Optimal Expression of the Envelope Glycoprotein of Orthobornaviruses Determines the Production of Mature Virus Particles

Most viruses causing persistent infection produce few infectious particles from the infected cells. Borna disease virus 1, a member of the genus Orthobornavirus, is an RNA virus that persistently infects the nucleus and has been applied to vectors for long-term gene expression.

ABSTRACT

An RNA virus-based episomal vector (REVec) whose backbone is Borna disease virus 1 (BoDV-1) can provide long-term gene expression in transduced cells. To improve the transduction efficiency of REVec, we evaluated the role of the viral envelope glycoprotein (G) of the genus Orthobornavirus, including that of BoDV-1, in the production of infectious particles. By using a G-pseudotype assay in which the lack of G in G-deficient REVec (ΔG-REVec) was compensated for the expression of G, we found that excess expression of BoDV-1-G does not affect particle production itself but results in uncleaved and aberrant mature G expression in the cells, leading to the production of REVec particles with low transduction titers. We revealed that the expression of uncleaved G in the cells inhibits the incorporation of mature G and viral genomic RNA into the particles. This feature of G was conserved among mammalian and avian orthobornaviruses; however, the cleavage efficacy of canary bornavirus 1 (CnBV-1)-G was exceptionally not impaired by its excess expression, which led to the production of the pseudotype ΔG-REVec with the highest titer. Chimeric G proteins between CnBV-1 and -2 revealed that the signal peptide of CnBV-1-G was responsible for the cleavage efficacy through the interaction with intracellular furin. We showed that CnBV-1-G leads to the development of pseudotyped REVec with high transduction efficiency and a high-titer recombinant REVec. Our study demonstrated that the restricted expression of orthobornavirus G contributes to the regulation of infectious particle production, the mechanism of which can improve the transduction efficiency of REVec.

IMPORTANCE Most viruses causing persistent infection produce few infectious particles from the infected cells. Borna disease virus 1, a member of the genus Orthobornavirus, is an RNA virus that persistently infects the nucleus and has been applied to vectors for long-term gene expression. In this study, we showed that, common among orthobornaviruses, excessive G expression does not affect particle production itself but reduces the production of infectious particles with mature G and genomic RNA. This result suggested that limited G expression contributes to suppressing abnormal viral particle production. On the other hand, we found that canary bornavirus 1 has an exceptional G maturation mechanism and produces a high-titer virus. Our study will contribute to not only understanding the mechanism of infectious particle production but also improving the vector system of orthobornaviruses.

INTRODUCTION

An engineered virus is a powerful tool for gene therapy (1, 2). We recently developed an RNA virus-based episomal vector (REVec) that is an artificially modified Borna disease virus 1 (BoDV-1) (3–5), a nonsegmented negative-strand RNA virus belonging to the genus Orthobornavirus. BoDV-1 characteristically replicates in the cell nucleus without cytotoxicity and maintains a long-lasting persistent infection by the chromosomal binding of viral ribonucleoproteins (vRNPs) throughout the cell cycle (6–9). This unique feature of BoDV-1 allows REVec to be a long-acting gene expression vector without DNA integration. In addition, REVec can transduce foreign genes into stem cells, including induced pluripotent stem cells (iPSCs), with high efficacy (10, 11), making REVec a potential gateway for gene therapy and regenerative medicine. To establish REVec as a breakthrough novel viral vector, however, there is a remaining problem to be resolved: producing REVec particles with a high transduction titer (11, 12). Previous studies revealed that BoDV-1 produces very few viral particles from persistently infected cells and that the culture supernatant contains only low infectious titers (13, 14). This feature may be a strategy of BoDV-1 to establish persistent infection in the nucleus, but it becomes a barrier to producing a high-titer REVec by reverse genetics. Furthermore, it is still largely unknown how BoDV-1 regulates the production of infectious particles from infected cells.

For enveloped RNA viruses, matrix protein (M) and glycoprotein (G) drive the formation of virus particles (15). M can solely form virus-like particles without G expression and is also involved in the incorporation of viral nucleocapsids into nascent particles (16–20). On the other hand, G is an envelope protein that plays roles in cell entry, including cellular receptor recognition and membrane fusion, of the particle. Although pivotal roles of envelope G in the release of virus particles have been demonstrated in many RNA viruses (16, 17, 21), the involvement of orthobornavirus G in the production of virions has not been well understood. For further improvement of the REVec system, therefore, we are focusing on the role of orthobornavirus G in infectious particle production.

In this study, we first show the expression profile of G in BoDV-1-infected cells and that the increased expression of G induces aberrant G expression patterns with increases in an uncleaved form, implying the involvement of restricted G expression in the production of mature virus particles. A G-pseudotype assay using G-deficient REVec (ΔG-REVec) (3) revealed that the elevated expression of BoDV-1-G increased the amount of uncleaved precursor G (pre-G) in the infected cells, leading to reduced production of infectious virus particles. We also show that in the infected cells, the uncleaved G decreases the cleavage efficacy of G and the incorporation of mature forms of G, GP1, and GP2 and viral genomic RNA (vgRNA) in the nascent particles. Furthermore, we demonstrate that the cleavage mechanism of G is conserved among mammalian and avian orthobornaviruses except canary bornavirus 1 (CnBV-1). The cleavage efficacy of CnBV-1-G was not impaired by the excess expression of G, leading to the production of the ΔG-REVec pseudotype with the highest titer of the orthobornavirus G pseudotypes. We further demonstrate that for a G chimera between the CnBV-1 and CnBV-2 Gs, the signal peptide sequence at the N terminus of CnBV-1-G is responsible for the cleavage efficacy in the cells. We finally demonstrate that ΔG-REVec pseudotyped with CnBV-1-G shows a high transduction efficiency for human cells, including iPSCs. This study suggests that the restricted expression of most orthobornavirus Gs serves as a mechanism to suppress aberrant particle production and that the enhanced cleavage efficacy of G contributes to the improvement of production of REVec with a high transduction titer.

RESULTS

Increased expression of G leads to aberrant modification of the protein.

BoDV-1 expresses polycistronic transcripts encoding M, G, and RNA-dependent RNA polymerase (L) proteins and regulates the expression levels of the encoded proteins by alternative splicing to accomplish its replication and/or persistent infection in the nucleus (22–25). We found, as shown in previous studies (26, 27), that the level of G expression in BoDV-1-infected cells varied compared to the nucleoprotein (N) expression level and that fewer than half of the infected cells were undetectable in the expression of G by immunofluorescence assay (IFA) (Fig. 1A and B), implying that the expression level of G may be associated with the low-infectivity particle production of BoDV-1.

FIG 1.

The expression of BoDV-1-G is restricted in vitro. (A) N and G expression in BoDV-GFP-infected 293T cells detected by IFA using an anti-N or anti-G antibody. The minimum thresholds for N or G detection were set to 46 or 13 using ImageJ, respectively. Scale bars, 10 μm. (B) The expression incidence of N and G among BoDV-GFP-infected 293T cells. The N- or G-positive cells were counted among GFP-expressing cells (n = 757 or 766). (C) Schematic representation of GspKO. These synonymous mutations are in the residues from 172 to 180 and from 1464 to 1472 around splicing donor and acceptor sites, respectively. (D) Western blot analysis of G expression in G-transfected 293T cells 48 h posttransfection and BoDV-GFP-infected 293T cells. A total of 3.0 × 10⁶ 293T cells was transfected with Gwt (1.5 μg) or GspKO (1.5 μg). Coomassie brilliant blue (CBB) stain was used as a loading control. (E) Comparison of the cleavage efficiencies of G. The efficiencies were calculated by dividing the sum of GP1 and GP2 by the total G (the sum of pre-G, GP1, and GP2) using ImageJ. The bars show the means ± the standard errors (SE) of three independent experiments. Statistical analysis was performed using a Fisher least significant difference (LSD) test. ****, P < 0.0001.

G is a type I surface glycoprotein that contains a signal peptide at the N terminus and a single transmembrane region at the C terminus (26). Pre-G (95 kDa) undergoes glycosylation and cleavage by furin, a ubiquitous subtilisin-like cellular proprotein convertase, which results in mature GP1 (51 to 60 kDa) and GP2 (43 kDa) being assembled into virus particles (26, 28, 29). The G open reading frame (ORF), which overlaps with the M and L ORFs, contains an intron spliced for L mRNA (23, 24). To evaluate the effect of the expression level of G on BoDV-1 particle formation, we first determined the expression profile of G in BoDV-1-infected and recombinant G-expressing cells. In addition to the wild-type plasmid construct encoding the G ORF (Gwt), we constructed a splicing knockout BoDV-1-G expression plasmid (GspKO) that has synonymous mutations around splicing acceptor and donor sites (30) to enhance the expression level of G (Fig. 1C). As shown in Fig. 1D and E, although pre-G and mature G were clearly detected in the cells transfected with Gwt or infected with BoDV-1 encoding GFP (BoDV-GFP), increased amounts of pre-G and abnormalities in modified pre-G and GPs were observed in the cells with GspKO. These observations indicated that the expression level of G can be involved in the maturation of infectious particles of BoDV-1.

Excess expression of G reduces infectious virus production of REVec.

To understand whether the expression level of G is linked to the efficiency of infectious particle production of BoDV-1, we next transfected different amounts of GspKO expression plasmids into 293T cells persistently infected with ΔG-REVec encoding GFP and harvested ΔG-REVec (BoDV-1G-REVec) enveloped with BoDV-1-G (Fig. 2A) (3). Vero cells were inoculated with BoDV-1G-REVec, and the GFP-expressing cells were counted 3 days after infection. Interestingly, although the titer of BoDV-1G-REVec was increased by 0.1-μg transfection with the GspKO expression plasmid, a 1.5-μg transfection reduced the transduction titer (Fig. 2B), suggesting that there is an optimal level of G in recovering a high titer of REVec.

FIG 2.

REVec titer and cleavage efficiency of G depend on the G expression level. (A) A schematic diagram of the method for collecting transiently infectious ΔG-REVec. BoDV-1G_REVec-GFP was collected by ectopically expressing BoDV-1-G in ΔG-REVec-infected 293T cells, which has vRNP due to persistent infection with ΔG-REVec. (B) Comparison of vector titers. A total of 3.0 × 10⁶ ΔG-REVec-infected 293T cells was transfected with 0.01, 0.1, or 1.5 μg of pCAG-GspKO. To promote particle production, 5 μg of M expression plasmid was cotransfected. After 48 h, a virus solution was obtained by ultracentrifugation from HEPES buffer containing MgCl₂. Titers were determined using Vero/Puro cells. Mock indicates ΔG-REVec-infected 293T cells that was not transfected G expression plasmids. (C, left) Western blot analysis of the G expression pattern in ΔG-REVec-infected 293T cells 48 h posttransfection. CBB staining was used as a loading control. (C, right) Comparison of cleavage efficiencies of G. The efficiency was calculated by dividing the sum of GP1 and GP2 by the total G (the sum of pre-G, GP1, and GP2) using ImageJ. (D) Western blot analysis of G and M in vector particles. Lysate of ΔG-REVec-infected 293T cells transfected with 0.1 μg of pCAG-GspKO from panel C was used for positive control. (E) Comparison of the expression levels of GP1 and GP2. The band intensities were calculated and normalized to that of M using ImageJ. (F) Comparison of the expression levels of M. The band intensities were calculated by ImageJ. (G) Western blot analysis of G and M in vector particles (bottom) and comparison of the expression levels of precursor G (pre-G) (top). A total of 3.0 × 10⁶ ΔG-REVec-infected 293T cells was transfected with 1.5 or 0.1 μg of BoDV-1-G expression plasmid and 0.025, 0.085, 0.25, 0.85, 2.5, or 8.5 μg of human furin expression plasmid. After 48 h, a virus solution was obtained from the supernatant of cells treated with HEPES buffer containing MgCl₂. Lysate of ΔG-REVec-infected 293T cells transfected with 0.1 μg of pCAG-GspKO was used for a positive control. The band intensities were calculated and normalized to that of M using ImageJ. (H, top) Comparison of titer of vectors collected in panel G. Titers were determined using Vero/Puro cells. (H, bottom) Western blot analysis of furin expression in ΔG-REVec-infected 293T cells at 48 h posttransfection. CBB staining was used as a loading control. (I) Electron microscopy analysis of vector particles. Ultracentrifuged vector particles were negatively stained. Scale bar, 25 nm. (J and K) Relative comparison of viral genomic RNA contained in ΔG-REVec-infected 293T cells at 48 h posttransfection (J) and in vector particles (K) as determined by RT-qPCR. In the graphs, the bars show the means ± the SE of three independent experiments. Statistical analysis was performed using a Fisher LSD test (B, D, E, F, G, H, J, and K) or Tukey’s multiple-comparison test (C). *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001; n.s., nonsignificant.

We next analyzed BoDV-1-G expression in transfected ΔG-REVec-infected 293T cells by Western blotting. GP1 and GP2 were clearly detected in cells that produced BoDV-1G-REVec with a high titer, while as shown in Fig. 1D, pre-G and aberrant G (GP1*) were dominantly expressed in the cells transfected with excess G expression plasmid, the latter of which could be glycosylation variants of GP1 (Fig. 2C) (31). Next, we evaluated G incorporation in particles released from the transfected ΔG-REVec-infected 293T cells. As shown in Fig. 2D, only GP1 and GP2 were detected in particles released from cells transfected with low and moderate amounts of G plasmid. On the other hand, when excess G plasmid was transfected into the cells, the amount of GP1 and GP2 incorporated into the particles markedly decreased, and pre-G became detectable (Fig. 2D and E). Note that the particle production evaluated by M expression was not affected even in cells expressing excess G (Fig. 2F). This observation confirmed that the excess expression of BoDV-1-G increases uncleaved G incorporated in virus particles. Since endogenous furin is present at low abundance in the cells (32), we evaluated the BoDV-1-G cleavage and the transduction titer of BoDV-1G-REVec under several levels of furin overexpression. As expected, furin overexpression decreased uncleaved G (Fig. 2G) in the particles and enhanced the transduction titer in a dose-dependent manner; however, the titers were lower than that for the optimal level (0.1 μg) of BoDV-1-G expression plasmid (Fig. 2H). These data suggested that not only furin but also the expression level of G are determinants of the transduction titer of BoDV-1G-REVec.

To confirm the particle production by REVec, negatively stained specimens were subjected to electron microscopy analysis. We observed particles in the supernatant of ΔG-REVec-infected 293T cells without G expression (Fig. 2I), indicating that M expression is enough to produce the particles. Furthermore, particles with membrane protrusions were observed in supernatants from the excess G-expressing cells, the BoDV-GFP-infected cells, and the optimal G-expressing cells (Fig. 2I). These data suggested that excess BoDV-1-G expression has no effect on REVec particle production and affects the assembly of GP1 and GP2 into the particles, leading to reduced infectious particle production.

In some RNA enveloped viruses, the interaction of envelope protein with vRNP facilitates virus particle production (33–35). To assess the incorporation of vRNA into BoDV-1G-REVec particles, we quantified vgRNA in the particles by reverse transcription-quantitative PCR (RT-qPCR). In the transfected cells, the amount of vgRNA was slightly increased by the excess G expression (Fig. 2J). As shown in Fig. 2K, vgRNA was detected in particles from the mock-transfected cells, indicating that M is sufficient for vgRNA incorporation into the particles. On the other hand, transfection of excess G, which induced the production of excess pre-G in the cells (Fig. 2C), significantly decreased the amount of vgRNA incorporated in the particles (Fig. 2K). Taken together, our results suggested that the expression level of BoDV-1-G in infected cells is involved in the incorporation of vgRNA and mature G into the particles of REVec.

Pre-G reduces the cleavage efficacy of G and vgRNA incorporation into particles.

We next evaluated whether excess uncleaved BoDV-1-G expression in infected cells affects infectious particle production. To this end, we generated a G mutant whose multibasic furin cleavage site (28) was replaced with a series of alanine residues (GspKO_R4A) (Fig. 3A). First, to assess the involvement of G cleavage in the cellular localization itself, Vero cells infected with ΔG-REVec were transfected with GspKO or GspKO_R4A plasmids and stained with anti-BoDV-1-G polyclonal antibody. As a result, the localization of GspKO_R4A was similar to that of GspKO (Fig. 3B), indicating that BoDV-1-G cleavage does not affect the cellular localization itself. Next, 293T cells persistently infected with ΔG-REVec were cotransfected with GspKO and GspKO_R4A, and the expression pattern of G was analyzed by Western blotting. Despite the constant transfection amount of GspKO, the cleavage efficacy of G decreased as the amount of uncleaved G increased (Fig. 3C). The expression of uncleaved G also reduced the amount of GP1 and GP2 assembled into the particles (Fig. 3D) and the transduction titer (Fig. 3E). Furthermore, the expression of uncleaved G decreased the incorporation of vgRNA into the particles (Fig. 3F), as shown by the excess G expression (Fig. 2K). Electron microscopy analysis showed particle production with membrane protrusions in the supernatant from the uncleaved G-expressing cells, which was similar to those from the BoDV-GFP-infected cells and the optimal G-expressing cells (Fig. 3G). These results indicated that the elevated uncleaved BoDV-1-G expression impairs the incorporation of cleaved G and vgRNA into particles, reducing the transduction titer of the particles.

FIG 3.

Expression of uncleaved G affects the efficiency of G cleavage and the production of mature virus particles. (A) Schematic representation of GspKO_R4A. The arginine at residues 246 to 249 of G, which is the recognition site for furin cleavage, was replaced with alanine. (B) Intracellular localization of GspKO or GspKO_R4A in Vero cells infected with G-deficient BoDV encoding GFP by IFA using an anti-G antibody. Scale bar, 15 μm (inset bar, 5 μm). (C, left) Western blot analysis of the G expression pattern in ΔG-REVec-infected 293T cells 48 h posttransfection. A total of 3.0 × 10⁶ ΔG-REVec-infected 293T cells was transfected with GspKO (0.1 μg), GspKO_R4A (0.1, 1.5 μg), and M expression plasmids (5 μg). CBB staining was used as a loading control. (C, right) Comparison of the expression levels of GP1 and GP2. The band intensities were calculated and normalized to that of CBB using ImageJ. (D, left) Western blot analysis of G and M in vector particles. (D, right) Comparison of the expression levels of GP1 and GP2. Lysate of ΔG-REVec-infected 293T cells transfected with 0.1 μg of pCAG-GspKO from panel C was used for positive control. The band intensities were normalized to that of M. (E) Comparison of vector titers. Titers were determined using Vero/puro. (F) Comparison of viral genomic RNA contained in vector particles by RT-qPCR. Mock indicates ΔG-REVec-infected 293T cells in which neither GspKO nor GspKO_R4A was transfected. (G) Electron microscopy analysis of vector particles. Ultracentrifuged vector particles were negatively stained. Scale bar, 25 nm. In the graphs, the bars show the means ± the SE of three independent experiments. Statistical analysis was performed using Tukey’s multiple-comparison test (C) or a Fisher LSD test (D, E, and F). *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001.

The cleavage mechanism of G is conserved among the orthobornaviruses except CnBV-1.

The genus Orthobornavirus comprises various viruses, including BoDV-1 (36). Since the cytoplasmic tail of G is highly conserved among the orthobornaviruses (Fig. 4A), we predicted that orthobornavirus G proteins can be compatibly assembled into ΔG-REVec, which was generated based on BoDV-1. By comparing orthobornavirus Gs derived from diverse host species, we further investigated the mechanism of G maturation and infectious virus particle production. We thus generated pseudotyped ΔG-REVec with Gs from nine genotypes in different species of orthobornavirus. The genotypes used in this study are as follows: variegated squirrel bornavirus 1 (VSBV-1; the species Mammalian 2 orthobornavirus), canary bornavirus 1 and 2 (CnBV-1 and -2; Passeriform 1 orthobornavirus), estrildid finch bornavirus 1 (EsBV-1; Passeriform 2 orthobornavirus), parrot bornavirus 2, 4, and 7 (PaBV-2, -4, and -7; Psittaciform 2 orthobornavirus), parrot bornavirus 5 (PaBV-5; Psittaciform 2 orthobornavirus), and Munia bornavirus 1 (MuBV-1; unclassified) (36). As expected, all tested Gs had compatibility with BoDV-1-G for the production of infectious particles (Fig. 4B and C). Among the examined vectors, REVec pseudotyped with CnBV-1-G (CnBV-1G-REVec) showed the highest titer in both Vero and quail-derived QT6 cells (Fig. 4B and C).

FIG 4.

CnBV-1-G cleavage efficiency is independent of its expression level and does not decrease the titer. (A) Multiple sequence alignment of the cytoplasmic region of G derived from several viruses belonging to the genus Orthobornavirus. Black highlights indicate different amino acids or gaps. (B and C) Comparison of pseudotype ΔG-REVec titers. A total of 1.2 × 10⁵ ΔG-REVec-infected 293T cells was transfected with 0.4 μg of one of several G expression plasmids. After 48 h, the vectors were recovered by the freeze-thaw method. Titers were determined using Vero/puro (B) or QT6 (C). (D) Changes in titer of pseudotype ΔG-REVec-GFP depending on the G expression level. A total of 1.2 × 10⁵ ΔG-REVec-infected 293T cells was transfected with 0.015 or 0.4 μg of G expression plasmid. The vector collection and titration were performed following the same method as in panel B. (E) Comparison of the titers of CnBV-1G_ΔG-REVec. A total of 1.2 × 10⁵ ΔG-REVec-infected 293T cells was transfected with 0.015, 0.05, 0.15, or 0.4 μg of CnBV-1G expression plasmid. The vector collection and titration were performed following the same method as in panel B. (F) Western blot analysis of the G expression pattern in ΔG-REVec-infected 293T cells 48 h posttransfection. The cell lysate was obtained from ΔG-REVec-infected 293T cells transfected following the same method as in panel E. CBB staining was used as a loading control. The bars show the means ± SE of three independent experiments. Statistical analysis was performed using Dunnett’s multiple-comparison test (B and C), an unpaired Student t test (D), or Tukey’s multiple-comparison test (E and F). *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001; n.s., nonsignificant.

To examine whether G cleavage of CnBV-1 is involved in the high transduction titer of the vector, the expression levels of each genotype of G were increased in 293T cells infected with ΔG-REVec. Intriguingly, although the elevated expression of G impaired the titers of most pseudotyped REVec produced from the cells, CnBV-1G-REVec was not affected by the excess G expression (Fig. 4D). We verified that various excess G expression levels had no effect on the production of CnBV-1G-REVec (Fig. 4E). Analysis of the CnBV-1-G expression pattern in the cells showed that pre-G was detected, but the cleavage efficacy of G was not decreased (Fig. 4F). These data demonstrated that the cleavage mechanism of G that determines infectious particle production is widely conserved in orthobornaviruses, except for CnBV-1.

The signal peptide of CnBV-1-G is involved in the cleavage efficacy that leads to the high transduction titer of REVec.

To identify the region responsible for the stable cleavage efficacy of CnBV-1-G, even with excessive expression, we compared the sequences of CnBV-1-G and CnBV-2-G, which is genetically related to CnBV-1 (Fig. 5A) (37). We focused on three unconserved regions between these two sequences. The first is the signal peptide (SP) at the N terminus, in which CnBV-1 has 9 fewer amino acids than CnBV-2. The second is the amino acid residue at position 121 (P), which is located around a fusion loop presumed to be important for membrane fusion function (38, 39). The third is the region just before the furin cleavage site (BC) (28). We generated chimeric G expression plasmids that are recombinations between CnBV-1 and CnBV-2 at these three regions (Fig. 5B) and collected pseudotyped REVec with chimeric CnBV-G proteins. As a result, replacement of the CnBV-1-G SP with the CnBV-2-G SP (CnBV-1_SP2) reduced REVec production (Fig. 5C). When CnBV-2-G was used as the backbone, the titer of REVec with G recombined with the SP of CnBV-1-G (CnBV-2_SP1) was increased (Fig. 5D). These data suggested that the SP of CnBV-1-G is involved in the high infectious particle production ability of CnBV-1-G.

FIG 5.

The CnBV-1G signal peptide is involved in cleavage efficiency. (A) Multiple sequence alignment of CnBV-1-G and CnBV-2-G. Black highlights indicate different amino acids or gaps. Colored highlights indicate the recombined regions. SP, signal peptide (orange); P, polar amino acid (yellow); BC, before cleavage (green). (B) Schematic diagram of the prepared chimeric CnBV-1-G and CnBV-2. (C and D) Comparison of titers of ΔG-REVec pseudotyped with chimeric CnBV-1-G (C) or chimeric CnBV-2-G (D) 1.2 × 10⁵ ΔG-REVec-infected 293T cells was transfected with 0.4 μg of chimeric G expression plasmid. After 48 h, the vector was recovered by the freeze-thaw method. Titers were determined by using Vero/Puro cells. (E) Western blot analysis of the G expression pattern in 293TΔG-GFP cells at 48 h posttransfection. A total of 1.2 × 10⁵ ΔG-REVec-infected 293T cells was transfected with 0.015 or 0.4 μg of chimeric G expression plasmid. CBB staining was used as a loading control. (F and G) Comparison of chimeric CnBV-1-G (F) and chimeric CnBV-2-G cleavage efficiency. The efficiency was calculated by dividing GP2 by the total G (the sum of pre-G and GP2). (H, top) Interaction between chimeric CnBV-1-G or -2-G and furin in transfected Vero cells detected by PLA. The interaction signals and cytoskeleton were stained with a probe molecule (red) and phalloidin (green). (H, bottom) Intracellular localization of chimeric CnBV-1-G or -2-G and furin in transfected Vero cells detected by IFA. Scale bar, 10 μm. (I) Comparison of interaction with G and furin by quantification of the signal area per cell from (H) using ImageJ. In the graphs, the bars show the means ± the SE of three independent experiments (C, D, F, and G). Statistical analysis was performed using Dunnett’s multiple-comparison test (C and D), Tukey’s multiple-comparison test (F and G), or the Fisher LSD test (I). *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001; n.s., nonsignificant.

We next examined whether the SP of CnBV-1-G also affects the cleavage efficacy of G in the cells. The cleavage efficacy of CnBV-1_SP2 was remarkably reduced compared to that of CnBV-1_Wt, and elevated G expression led to a further reduction in the efficacy (Fig. 5E and F). On the other hand, CnBV-2_SP1 showed high cleavage efficacy, and the elevated G expression maintained the efficacy at the same level as that of CnBV-2_Wt (Fig. 5E and G). Then, to investigate how SP of G affects the cleavage efficacy, we assessed the interaction of the chimeric Gs with the intracellular furin by proximity ligation assay (PLA). In the cells expressing CnBV-1-G and CnBV-2_SP1, more PLA signals that indicate the interaction between Gs and furin were detected than in the cells expressing CnBV-2-G and CnBV-1_SP2, while cellular localization of Gs and furin were not different among the G-expressing cells (Fig. 5H and I). These results showed that the SP of CnBV-1-G is involved in the cleavage efficiency of pre-G through the interaction with intracellular furin, resulting in high-titer REVec production.

REVec with CnBV-1-G shows a high transduction titer.

Although CnBV-1 has bird hosts (40, 41), CnBV-1-G can be efficiently cleaved and confer ΔG-REVec infectivity even in mammalian cells (Fig. 4B, E, and F). Therefore, we assessed the transduction efficiency of CnBV-1-G-REVec in human-derived glial cells and iPSC lines (201B7 and 409B2), which are targets of gene cell therapy (42–44). In human oligodendroglioma (OL) cells, CnBV-1-G enhanced REVec infectivity by 15-fold compared to the infectivity with BoDV-1-G (Fig. 6A). The transduction titer of CnBV-1-G was 5.8- and 13.6-fold higher than those of BoDV-1-G in 201B7 and 409B2, respectively (Fig. 6B). These results indicated that CnBV-1-G-REVec can efficiently transduce a foreign gene in human-derived cells, including iPSCs.

FIG 6.

ΔG-REVec pseudotyped with CnBV-1-G has high transduction efficiency for human-derived cells. (A) Comparison of the titers of BoDV-1G and CnBV-1G_ΔG-REVec. A total of 1.2 × 10⁵ ΔG-REVec-infected 293T cells was transfected with 0.4 μg of several G expression plasmids. After 48 h, the vectors were recovered by the freeze-thaw method. The titer was determined using OL cells. (B, left) Comparison of GFP transduction efficiencies of BoDV-1G_ and CnBV-1G_ΔG-REVec in human iPSCs. At 5 days postinoculation, GFP fluorescence was observed. (B, right) Expression analysis of transduced GFP expression in iPSCs. The area where GFP was expressed was calculated using ImageJ. (C) Schematic representation of the genome structure of REVec-GFP and G recombinant ΔM-REVec. Pink highlights indicate G gene overlap region with M or L gene. (D) A schematic diagram of the method for recovering G recombinant ΔM-REVec. (E) Comparison of titers of G recombinant ΔM-REVec. The vectors were recovered by sonication method and titers were determined using Vero/puro. In the graphs, the bars show the means ± the SE of three independent experiments. Statistical analysis was performed using Bonferroni’s multiple-comparison test (A and B) or the Fisher LSD test (E). **, P < 0.01; ****, P < 0.0001.

We finally generated REVec possessing CnBV-1 in its genome and compared the titer with REVec with BoDV-1-G or CnBV-2-G. Since G gene overlaps with M and L genes in the REVec genome (Fig. 6C), the ORF alone cannot be deleted from the genome. Therefore, we employed ΔMG-REVec-GFP (4), which lacks M and G genes and the intron of L gene, and inserted G gene between GFP and L gene (Fig. 6C). The G recombinant REVec was rescued from 293T cells transfected with the constructed REVec and helper plasmids (N, X, P, M, and L) (Fig. 6D) and transduced into Vero cells to determine the viral titers. As a result, CnBV-1-G led to the highest titer of REVec compared to BoDV-1-G and CnBV-2-G (Fig. 6E). Our results indicated that CnBV-1-G can facilitate REVec production not only in pseudotype system but also as recombinant virus.

DISCUSSION

In this study, to improve the REVec system, we focused on the role of orthobornavirus G in the formation of infectious particles. We showed here that the excess expression of BoDV-1-G resulted in an increased level of pre-G (Fig. 1D and 2C), leading to the impairment of G cleavage and the incorporation of mature G and vgRNA into the particles (Fig. 2C and K). The presence of such aberrant particles reduced the viral titer of released virions (Fig. 2B, 2D, 3D, and 3E). A low abundance of endogenous furin may conceivably prevent the efficient cleavage of BoDV-1-G (32). In fact, the titer of BoDV-1G-REVec was increased with increasing amount of transfected furin expression plasmid (Fig. 2H). However, infectious particle production by the overexpression of furin could not exceed the efficiency resulting from optimal G expression without exogenous furin expression (Fig. 2H). In addition, we also found that excess G expression did not affect the cleavage efficacy of CnBV-1-G (Fig. 4F). These data imply that other cellular proteases or factors, such as those that influence cleavage or cellular localization of G, are required for the efficient maturation of orthobornavirus G.

It has been reported that the maturation of viral envelope proteins through cleavage is involved in their assembly into particles and the intracellular transport of proteins (45–49). In BoDV-1-G, the intracellular localization of uncleaved G was similar to that of G (Fig. 3B), indicating that the cleavage of G does not affect its intracellular transport. On the other hand, we showed that the accumulation of uncleaved G in the cells decreases the cleavage of G (Fig. 3C) and impairs vgRNA incorporation into the viral particles (Fig. 3F). Little is known about how uncleaved G induces such perturbations in virion formation, and uncleaved G can be an indicator of excess G expression and function as negative feedback for infectious particle production. Further investigations are required to assess the interaction of G with M or vRNP and identify host proteases for efficient G cleavage.

Our data suggested that restricted BoDV-1-G expression serves as an exclusive mechanism for mature infectious particle production. In BoDV-1-infected cells, the expression of BoDV-1-G is posttranscriptionally regulated by the host splicing machinery in infected cells (22, 23). G is translated from unspliced (2.8 and 7.2 kb) and spliced mRNAs (2.7 and 7.1 kb) through leaky ribosomal scanning and ribosomal reinitiation promoted by the upstream minicistron, respectively (23), since mRNAs have several alternative start codons upstream of the G ORF. Furthermore, the ORF of BoDV-1-G contains an intron for L mRNA (24). Previous studies revealed that restricted G production with immature G mRNA expression was detected in the late phase of BoDV-1 infection in vivo (50). In some enveloped viruses, it has been demonstrated that excess expression of envelope proteins can promote mature virus particle production, but in turn, it strongly stimulates the host innate immune response by producing immature envelope protein (51, 52). These observations indicate a trade-off between evasion of the innate immune response and effective production of mature virus particles. BoDV-1 might use the strategy for stealth production of the mature virus to maintain persistent infection in the nucleus.

We revealed that the mechanism of mature particle production, which depends on the expression level of G, was conserved among most viruses belonging to the genus Orthobornavirus (Fig. 4D). Intriguingly, however, CnBV-1-G exceptionally maintained its cleavage efficacy and particle production despite excess G expression (Fig. 4E and F). CnBV-1 was identified in 2009 from a canary (Serinus canaria) that suffered from proventricular dilatation disease (PDD) (40), and experimental infection reproduced pathogenicity (41, 53), indicating that the virus is a causative agent of PDD. In Germany, CnBV was detected in 40% of captive canaries, and CnBV-1 to -3 have been identified, but the differences in virological properties and pathogenicity among the genotypes is unknown (41). In this study, by using chimeric CnBV-1-G recombined with CnBV-2-G, we showed that the SP is responsible for vector production and G cleavage efficiency through the interaction with intracellular furin (Fig. 5C to I). Although the SP is a sequence for directing intracellular transport involved in protein secretion and maturation, the viral envelope protein SP has additional roles (45, 54–57). For example, the SP of the HIV-1 envelope protein affects its expression level, transport efficiency, glycosylation, folding, and infection efficiency (55, 56, 58). The SP of the lymphocytic choriomeningitis virus envelope glycoprotein (GP) forms a complex with itself after cleavage by signal peptidase and is involved in the cleavage of GP during maturation and its expression on the cell surface (45). Our data show that the SP of CnBV-1-G is also involved in the interaction with cellular compartment affecting the G cleavage efficacy (Fig. 5H and I). Note that CnBV-1 develops persistent infection in vitro (41) and shows a low titer in vivo (53). This may indicate that the other viral components of CnBV-1 can restrict the high cleavage and virion maturation efficiencies of CnBV-1-G in infected cells.

Our findings about the role of G in the infectious particle production of orthobornavirus could strongly contribute to improvement of the REVec system. Improvement of the virus vector system was carried out through modification of envelope proteins. Currently, lentivirus and retrovirus vectors, which are widely approved for gene therapy (2), are enveloped with the vesicular stomatitis virus (VSV) envelope glycoprotein G (59, 60). VSV-G expands the susceptible cell types and promotes the stability of particles so that they tolerate the purification process (61–63). Our previous study showed that VSV-G has no compatibility with BoDV-1-G for ΔG-REVec production and that the cytoplasmic region of BoDV-1-G is necessary for envelope protein assembly into particles (3). Consistent with the report, all the Gs derived from the genus Orthobornavirus that have the conserved cytoplasmic region showed compatibility with BoDV-1-G (Fig. 4A to C). CnBV-1G-REVec achieved efficient transduction of a foreign gene in human-derived cells, including iPSCs (Fig. 6A and B) and CnBV-1-G recombinant REVec showed highest titer compared to recombinant REVec with BoDV-1-G and CnBV-2-G (Fig. 6E), indicating a potential for gene cell therapy applications. However, in vivo characterization of CnBV-1G-REVec, such as determining its immunogenicity and distribution, is needed for clinical application as a vector in the future. This study clarifies that a new mechanism for stealth mature infectious particle production is regulated through the restricted expression of envelope proteins and will contribute to drastic improvement of the REVec production system.

MATERIALS AND METHODS

Cell culture.

Human embryonic kidney cells (293T), 293T cells infected with recombinant BoDV-1 encoding GFP, and African green monkey kidney cells expressing the puromycin resistance gene (Vero/puro) were cultured in Dulbecco modified Eagle medium (DMEM; 1.0 g/liter glucose; Nacalai Tesque, Kyoto, Japan) supplemented with 10% fetal calf serum (FCS). Vero cells infected with G-deficient BoDV encoding GFP were cultured in DMEM (1.0 g/liter glucose; Nacalai) supplemented with 2% FCS. Human OL cells and 293T cells infected with ΔG-REVec were cultured in DMEM (4.5 g/liter glucose; Thermo Fisher Scientific, Waltham, MA) supplemented with 5 or 10% FCS, respectively. Quail fibroblasts (QT6) were cultured in DMEM-F12 (Thermo Fisher Scientific) supplemented with 5% FCS. Human iPSCs 201B7 and 409B2 were maintained as previously described (10).

Immunofluorescence assay.

This was performed as previously reported (64). Briefly, the cells were fixed with 4% paraformaldehyde (Wako Pure Chemical Industries, Osaka, Japan) at room temperature and treated with phosphate-buffered saline (PBS) containing 0.5% Triton X-100 and 2% FCS for 15 min, followed by incubation with anti-N antibody (HN132; 1:4) anti-G antibody (1:750) (65), anti-FLAG M2 (1:5,000; Merck, Darmstadt, Germany), or anti-furin antibody (ab183495; 1:750; Abcam, Cambridge, UK) for 1 h. After washing, the cells were reacted with a 1,000-fold,diluted Alexa Fluor rabbit 555-conjugated or mouse 488-conjugated secondary antibody (Thermo Fisher Scientific) and 300 nM DAPI (4′,6′-diamidino-2-phenylindole; Merck) for 1 h. After a wash with PBS, the cells were mounted with Fluoro-KEEPER Antifade Reagent, nonhardening type (Nacalai) or Prolong Gold (Thermo Fisher Scientific). Fluorescence microscopy was performed using a Ti-E inverted microscope with a C1 confocal laser scanning system (Nikon, Tokyo, Japan) or DeltaVision Elite (Cytiva, Tokyo, Japan).

Plasmid construction.

To prepare the GspKO expression plasmid, a sequence mutated at both the splicing donor (SD) site and splicing acceptor (SA) site was inserted into pCAGGS. SD and SA sites introduced nonsynonymous substitutions from GAG GTT AGT to GAA GTC TCC and from CCA GCC TCC to CCC GCA AGC, respectively (30). Sequences of G mutants whose furin cleavage site was replaced with alanine (GspKO_R4A) were generated by PCR-based mutagenesis and cloned into pCAGGS. To prepare EsBV-1-G (GenBank accession no. KF680099), CnBV-1-G (GenBank accession no. KC464471), CnBV-2-G (GenBank accession no. KC464478), and PaBV-7-G (GenBank accession no. JX065210) expression plasmids, these fragments were obtained from an artificial gene synthesis service (Fasmac, Kanagawa, Japan). The VSBV-1-G fragment was obtained from gBlocks gene fragments (Integrated DNA Technologies, Coralville, IA). PaBV-5-G (GenBank accession no. LC120625), MuBV-1-G, PaBV-2-G (66), and PaBV-4-G (66) fragments were amplified by RT-PCR from the feces of PaBV-5-infected Eclectus roratus and QT6 cells infected with MuBV-1, PaBV-2, or PaBV-4, respectively. All of these sequences underwent nonsynonymous substitution at the SA site, whose AG sequence was inserted into pCAGGS. Chimeric CnBV-1-G and CnBV-2 were generated by PCR-based mutagenesis. The SP regions were determined using SignaIP-5.0 (https://www.cbs.dtu.dk/services/SignalP/). The chimeric fragments were inserted into pCAGGS. This backbone vector was kindly provided by Yayoi Otsuka, Iwate University. Human furin cDNA was synthesized from RNA extracted from 293T cells by RT-PCR and cloned into pCAGGS. To prepare BoDV-1-G-, CnBV-1-G-, or CnBV-2G-ΔM-REVec plasmids, restriction enzyme sites (AscI and AsiSI) were inserted between P and GFP gene based on pFct-BoDV ΔMG-GFP (4). GFP gene and each G gene were inserted between AscI/AsiSI, and BstBI/PacI sites, respectively.

Western blotting.

Cell lysates were prepared as previously described (64). To prepare protein samples from ultracentrifuged virus solutions for Western blotting, 2× SDS sample buffer was used. Proteins were separated by SDS-PAGE using e-PAGEL (ATTO Corporation, Tokyo, Japan) and transferred to polyvinylidene difluoride membranes from a Trans-Blot Turbo polyvinylidene difluoride transfer pack (Bio-Rad, Richmond, CA). The membranes were blocked with 5% skim milk (Wako Pure Chemical Industries) in TBS-T (Tris-buffered saline, 0.1% Tween 20) and incubated with anti-BoDV-1-G antibody (1:1,000) (65), custom-made anti-CnBV-1-G antibody (1:1,000; Eurofins Genomics K.K., Tokyo, Japan), anti-M antibody (1:500) (67), or anti-furin antibody (Abcam, ab183495; 1:20,000) diluted with Can Get Signal Immunoreaction Enhancer Solution 1 (Toyobo, Osaka, Japan) at 4°C overnight. After a washing step with TBS-T, the membranes were incubated with a 50,000-fold-diluted horseradish peroxidase-labeled anti-mouse or rabbit IgG antibody (Merck) at room temperature for 2 h or more. After a washing step with TBS-T, Clarity Western ECL substrate (Bio-Rad) was used for detection by a chemiluminescence reaction. Bands were detected and photographed by a Fusion Solo instrument (Vilber-Lourmat, Marne-la-Vallée Cedex, France). Band intensities were analyzed using ImageJ (68).

Preparation of cell-free virus and titration.

ΔG-REVec-infected 293T cells were seeded in cell matrix (Nitta Gelatin, Inc., Osaka, Japan)-coated dishes and transfected with GspKO, GspKO_R4A, several G orthobornaviruses, M, or furin expression plasmids using TransIT-293 (TaKaRa, Shiga, Japan) according to the manufacturers’ protocols. At 48 h posttransfection, after a wash with 20 mM HEPES (pH 7.5), 1.5 ml of 20 mM HEPES containing 250 mM MgCl₂ and 1% FCS solution was added. After 90 min of incubation at 37°C, supernatants were filtered through 0.22-μm membrane filters (Merck). Using 20% sucrose cushion in 20 mM HEPES containing 1% FBS, the filtered supernatant was ultracentrifuged at 80,000 × g for 1 h at 4°C, and the pellet was suspended in PBS (69). Alternatively, at 48 h posttransfection, the virus solution was collected by the freeze-thaw method. After a washing step with FCS-free conditioned medium, the same medium was added and freeze-thawed twice. The supernatant was centrifuged at 3,000 rpm for 5 min and collected. The virus solutions were stored at –80°C. The infectious titer was determined by inoculating Vero, QT6, or OL cells with a 10-fold serially diluted virus solution and counting the number of cells expressing GFP. After 3 days of inoculation, a TE-2000-U inverted microscope (Nikon) was used to count GFP-expressing cells as virus-infected cells.

Quantitative reverse transcription-PCR of viral genomic RNA.

Total RNA was extracted from the virus solution after ultracentrifugation using the NucleoSpin RNA virus kit (Macherey-Nagel, Düren, Germany) according to the manufacturers’ protocols. cDNA was synthesized from 7 μl of RNA sample using SuperScript IV reverse transcriptase (Thermo Fisher Scientific) and a BoDV-1 genome-specific primer (5′-GTT GCG TTA ACA ACA AAC CAA TCA T-3′) (70). qPCR was performed using 1 μl of the synthesized cDNA as a template and Luna Universal qPCR Master Mix (New England Biolabs, Ipswich, MA). The conditions were 95°C for 30 s and then 40 cycles at 95°C for 5 s and 60°C for 30 s for detection by a CFX Connect real-time system (Bio-Rad). BoDV-1 genome-specific primers (forward, 5′-ATG CAT TGA CCC AAC CGG TA-3′, and reverse, 5′-ATC ATT CGA TAG CTG CTC CCT TC-3′) were used for the detection of viral genomic RNA (71). By measuring the C_T value, the amounts of each RNA were compared by a relative quantification method using a standard curve.

Electron microscopy analysis.

The purified virions were fixed in 2% paraformaldehyde and then negatively stained with 2% uranyl acetate. Transmission electron microscope images were taken by an HT7700 (Hitachi High-Tech, Tokyo, Japan) operating at 80 kV.

Multiple sequence alignment.

Using CLC Genomics Workbench Version 20.0.4w, the cytoplasmic region of Gs derived from the viruses of the genus Orthobornavirus and CnBV-1-G and CnBV-2-G were aligned. The TMHMM Server v2.0 (https://www.cbs.dtu.dk/services/TMHMM/) was used for the determination of the cytoplasmic region.

In situ proximity ligation assay.

A total of 5.0 × 10⁴ Vero/Puro cells was cotransfected with FLAG-tagged CnBV-1-G, CnBV-1_SP2, CnBV-2-G, or CnBV-2_SP1 (0.4 μg), and human furin (0.1 μg) using Lipofectamine 2000 (Thermo Fisher Scientific) according to the manufacturers’ protocols. At 48 h posttransfection, the cells were fixed with 4% paraformaldehyde (Wako Pure Chemical Industries) at room temperature and treated with PBS containing 0.5% Triton X-100 and 2% FCS for 15 min, followed by incubation with anti-FLAG M2 (1:5,000; Merck), or anti-furin antibody (1:750; Abcam, ab183495) for 1 h. The following process followed the manufacturers’ protocols using DuoLink PLA technology probes and reagents (Merck). Anti-mouse PLUS and anti-rabbit MINUS were used as PLA probes. After an amplification step, the cells were incubated with 100 nM Acti-Stain 488 fluorescent phalloidin (Cytoskeleton, Denver, CO) for 30 min at room temperature. After a wash with PBS, the cells were mounted with Duolink in situ mounting medium containing DAPI. Fluorescence microscopy was performed using DeltaVision Elite (Cytiva).

Statistical analysis.

Statistical significance values were obtained using GraphPad Prism 8. The statistical tests used for the calculations are indicated in the figure legends.

Transduction efficiency of ΔG-REVec for iPSCs.

ΔG-REVec-infected 293T cells were seeded in cell matrix (Nitta Gelatin, Inc.)-coated dishes and transfected with BoDV-1-G or CnBV-1-G expression plasmids using TransIT-293 (TaKaRa) according to the manufacturers’ protocols. At 48 h posttransfection, the vector was obtained by sonication. Briefly, the cells were suspended in 200 μl of DMEM with 2% FCS and disrupted by sonication. The supernatant was collected by centrifugation at 1,200 × g at 4°C for 25 min. Inoculation of ΔG-REVec into 201B7 or 409B2 iPSCs was performed as described previously (10). At 5 days postinoculation, fluorescence microscopy was performed using an Eclipse TE-2000-U inverted microscope (Nikon), and the area of the GFP expression region was analyzed by ImageJ (68).

Rescue of G recombinant ΔG-REVec-GFP.

293T cells (6.0 × 10⁵) was cotransfected with pCAG-BoDV-2-N (500 ng), BoDV-1-P (25 ng), BoDV-1-X (5 ng), BoDV-1-M (20 ng), BoDV-1-L (250 ng), and BoDV-1-G-, CnBV-1-G-, or CnBV-2-G-ΔM-REVec (2 μg) using TransIT-293 (TaKaRa) according to the manufacturers’ protocols. At 3 days posttransfection, transfected cells (4.0 × 10⁶) were harvested, and cell-free virus was obtained by sonication.