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Mutations in the 5' NTR and the Non-Structural Protein 3A of the Coxsackievirus B3 Selectively Attenuate Myocarditogenicity.

Massilamany C, Gangaplara A, Basavalingappa RH, Rajasekaran RA, Vu H, Riethoven JJ, Steffen D, Pattnaik AK, Reddy J - PLoS ONE (2015)

Bottom Line: The 5' non-translated region (NTR) is an important molecular determinant that controls replication and virulence of coxsackievirus B (CVB)3.We noted that the disease-inducing ability of the infectious clone-derived virus in three mouse strains was restricted to pancreatitis alone, and the incidence and severity of myocarditis were significantly reduced.We then reversed the mutations by creating three new clones, representing 1) U97C; 2) G4327A and U5088C; and 3) their combination together in the third clone.

View Article: PubMed Central - PubMed

Affiliation: School of Veterinary Medicine and Biomedical Sciences, University of Nebraska-Lincoln, Lincoln, Nebraska, United States of America.

ABSTRACT
The 5' non-translated region (NTR) is an important molecular determinant that controls replication and virulence of coxsackievirus B (CVB)3. Previous studies have reported many nucleotide (nt) sequence differences in the Nancy strain of the virus, including changes in the 5' NTR with varying degrees of disease severity. In our studies of CVB3-induced myocarditis, we sought to generate an infectious clone of the virus for routine in vivo experimentation. By determining the viral nt sequence, we identified three new nt substitutions in the clone that differed from the parental virus strain: C97U in the 5' NTR; a silent mutation, A4327G, in non-structural protein 2C; and C5088U (resulting in P1449L amino acid change) in non-structural protein 3A of the virus leading us to evaluate the role of these changes in the virulence properties of the virus. We noted that the disease-inducing ability of the infectious clone-derived virus in three mouse strains was restricted to pancreatitis alone, and the incidence and severity of myocarditis were significantly reduced. We then reversed the mutations by creating three new clones, representing 1) U97C; 2) G4327A and U5088C; and 3) their combination together in the third clone. The viral titers obtained from all the clones were comparable, but the virions derived from the third clone induced myocarditis comparable to that induced by wild type virus; however, the pancreatitis-inducing ability remained unaltered, suggesting that the mutations described above selectively influence myocarditogenicity. Because the accumulation of mutations during passages is a continuous process in RNA viruses, it is possible that CVB3 viruses containing such altered nts may evolve naturally, thus favoring their survival in the environment.

No MeSH data available.


Related in: MedlinePlus

Derivation of infectious CVB3 cDNA clone.Using cDNA synthesized from the Wt CVB3 (Nancy) virus RNA, overlapping fragments 1 and 2 were amplified by PCR to generate a full-length cDNA clone. While RsrII and T7 RNA polymerase promoter sequences were inserted upstream of the 5’ NTR of fragment 1, the poly(A) tail (59 adenosine residues) and PacI sequences were inserted downstream to the 3’ NTR of fragment 2. The fragments were cloned into the pBR322 vector and sequenced. Vector map was derived using SimVector software.
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pone.0131052.g001: Derivation of infectious CVB3 cDNA clone.Using cDNA synthesized from the Wt CVB3 (Nancy) virus RNA, overlapping fragments 1 and 2 were amplified by PCR to generate a full-length cDNA clone. While RsrII and T7 RNA polymerase promoter sequences were inserted upstream of the 5’ NTR of fragment 1, the poly(A) tail (59 adenosine residues) and PacI sequences were inserted downstream to the 3’ NTR of fragment 2. The fragments were cloned into the pBR322 vector and sequenced. Vector map was derived using SimVector software.

Mentions: To generate the infectious clone, we first generated a stock virus (passage 1) from a well-isolated plaque of CVB3 (Nancy strain, ATCC) and then prepared RNA from the stock virus using Trizol LS reagent (Invitrogen, Carlsbad, CA). We synthesized full-length cDNA using Moloney murine leukemia virus-reverse transcriptase in a reaction mixture containing oligo-dT (Invitrogen) and performed PCR with this cDNA to amplify two fragments using high-fidelity Pfu polymerase (Agilent Technologies, Santa Clara, CA). The primer sets used in the preparation of these amplicons are described in Table 1; the amplicons were cloned into plasmid pBR322 in a stepwise manner (Fig 1). To facilitate in vitro RNA transcription, bacteriophage T7 RNA polymerase promoter was inserted in the 5’ end of P1 primer next to the RsrII site (Fig 1 and Table 1). The first fragment was digested with RsrII and PacI and cloned into a modified pBR322 vector as described previously [30]. We digested this vector with SpeI and PacI and gel-purified the plasmid DNA, into which we then cloned the second PCR fragment digested with SpeI and PacI. This vector containing the full-length cDNA clone, hereafter designated, pBRCVB3 was sequenced leading us to identify three new nt changes that were not reported previously (Table 2). For in vitro transcription, the pBRCVB3 vector was linearized with PacI, and the transcription reaction was performed using T7 RNA polymerase at 37°C as recommended by the manufacturer (Promega, Madison, WI).


Mutations in the 5' NTR and the Non-Structural Protein 3A of the Coxsackievirus B3 Selectively Attenuate Myocarditogenicity.

Massilamany C, Gangaplara A, Basavalingappa RH, Rajasekaran RA, Vu H, Riethoven JJ, Steffen D, Pattnaik AK, Reddy J - PLoS ONE (2015)

Derivation of infectious CVB3 cDNA clone.Using cDNA synthesized from the Wt CVB3 (Nancy) virus RNA, overlapping fragments 1 and 2 were amplified by PCR to generate a full-length cDNA clone. While RsrII and T7 RNA polymerase promoter sequences were inserted upstream of the 5’ NTR of fragment 1, the poly(A) tail (59 adenosine residues) and PacI sequences were inserted downstream to the 3’ NTR of fragment 2. The fragments were cloned into the pBR322 vector and sequenced. Vector map was derived using SimVector software.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4476614&req=5

pone.0131052.g001: Derivation of infectious CVB3 cDNA clone.Using cDNA synthesized from the Wt CVB3 (Nancy) virus RNA, overlapping fragments 1 and 2 were amplified by PCR to generate a full-length cDNA clone. While RsrII and T7 RNA polymerase promoter sequences were inserted upstream of the 5’ NTR of fragment 1, the poly(A) tail (59 adenosine residues) and PacI sequences were inserted downstream to the 3’ NTR of fragment 2. The fragments were cloned into the pBR322 vector and sequenced. Vector map was derived using SimVector software.
Mentions: To generate the infectious clone, we first generated a stock virus (passage 1) from a well-isolated plaque of CVB3 (Nancy strain, ATCC) and then prepared RNA from the stock virus using Trizol LS reagent (Invitrogen, Carlsbad, CA). We synthesized full-length cDNA using Moloney murine leukemia virus-reverse transcriptase in a reaction mixture containing oligo-dT (Invitrogen) and performed PCR with this cDNA to amplify two fragments using high-fidelity Pfu polymerase (Agilent Technologies, Santa Clara, CA). The primer sets used in the preparation of these amplicons are described in Table 1; the amplicons were cloned into plasmid pBR322 in a stepwise manner (Fig 1). To facilitate in vitro RNA transcription, bacteriophage T7 RNA polymerase promoter was inserted in the 5’ end of P1 primer next to the RsrII site (Fig 1 and Table 1). The first fragment was digested with RsrII and PacI and cloned into a modified pBR322 vector as described previously [30]. We digested this vector with SpeI and PacI and gel-purified the plasmid DNA, into which we then cloned the second PCR fragment digested with SpeI and PacI. This vector containing the full-length cDNA clone, hereafter designated, pBRCVB3 was sequenced leading us to identify three new nt changes that were not reported previously (Table 2). For in vitro transcription, the pBRCVB3 vector was linearized with PacI, and the transcription reaction was performed using T7 RNA polymerase at 37°C as recommended by the manufacturer (Promega, Madison, WI).

Bottom Line: The 5' non-translated region (NTR) is an important molecular determinant that controls replication and virulence of coxsackievirus B (CVB)3.We noted that the disease-inducing ability of the infectious clone-derived virus in three mouse strains was restricted to pancreatitis alone, and the incidence and severity of myocarditis were significantly reduced.We then reversed the mutations by creating three new clones, representing 1) U97C; 2) G4327A and U5088C; and 3) their combination together in the third clone.

View Article: PubMed Central - PubMed

Affiliation: School of Veterinary Medicine and Biomedical Sciences, University of Nebraska-Lincoln, Lincoln, Nebraska, United States of America.

ABSTRACT
The 5' non-translated region (NTR) is an important molecular determinant that controls replication and virulence of coxsackievirus B (CVB)3. Previous studies have reported many nucleotide (nt) sequence differences in the Nancy strain of the virus, including changes in the 5' NTR with varying degrees of disease severity. In our studies of CVB3-induced myocarditis, we sought to generate an infectious clone of the virus for routine in vivo experimentation. By determining the viral nt sequence, we identified three new nt substitutions in the clone that differed from the parental virus strain: C97U in the 5' NTR; a silent mutation, A4327G, in non-structural protein 2C; and C5088U (resulting in P1449L amino acid change) in non-structural protein 3A of the virus leading us to evaluate the role of these changes in the virulence properties of the virus. We noted that the disease-inducing ability of the infectious clone-derived virus in three mouse strains was restricted to pancreatitis alone, and the incidence and severity of myocarditis were significantly reduced. We then reversed the mutations by creating three new clones, representing 1) U97C; 2) G4327A and U5088C; and 3) their combination together in the third clone. The viral titers obtained from all the clones were comparable, but the virions derived from the third clone induced myocarditis comparable to that induced by wild type virus; however, the pancreatitis-inducing ability remained unaltered, suggesting that the mutations described above selectively influence myocarditogenicity. Because the accumulation of mutations during passages is a continuous process in RNA viruses, it is possible that CVB3 viruses containing such altered nts may evolve naturally, thus favoring their survival in the environment.

No MeSH data available.


Related in: MedlinePlus