Suppression of Borna Disease Virus Replication during Its Persistent Infection Using the CRISPR/Cas13b System

Abstract

Borna disease virus (BoDV-1) is a bornavirus that infects the central nervous systems of various animal species, including humans, and causes fatal encephalitis. BoDV-1 also establishes persistent infection in neuronal cells and causes neurobehavioral abnormalities. Once neuronal cells or normal neural networks are lost by BoDV-1 infection, it is difficult to regenerate damaged neural networks. Therefore, the development of efficient anti-BoDV-1 treatments is important to improve the outcomes of the infection. Recently, one of the clustered regularly interspaced short palindromic repeats (CRISPRs) and CRISPR-associated (Cas) systems, CRISPR/Cas13, has been utilized as antiviral tools. However, it is still unrevealed whether the CRISPR/Cas13 system can suppress RNA viruses in persistently infected cells. In this study, we addressed this question using persistently BoDV-1-infected cells. The CRISPR/Cas13 system targeting viral mRNAs efficiently decreased the levels of target viral mRNAs and genomic RNA (gRNA) in persistently infected cells. Furthermore, the CRISPR/Cas13 system targeting viral mRNAs also suppressed BoDV-1 infection if the system was introduced prior to the infection. Collectively, we demonstrated that the CRISPR/Cas13 system can suppress BoDV-1 in both acute and persistent infections. Our findings will open the avenue to treat prolonged infection with RNA viruses using the CRISPR/Cas13 system.

Keywords: Borna disease virus; CRISPR/Cas13b; antiviral; antivirals; persistent infection.

Figure 1

CRISPR RNAs (crRNAs) designed to target BoDV-1 mRNAs. (A) Schematic view of the N mRNA with the designed crRNAs (N#1–#6). Alignment of the N mRNA sequences of various BoDV-1 and BoDV-2 strains targeted by the designed crRNAs. (B) Schematic view of the M/G/L mRNA with the designed crRNAs (L#1–#6). Alignment of the M/G/L mRNA sequences of various BoDV-1 and BoDV-2 strains potentially targeted by the designed crRNAs. The dotted line indicates a major splicing of the M/G/L mRNA.

Figure 2

Decrease in the BoDV-1 mRNAs by the CRSISPR/Cas13b system targeting the N or the M/G/L mRNAs in persistently BoDV-1-infected cells. (A) Schematic representation of the protocol to evaluate the decrease in BoDV-1 RNAs using the CRISPR/Cas13b system in 293T/BoDV cells. 293T/BoDV cells were transfected with the plasmid expressing Cas13b and the indicated crRNA and then incubated for 2 days. The level of BoDV-1 RNAs in 293T/BoDV cells was determined by RT-qPCR assays. (B,C) Effects of the CRISPR/Cas13b system targeting the N (B) and the M/G/L (C) mRNAs on the level of the respective mRNAs. The levels of the N (B) and the M/G/L (C) mRNAs in 293T/BoDV cells were determined by RT-qPCR assays. (D,E) Effects of the CRISPR/Cas13b system targeting the N (D) and the M/G/L (E) mRNAs on the level of BoDV-1 gRNAs. (F) Collateral RNA degradation activity of Cas13b by crRNAs targeting BoDV-1 mRNAs in persistently infected cells. 293T/BoDV and uninfected 293T cells were transfected with the plasmid expressing Cas13b and the indicated crRNA and incubated for 2 days. The expression level of the GAPDH mRNA in 293T/BoDV and uninfected 293T cells expressing Cas13b and the indicated crRNA was determined by RT-qPCR assays. Values are expressed as the mean ± S.E. of at least three independent experiments. Cont, a non-target control crRNA designed in [35]; N#1–#6, crRNAs targeting different region in the N mRNA shown in Figure 1A; L#1–#6, crRNAs targeting different region in the M/G/L mRNA shown in Figure 1B. ns: not significant, *, p < 0.05; **, p < 0.01; ***, p < 0.005; ****, p < 0.001, (vs. Cont in 293T/BoDV; a two-tailed Student’s t-test).

Figure 3

Decrease in the BoDV-1 load by the CRISPR/Cas13b system targeting the N and/or the M/G/L mRNAs in persistently BoDV-1-infected cells. (A,B) Effects of the CRISPR/Cas13b system using a combination of multiple crRNAs targeting BoDV-1 mRNAs on the level of BoDV-1 mRNAs. 293T/BoDV cells were transfected with the plasmid expressing Cas13b and the indicated single or multiple crRNAs and incubated for 2 days. The levels of the N (A) and the M/G/L (B) mRNAs in 293T/BoDV cells were determined by RT-qPCR assays. (C) Effects of the CRISPR/Cas13b system using a combination of multiple crRNAs targeting BoDV-1 mRNAs on the BoDV-1 load. The level of BoDV-1 gRNA in 293T/BoDV cells was determined by RT-qPCR assays. (D–F) Collateral RNA degradation activity of Cas13b by a combination of multiple crRNAs targeting BoDV-1 mRNAs in persistently infected cells. 293T/BoDV and uninfected 293T cells were transfected with the plasmid expressing Cas13b and the indicated crRNA and incubated for 2 days. The expression level of the GAPDH (D), the HPRT1 (E), and the L3MBTL1 (F) mRNAs in 293T/BoDV and uninfected 293T cells expressing Cas13b and the indicated crRNA was determined by RT-qPCR assays. (G) Cell viability of 293T/BoDV and uninfected 293T cells expressing Cas13b and the indicated crRNA. Values are expressed as the mean ± S.E. of at least three independent experiments. Cont, a control crRNA designed in [35]; N#3, a crRNA targeting the N mRNA shown in Figure 1A; L#6, a crRNA targeting the M/G/L mRNA shown in Figure 1B; N#mix, crRNAs targeting the N mRNA (a combination of crRNA N#1–N#6 in Figure 1A); L#mix, crRNAs targeting the M/G/L mRNA (a combination of crRNA L#1–L#6 in Figure 1B); NL#mix, crRNAs targeting both the N and the M/G/L mRNAs (a combination of crRNA N#1–N#6 and L#1–L#6 in Figure 1). ns: not significant, *, p < 0.05; **, p < 0.01; ***, p < 0.005; ****, p < 0.001 (vs. Cont in 293T/BoDV; a two-tailed Student’s t-test).

Figure 4

Effect of the CRISPR/Cas13b system on de novo BoDV-1 infection. (A) Schematic representation of the protocol to evaluate the effect of the CRISPR/Cas13b system targeting the N and/or the M/G/L mRNAs on de novo BoDV-1 infection. Uninfected 293T cells were transfected with the plasmid expressing Cas13b and the indicated crRNA. At 2 days post-transfection, the cells were infected with BoDV-1/GLuc at an MOI of 0.25 or 0.01 and further incubated for 2 and 3 days for GLuc assay and Western blot, respectively. (B,C) Effects of the CRISPR/Cas13b system targeting the N and/or the M/G/L mRNAs on de novo BoDV-1 infection. Viral infections at an MOI of 0.25 (B) and 0.01 (C) were evaluated by measuring the activity of Gaussia luciferase (GLuc) expressed from BoDV-1/GLuc-infected 293T cells. Cont, a control crRNA designed in [35]; N#3, a crRNA targeting the N mRNA shown in Figure 1A; L#6, a crRNA targeting the M/G/L mRNA shown in Figure 1B; NL#mix, crRNAs targeting both the N and the M/G/L mRNAs (a combination of crRNA N#1–N#6 and L#1–L#6 in Figure 1). Values are expressed as the mean ± S.E. of at least three independent experiments. **, p < 0.01; ***, p < 0.005; ****, p < 0.001 (vs. Cont; a two-tailed Student’s t-test).