Essential role of cis-encoded mature NS3 in the genome packaging of classical swine fever virus

Abstract

Classical swine fever virus (CSFV) is a member of the genus Pestivirus within the family Flaviviridae. The enveloped particles contain a plus-stranded RNA genome encoding a single large polyprotein. The processing of this polyprotein undergoes dynamic changes throughout the infection cycle. The release of mature NS3 from the polyprotein is mediated and regulated by the NS2 autoprotease and a cellular co-factor, restricting efficient cleavage to the early phases of infection. NS3 is a multifunctional viral enzyme exhibiting helicase, NTPase, and protease activities pivotal for viral replication. Hence, the release of mature NS3 fuels replication, whereas unprocessed NS2-3 precursors are vital for progeny virus production in later phases of infection. Thus far, no packaging signals have been identified for pestivirus RNA. To explore the prerequisites for particle assembly, trans-packaging experiments were conducted using CSFV subgenomes and coreless CSFV strains. Intriguingly, we discovered a significant role of mature NS3 in genome packaging, effective only when the protein is encoded by the RNA molecule itself. This finding was reinforced by employing artificially engineered CSFV strains with duplicated NS3 genes, separating uncleavable NS2-3 precursors from mature NS3 molecules. The model for NS2-3/NS3 functions in genome packaging of pestiviruses appears to be much more complicated than anticipated, involving distinct functions of the mature NS3 and its precursor molecule NS2-3. Moreover, the reliance of genome packaging on cis-encoded NS3 may act as a downstream quality control mechanism, averting the packaging of defective genomes and coordinating the encapsidation of RNA molecules before membrane acquisition.

Importance: Pestiviruses are economically significant pathogens in livestock. Although genome organization and non-structural protein functions resemble those of other Flaviviridae genera, distinct differences can be observed. Previous studies showed that coreless CSFV strains can produce coreless virions mediated by single compensatory mutations in NS3. In this study, we could show that only RNA molecules encoding these mutations in the mature NS3 are packaged in the absence of the core protein. Unlike this selectivity, a pool of structural proteins in the host cell was readily available for packaging all CSFV genomes. Similarly, the NS2-3-4A precursor molecules required for packaging could also be provided in trans. Consequently, genome packaging in pestiviruses is governed by cis-encoded mature NS3. Reliance on cis-acting proteins restricts the acceptance of defective genomes and establishes packaging specificity regardless of RNA sequence-specific packaging signals. Understanding the role of NS3 in pestiviral genome packaging might uncover new targets for antiviral therapies.

Keywords: CSFV; NS3; classical swine fever virus; coreless virus; cytopathogenicity; genome packaging; morphogenesis; pestivirus; trans-complementation; virus assembly.

Fig 1

Recombinant viral genomes used in this study. (A) The genome organization of wtCSFV is illustrated. Captions encompass the 5′-UTR, the ORF including N^pro, core, E^rns, E1, E2, P7, NS2, NS3, NS4A, NS4B, NS5A, NS5B, and the 3′-UTR. A star highlights the mutation N₂₁₇₇Y, which can compensate for the lack of core protein. (B) Genome organization of a coreless CSFV (CSFVΔcore) including corresponding captions. (C) Genome organization of pestiviral replicons. A reporter gene (mCherry) was positioned after amino acid 15 of N^pro, whereas the core to NS2 genes is deleted in the CSFV-DI-mCherry and the Linda-DI-mCherry. The BVDV-1 DI9 was cloned many years ago from a field case of mucosal disease, and we only had to insert a reporter gene to generate BVDV-DI-mCherry. (D) Genome organization of a cytopathogenic CSFV (CSFV-Ubi) with duplicated NS3–NS4B genes. The active cysteine (C₁₅₁₂) was exchanged against an alanine to prevent NS2-3 maturation. The mutation N₂₁₇₇Y, compensating for the lack of core protein, was introduced into either NS2-3 or NS3 genes. (E) Genome organization of a coreless cytopathogenic CSFV. Alongside the designation of the different virus genomes, the genome size is specified in parentheses.

Fig 2

Transfection of SK-6 cells with wtCSFV and DIs. Synthetic RNA of wtCSFV and the reporter DIs CSFV-DI-mCherry-N₂₁₇₇Y (with a compensatory NS3 mutation) and CSFV-DI-mCherry (without the mutation) were transfected via electroporation. After 24 hours, the cells were fixed and immunostained for the E2 protein. Nuclei were counterstained with DAPI, and fluorescence was captured using a monochromatic camera at 20-fold magnification. To enhance visualization, images were overlaid with DAPI in blue, mCherry in red, and E2 in green. Notably, double-transfected cells exhibited yellow staining. Naïve SK-6 cells served as a control for the E2 staining. A size marker of 100 µm length was included in the upper left corner for reference.

Fig 3

Infecting SK-6 cells with cell culture supernatants from wtCSFV and DI transfected cells. Naïve SK-6 cells were inoculated with 100 µL of sterile filtered cell culture supernatant harvested 48 hours post-transfection. After a 48-hour incubation period, the cells were fixed and immunostained for the E2 protein, with nuclei counterstained using DAPI. Fluorescence was captured via a monochromatic camera at 20-fold magnification. For enhanced visualization, images were overlaid, presenting DAPI in blue, mCherry in red, and E2 in green. Noteworthy is the discernible focus formation of DIs, evident after co-packaging with the helper virus. A size marker, 100 µm in length, was incorporated in the upper left corner for reference.

Fig 4

Western blot analyses conducted on cells transfected or infected with wtCSFV and DIs. (A) E2 expression was examined using A18 anti-CSFV E2. No signal was detected in naïve SK-6 cells or cells solely transfected with DIs or infected with cell culture supernatant from DI transfection. However, transfection of wtCSFV RNA and infection with the supernatant produced characteristic bands of E2 at 55 kDa (monomer), 80 kDa (heterodimer), and 120 kDa (homodimer). The same band pattern was observed after co-transfection with the DIs and infection. (B) NS3 expression was analyzed using 8.12.7 anti-NS3. Transfection of wtCSFV and infection with the supernatant resulted in typical NS2-3 bands at 125 kDa. The NS2-3 band was also present after co-transfection and infection with the DIs. Transfection of DI-RNA alone yielded a band at 80 kDa (mature NS3), which was also observed after trans-packaging with the helper virus but was absent when naïve cells were inoculated with supernatant from the DIs alone. (C) Box and whisker plots depicting progeny virus production and trans-packaging. The 50% tissue culture infection dose (TCID₅₀) was determined for both the helper virus (blue box) and the trans-packaged DIs (red box). Forty-eight hours post-transfection, the supernatants were harvested and used to infect naïve cells in dilution series, followed by immunostaining against the E2 protein to quantify the helper virus. The fluorescence of the mCherry reporter was used to quantify DI production in cultures superinfected with wtCSFV to allow DI-plaque formation. Titers were calculated using the Spaerman-Kärber method based on the results of three independent RNA transfections, each analyzed in three dilution series.

Fig 5

Transfection of SK-6 cells with a coreless CSFV bearing compensatory mutation N₂₁₇₇Y (CSFV-Δcore-N₂₁₇₇Y) and DIs. Synthetic RNA of CSFV-Δcore-N₂₁₇₇Y and the reporter DIs CSFV-DI-mCherry-N₂₁₇₇Y (also containing the compensatory NS3 mutation) and CSFV-DI-mCherry (bearing wild-type NS3 sequences) were transfected via electroporation. Following a 24 hour incubation period, the cells were fixed and subjected to immunostaining for the E2 protein. Nuclei were counterstained with DAPI, and fluorescence signals were captured using a monochromatic camera at 20-fold magnification. For visualization, the images were superimposed with DAPI in blue, mCherry in red, and E2 in green. Notably, a greater number of cells exhibited mCherry fluorescence in the case of CSFV-DI-mCherry-N₂₁₇₇Y. A size marker of 100 µm length was included in the upper left corner for scale reference.

Fig 6

Inoculating naïve SK-6 cells with cell culture supernatants from transfections of coreless CSFV bearing compensatory mutation N₂₁₇₇Y (CSFV-Δcore-N₂₁₇₇Y) and from DI co-transfections. After a 48 hour post-transfection incubation, 100 µL of sterile filtered cell culture supernatant was used for inoculation of naïve cells. After another 48 hour incubation, the cells were fixed and immunostained for the E2 protein, whereas nuclei were counterstained using DAPI. Captured at 20-fold magnification with a 100 µm size marker, the images were overlaid, showing DAPI in blue, mCherry in red, and E2 in green. Notably, only CSFV-DI-N₂₁₇₇Y-mCherry was trans-packaged by the CSFV-Δcore-N₂₁₇₇Y helper virus.

Fig 7

Western blot analyses performed on cells transfected or infected with CSFV-Δcore-N₂₁₇₇Y and DIs. (A) Expression of CSFV E2, marked by bands at 55, 80, and 120 kDa, was consistently observed post-transfection and infection of/with CSFV-Δcore-N₂₁₇₇Y, regardless of DIs co-transfection. (B) NS2-3 expression, with a distinctive band at 125 kDa, was evident after transfection and infection of/with CSFV-Δcore-N₂₁₇₇Y, irrespective of DIs co-transfection. The mature NS3, represented by an 80 kDa band, emerged following DIs co-transfection. However, this band only appeared upon infection of naïve cells in the case of CSFV-DI-N₂₁₇₇Y-mCherry, indicating successful trans-packaging by the CSFV-Δcore-N₂₁₇₇Y helper virus. (C) Box and whisker plots depicting progeny virus production and trans-packaging in the absence of core protein. The 50% tissue culture infection dose (TCID₅₀) was determined for both the helper virus (blue box) and the trans-packaged DI (red box). Supernatants were applied to naïve cells in a dilution series 48 hours post-transfection, followed by immunostaining against the E2 protein to quantify the helper virus. A measurement of mCherry fluorescence after superinfection of the cultures with wtCSFV was used to quantify DI production. Titers were calculated using the Spaerman-Kärber method based on the results of three independent RNA transfections, each analyzed in three dilution series. The abbreviation n.d. means not detected.

Fig 8

SK-6 cells transfected with RNAs of CSFV-Δcore and DIs. Synthetic RNA of CSFV-Δcore lacking compensatory NS3 mutations and the reporter DIs CSFV-DI-mCherry-N₂₁₇₇Y (bearing the compensatory NS3 mutation) and CSFV-DI-mCherry (without NS3 mutation) were co-transfected via electroporation. After 24 hours, the cells were fixed and immunostained for the E2 protein, with nuclei counterstained using DAPI. Fluorescence microscopy was performed at 20-fold magnification, with overlay images displaying DAPI in blue, mCherry in red, and E2 in green, alongside a size marker of 100 µm.

Fig 9

Inoculation of SK-6 cells with cell culture supernatants from CSFV-Δcore and DI transfected cells. SK-6 cells were infected with 100 µL cell culture supernatant, followed by 48 hours of incubation. After fixing, the cells were immunostained for the E2 protein and counterstain using DAPI. The results showed that CSFV-Δcore failed to produce progeny virus, regardless of DI co-transfection. The co-transfection of CSFV-DI-mCherry resulted in no mCherry-fluorescence positive cells. However, individual red cells emerged post-infection with supernatants of CSFV-DI-N₂₁₇₇Y-mCherry co-transfections indicating successful trans-packaging by the coreless helper virus.

Fig 10

Western blot analyses conducted on cells transfected or infected with CSFV-Δcore and DIs. (A) Expression of CSFV E2, evident through bands at 55, 80, and 100 kDa, was consistently observed post-transfection. However, in infection experiments, E2 expression was lacking, indicating no progeny virus production of CSFV-Δcore regardless of DI co-transfection. (B) NS2-3 expression, marked by a band at 125 kDa, was evident after transfection of CSFV-Δcore but absent in all infection experiments. Mature NS3, characteristic of DIs, represented by an 80 kDa band, was present following DIs transfection. However, this band was also not visible following infection. Successful trans-packaging was documented by the fluorescence of mCherry. The absence of the NS3 band in CSFV-DI-N2177Y infected cells might be attributed to the overall low number of infected cells and the absence of a helper virus, which might have increased the number of DI-infected cells. (C) Box and whisker plots depicting progeny virus production and trans-packaging in the absence of core protein and compensatory mutation in the NS2-3 of the helper virus. Packaging of helper viruses was not detected (n.d.). The 50% tissue culture infection dose (TCID50) was determined for the trans-complemented DI (red box). A measurement of mCherry fluorescence after superinfection of the cultures with wtCSFV was used to quantify DI production. Titers were calculated using the Spaerman-Kärber method based on the results of three independent RNA transfections, each analyzed in three dilution series.

Fig 11

SK-6 cells were transfected with RNAs from coreless CSFVs harboring uncleavable NS2-3 and a genomic duplication of NS3 to NS4B. Synthetic RNAs of the CSFV-Ubi-C₁₅₁₂A-Δcore, CSFV-Ubi-C₁₅₁₂A-Δcore-NS2-3-N₂₁₇₇Y, CSFV-Ubi-C₁₅₁₂A-Δcore-NS3-N₂₁₇₇Y, and CSFV-Ubi-C1512A-Δcore-NS2-3-N₂₁₇₇Y-NS3-N₂₁₇₇Y were transfected via electroporation. Following a 24 hour incubation period, the cells were fixed and subjected to immunostaining targeting the E2 protein with DAPI used for nuclei counterstaining. Fluorescence microscopy analysis was conducted at 20-fold magnification accompanied by a 100 µm size marker. An overlay is shown with DAPI displayed in blue and E2 in green. Although CSFV-Ubi-C₁₅₁₂A-Δcore and CSFV-Ubi-C₁₅₁₂A-Δcore-NS2-3-N₂₁₇₇Y exhibited predominantly single transfected cells and doublets, CSFV-Ubi-C₁₅₁₂A-Δcore-NS3-N₂₁₇₇Y and CSFV-Ubi-C₁₅₁₂A-Δcore-NS2-3-N₂₁₇₇Y-3-N₂₁₇₇Y displayed an increased number of positive cells with formation of small foci.

Fig 12

Inoculation of SK-6 cells with cell culture supernatants from coreless CSFVs harboring uncleavable NS2-3 and a genomic duplication of NS3 to NS4B. Following inoculation with 100 µL of cell culture supernatant from CSFV-Ubi-C₁₅₁₂A-Δcore, CSFV-Ubi-C₁₅₁₂A-Δcore-NS2-3-N₂₁₇₇Y, CSFV-Ubi-C₁₅₁₂A-Δcore-NS3-N₂₁₇₇Y, and CSFV-Ubi-C₁₅₁₂A-Δcore-NS2-3-N₂₁₇₇Y-NS3-N₂₁₇₇Y transfected cells, naïve SK-6 cells were incubated for 48 hours. Subsequently, the cells were fixed and subjected to immunostaining for the expression of E2 protein. The resulting images were supplemented with a DAPI counterstain and included a 100 µm size marker. In the overlay, nuclei are highlighted in blue, whereas E2 immunofluorescence is depicted in green. The absence of green staining indicated the failure of CSFV-Ubi-C₁₅₁₂A-Δcore and CSFV-Ubi-C₁₅₁₂A-Δcore-NS2-3-N₂₁₇₇Y to produce progeny virus. Conversely, CSFV-Ubi-C₁₅₁₂A-Δcore-NS3-N₂₁₇₇Y and CSFV-Ubi-C₁₅₁₂A-Δcore-NS2-3-N₂₁₇₇Y-3-N₂₁₇₇Y exhibited focus formation and a cytopathogenic phenotype.

Fig 13

Western blot analyses were conducted on cells transfected or infected with cytopathogenic CSFV-Ubi strains. (A) Expression of E2, evident through strong bands at 80 kDa and additional bands at 55 as well as 100 kDa, was consistently observed post-transfection. However, in infection experiments, E2 expression was solely visible in the case of CSFV-Ubi-C₁₅₁₂A-Δcore-NS3-N₂₁₇₇Y and CSFV-Ubi-C₁₅₁₂A-Δcore-NS2-3-N₂₁₇₇Y-NS3-N₂₁₇₇Y. (B) Simultaneous expression of NS2-3 and NS3, marked by bands at 125 and 80 kDa, was evident after transfection of all coreless CSFV-Ubi clones. However, NS2-3 and NS3 expressions were solely detectable in case of infection with CSFV-Ubi-C₁₅₁₂A-Δcore-NS3-N₂₁₇₇Y and CSFV-Ubi-C₁₅₁₂A-Δcore-NS2-3-N₂₁₇₇Y-NS3-N₂₁₇₇Y. (C) It was possible to detect progeny virus production only in the case of the two genomes with the compensatory mutation in mature NS3 presenting titers that exceeded 1 × 10³ TCID₅₀/mL.

Fig 14

Transfection of SK-6 cells with wtCSFV and DIs of BVDV-1 and Linda pestivirus. Synthetic RNA of wtCSFV and the reporter DIs BVDV-DI9-mCherry and Linda-DI-mCherry were transfected via electroporation. After 24 hours, the cells were fixed and immunostained for the E2 protein. Nuclei were counterstained with DAPI, and fluorescence was captured using a monochromatic camera at 20-fold magnification. To enhance visualization, the images were overlaid with DAPI in blue, mCherry in red, and E2 in green. A size marker of 100 µm length was included in the upper left corner for reference.

Fig 15

Infecting SK-6 cells with cell culture supernatants from wtCSFV and BVDV-DI9-mCherry as well as wtCSFV and Linda-DI-mCherry transfected cells. Naïve SK-6 cells were inoculated with 100 µL of sterile filtered cell culture supernatant harvested 48 hours post-transfection. After a 48 hour incubation period, the cells were fixed and immunostained for the E2 protein, with nuclei counterstained using DAPI. Fluorescence was captured via a monochromatic camera at 20-fold magnification. For enhanced visualization, the images were overlaid, presenting DAPI in blue, mCherry in red, and E2 in green. No mCherry signals were found. A size marker, 100 µm in length, was incorporated in the upper left corner for reference.

Fig 16

Model of genome packaging in wild-type and core-free CSFVs. The viral RNA is replicated from plus-minus strand intermediates in membranous invaginations of the endoplasmic reticulum (ER), likely double-membrane vesicles. The replication machinery consists of the non-structural proteins NS3, NS4A, NS4B, NS5A, and NS5B. NS4B is responsible for the acquisition of membranes, whereas the other non-structural proteins are actively involved in RNA replication, with the RNA-dependent RNA polymerase NS5B playing a crucial role. No differences in RNA replication were observed between core-coding and coreless CSFV genomes. The replicated genomic plus strands are transported outside these compartments and translated at ER membranes. The newly synthesized NS3 or a larger complex containing NS3 maintains contact with the coding RNA molecule (cis-activity). Genome-bound NS3 loads core protein onto the RNA and directs the nucleocapsid to the assembly site, where membrane acquisition occurs at pre-formed pits. Only mutant NS3 (NS3-N₂₁₇₇Y) can envelop the RNA at assembly sites in the absence of the core. Uncleaved NS2-3-4A precursor molecules, which are also essential for the packaging process, are available for packaging genomes, regardless of the N₂₁₇₇Y mutation. The pool of glycoproteins in host cell membranes is also freely available. Finally, the virions are released by cellular exocytosis. Hence, RNA packaging in CSFV is controlled by the mature cis-active NS3.