in NS5B (T11992C; Fig 3B). Each step added one missense mutation and transcripts containing each of the individual changes (Ros_S1359N and Ros_A2668T) were found to be infectious in PK-15 cells (data not shown). The final construct Ros_S1359N_A2668T also proved to be infectious in cell culture. As both ancestral reconstructions led to infectious viruses, we further tested their replication efficiency in cell culture. PK-15 cells were infected with the same infectious dose; RNA was extracted at 2, 8 and 12 hours, and the level of CSFV genomes then measured by RT-qPCR. This analysis showed that the ancestor at the red node, vRos_S1359N_A2668T, replicated significantly faster than the ancestor at the black node, vRos (Fig 5). This was seen at both 8 (t-test, p = 0.003) and 12 hours post infection (t-test, p = 0.008).

Bottom Line: To test this we used phylogenetic methods to infer two ancestral sequences, which were then reconstructed as cDNA clones.Both reconstructed ancestral genomes proved functional, and displayed distinct phenotypes in vitro and in vivo.Importantly, ancestral reconstruction can be done even on the basis of a set of sequences that all correspond to non-functional variants.

View Article: PubMed Central - PubMed

Affiliation: DTU National Veterinary Institute, Technical University of Denmark, Lindholm, Kalvehave, Denmark; Center for Biological Sequence Analysis, DTU Systems Biology, Technical University of Denmark, Kgs. Lyngby, Denmark.

ABSTRACT
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.

No MeSH data available.


Related in: MedlinePlus