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. 2012 Oct 25;432(2):460-9.
doi: 10.1016/j.virol.2012.07.004. Epub 2012 Jul 24.

A single amino acid change resulting in loss of fluorescence of eGFP in a viral fusion protein confers fitness and growth advantage to the recombinant vesicular stomatitis virus

Phat X Dinh  1 , Debasis Panda, Phani B Das, Subash C Das, Anshuman Das, Asit K Pattnaik
Affiliations

A single amino acid change resulting in loss of fluorescence of eGFP in a viral fusion protein confers fitness and growth advantage to the recombinant vesicular stomatitis virus

Phat X Dinh et al. Virology. .

Abstract

Using a recombinant vesicular stomatitis virus encoding eGFP fused in-frame with an essential viral replication protein, the phosphoprotein P, we show that during passage in culture, the virus mutates the nucleotide C289 within eGFP of the fusion protein PeGFP to A or T, resulting in R97S/C amino acid substitution and loss of fluorescence. The resultant non-fluorescent virus exhibits increased fitness and growth advantage over its fluorescent counterpart. The growth advantage of the non-fluorescent virus appears to be due to increased transcription and replication activities of the PeGFP protein carrying the R97S/C substitution. Further, our results show that the R97S/C mutation occurs prior to accumulation of mutations that can result in loss of expression of the gene inserted at the G-L gene junction. These results suggest that fitness gain is more important for the recombinant virus than elimination of expression of the heterologous gene.

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Figures

Fig. 1
Fig. 1
Identification of a mutational hotspot within eGFP coding sequence in PeGFP fusion protein of VSV-PeGFP virus. (A) Recombinant VSV genome constructs used in this study. VSVwt, the wt VSV genome, with N, P, M, G and L shown in rectangular boxes; VSV-PeGFP, genome encoding PeGFP in place of wt P; VSV-eGFP, eGFP was inserted as an independent cistron at the G-L junction; VSV-PeGFP-M-MmRFP, genome encoding PeGFP in place of wt P and MmRFP as an extra cistron at the G-L junction. (B) Emergence and dominance of VSV-PeGFPng from the culture supernatant of plasmid recovered population of recombinant VSV-PeGFP. The supernatant containing recovered VSV-PeGFP virus (passage 0, P0) was passaged at 0.01 MOI on BHK-21 cells and the percentage of VSV-PeGFPng virus was counted by plaque assay and counting of fluorescent and non-fluorescent viruses in various passages. Histograms show the average data from three independent experiments. (C) C289A is the only mutation found in full-length genome of VSV-PeGFPngR97S compared to VSV-PeGFP virus. RNA genome of VSV-PeGFP and VSV-PeGFPngR97S viruses was isolated directly from P0 supernatant (as in B). The viral RNAs were subjected to RT-PCR, followed by nucleotide sequencing for the whole genome using the primers shown in Table 2.
Fig. 2
Fig. 2
R97S/C mutation in eGFP confers growth advantage to VSV-PeGFPng virus. (A) One-step growth curve of wt VSV, VSV-PeGFP, VSV-PeGFPng containing C289A (VSV-PeGFPngR97S) or C289T (VSV-PeGFPngR97C) mutation. Infection was performed in BHK-21 cells at MOI of 5, supernatants were collected at indicated time points and viral yields were determined by plaque assay in fresh BHK-21 cells. Error bars represent the standard error of mean from 3 independent experiments. (B) Multistep growth curve. Viruses shown in A were allowed to infect BHK-21 cells separately at MOI of 0.01. Viral titers were determined and shown as described in A.
Fig. 3
Fig. 3
Increased fitness of VSV-PeGFPng over VSV-PeGFP in competition assay. (A) Competition assay at low MOI. BHK-21 cells were co-infected at an MOI of 0.01 with a 1:1 mixture of plaque-purified VSV-PeGFP and VSV-PeGFPng (P0). At 16 hpi, supernatant (P1) was collected and titers VSV-PeGFP and VSV-PeGFPng viruses were determined in fresh BHK-21. Supernatant of P1 and subsequent passages were used to infect new BHK-21 cells at the same MOI of 0.01 to produce P2 to P5. Histograms show the average relative levels of VSV-PeGFP and VSV-PeGFPng viruses from three independent experiments. The numbers indicate the average ratio of the two viruses as determined by plaques assay in BHK-21 cells for each passage. (B) Competition assay at high MOI. The assay was done at MOI of 5 and the data were obtained as in A. (C) Kinetics of loss of green fluorescence in clonally purified VSV-PeGFP. Isolated pure green VSV-PeGFP was used to infect and passage in BHK-21 cells at MOI of 0.01. Percentage of green over non-green virus was determined as described in Fig. 1B.
Fig. 4
Fig. 4
Mutation at C289 of eGFP occurs first in the context of VSV-PeGFP-M-MmRFP encoding two fluorescent fusion proteins. (A) Kinetics of the emergence of viruses with all possible fluorescence phenotypes from plasmid-recovered recombinant green-red (GR) VSV-PeGFP-M-MmRFP. Culture supernatant of plasmid-recovered VSV-PeGFP-M-MmRFPgr (considered as P0) was used to infect and serially passage at an MOI of 0.01 up to passage 12. The percentage of each phenotype virus was determined by plaque counting on BHK-21 under fluorescent microscope. Histograms show the average data from two independent experiments. (B) Analysis of viral RNA in cells infected with recombinant viruses. BHK-21 was infected with 5 MOI of wt VSV (lane 1), GR (VSV-PeGFP-M-MmRFP) (lane 2), nGR (VSV-PeGFP-M-MmRFPngr) (lane 3), GnR with Stop codon in MmRFP region (VSV-PeGFP-M-MmRFPStopgnr)(lane 4), GnR with bicistronic G-MmRFP (VSV-PeGFP-M-MmRFPBicisgnr) (lane 5) and nGnR carrying C289T together with bicistronic G-MmRFP (VSV-PeGFP-M-MmRFPBicisngnr) or with stop codon in MmRFP region (VSV-PeGFP-M-MmRFPStopngnr) (lane 6 and 7). RNAs were labeled with 3H-uridin at 5 hpi for 6 hr. M and P mRNAs migrate together, PeGFP and MmRFP also have the same mobility; G mRNA in VSV-PeGFP-M-MmRFP is shortened in length compared to wt VSV, due to the insertion of MmRFP at G-L junction. (C) Analysis of viral proteins in cell infected with recombinant viruses. Infection conditions and viruses used in lane 2 to 8 are the same as in panel A, lane 1 to 7. Lane 1 contains proteins from uninfected cells. Proteins were labeled with 35S-Met/Cys at 5 hpi for 4 h. Similar amounts of infected cells extracts were analyzed. Viral proteins and size markers (in kDa) are shown.
Fig. 5
Fig. 5
Replication and transcription activity of PeGFPngR97S mutant protein. (A) DI particle RNA replication. BHK-21 cells were infected with vTF7-3 and co-transfected with plasmids encoding VSV N, L, and PeGFP (WT), PeGFPngR97S (nG) or pGEM3empty vector (EV). At 10-12 hpt, cells were infected with DI particles, followed by labeling with 3H-uridine for 12 h. Labeled RNAs were immunoprecipitated by anti-N antibody (1:100), separated on urea-agarose gel and detected by fluorography (A, top panel). The products were detected as a doublet representing the +ve and −ve sense DI RNA replication products. Histogram (bottom panel) shows the quantitative levels of DI replication products. Error bars represent the standard error of mean from 3 independent experiments. (B) Transcription of VSV minigenome. 10μg of p10BN encoding a VSV minireplicon was co-transfected with pN, pL, and pPeGFP (WT), pPeGFPngR97S (nG) or pGEM3 empty vector (EV). At 8-10 hpt, cells were labeled with 3H-uridine for 12 hr in the presence of actinomycin D. Labeled transcription products were extracted and analyzed by electrophoresis and detected as in A (top panel). Histogram (bottom panel) shows the quantitative levels of transcription products. Error bars represent the standard error of mean from 3 independent experiments. Expression level of PeGFP (WT) or PeGFPngR97S (nG) in the transfected cells was examined by Western blotting. Levels of β-actin served as loading control.
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