YBX1 is required for assembly of viral replication complexes of chikungunya virus and replication of multiple alphaviruses

Abstract

Chikungunya virus (CHIKV), an enveloped positive-sense RNA virus, is a member of the alphaviruses and cause fever and arthralgia in humans. We performed genome-wide CRISPR/Cas9-based screens and identified Y-box binding protein 1 (YBX1) as an essential cellular factor for CHIKV. Deficiency of YBX1 inhibited CHIKV RNA replication and impaired virus production. Upon CHIKV infection, YBX1 showed a striking re-localization to viral replication complexes (vRCs), where it co-localized with CHIKV nsP3 and dsRNA intermediates. YBX1 directly interacted with CHIKV nsP3, and mutation of the YBX1-binding motif in CHIKV nsP3 suppressed viral replication in host cells. Furthermore, YBX1 bound to viral RNA and increased the viral RNA-binding activity of CHIKV nsP3. Consistently, the RNA-binding activity of YBX1, as well as the ability of nsP3 to bind to YBX1, was required for efficient CHIKV replication. In addition to CHIKV, YBX1 was also essential for replication of all examined alphaviruses including the prototypic alphavirus. Our findings suggest that YBX1 acts as a scaffold for assembly of chikungunya vRCs and an important factor for replication of multiple alphaviruses, which may serve as a potential target for the development of anti-alphavirus therapies.IMPORTANCEAlphaviruses are a group of mosquito-transmitted, enveloped, positive-strand RNA viruses in the Togaviridae family. Most alphaviruses are important pathogens that continue to cause human disease ranging from severe and potentially fatal neurological disease to chronic arthritic disease on a global scale. Here, we found that YBX1 promotes binding of CHIKV genomic RNA to nsP3, which is a key component of the replication complex, and is therefore pivotal for CHIKV replication. Deficiency of YBX1 results in reduced replication of multiple alphaviruses, including arthritogenic and encephalitic alphaviruses. These findings suggest that YBX1 is an important cellular factor for multiple alphaviruses and a potential target for preventing alphavirus infections.

Keywords: YBX1; alphaviruses; cellular factor; viral replication.

Fig 1

Identification of YBX1 as an important host factor for CHIKV/SINV by genome-wide CRISPR/Cas9 screens. (A) A schematic diagram of the genome-wide CRISPR/Cas9 screen process. (B) Statistical chart of the screen results of CHIKV and SINV infection. The top 20 candidates ranked by the MAGeCK score are shown in the table. (C–G) Effects of YBX1 deficiency on CHIKV or SINV infection in various cells. Huh7 (C), SW13 (D), HeLa (E), HepG2 (F), and HEK293T (G) cells were edited with a control gRNA or the indicated numbers of gRNAs targeting the YBX1 gene. Cells were infected with CHIKV (multiplicity of infection [MOI] = 1) or SINV (MOI = 1) for 12 h before reverse transcription quantitative PCR was performed. Data are normalized to the CHIKV RNA level or SINV RNA level in the control gRNA-edited cells. Data are represented as mean ± SD. *P < 0.05, **P < 0.01, ***P < 0.001.

Fig 2

YBX1 is required for the efficient infection of multiple alphaviruses. (A and B) CHIKV infectivity in YBX1-knockout HeLa cells. Single clones of YBX1-knockout HeLa cells were isolated and confirmed by immunoblots (A, left panel). The control (Con) or YBX1-deficient (YBX1-KO) clones were infected with CHIKV (MOI = 1) for the indicated times. RNA level of CHIKV (A, right panel; 12 hpi) and CHIKV protein expression (B; 12, 24, and 36 hpi) were measured by RT-qPCR and immunoblots, respectively. (C and D) Reconstitution of YBX1-deficient cells with YBX1 restores CHIKV infection. The Con or YBX1-KO HeLa cells reconstituted with empty vector or YBX1-Flag were infected with CHIKV (MOI = 1) for the indicated times. The expression of YBX1, RNA level of CHIKV, and production of CHIKV progeny viruses were measured by immunoblots (C, left panel), RT-qPCR (C, right panel), and plaque assay (D), respectively. (E) Effects of YBX1 deficiency on CHIKV-KC488650, CHIKV-MF740874, SINV, SFV, VEEV, ONNV, EMCV, and HSV-1 infection. The Con or YBX1-KO HeLa cells were inoculated with the indicated viruses at an MOI of 1 for 12 h (SINV, SFV, VEEV, ONNV, EMCV, and HSV-1) or 24 h (CHIKV-KC488650 and CHIKV-MF740874) before RT-qPCR was performed. Data are normalized to the viral RNA level in the control cells. Data are represented as mean ± SD. *P < 0.05, **P < 0.01, ***P < 0.001. ns, not significant.

Fig 3

YBX1 is essential for viral RNA replication. (A) Effects of YBX1 on CHIKV binding and internalization. The control (Con) or YBX1-deficient (YBX1-KO) HeLa cells were incubated with CHIKV (MOI = 10) on ice (binding) or at 37°C (internalization) as described in Materials and Methods. The CHIKV RNA levels were measured by RT-qPCR. Data are normalized to the CHIKV RNA level in the control cells. (B and C) Effects of YBX1 deficiency on CHIKV total RNA or (−) RNA levels produced during virus replication. The Con or YBX1-KO HeLa cells were infected with CHIKV (MOI = 1) for the indicated times. The cells were collected, and CHIKV total RNA (B) or (−) RNA (C) level was measured by RT-qPCR. (D) Effects of YBX1 deficiency on CHIKV dsRNA intermediates produced during virus replication. The Con or YBX1-KO HeLa cells were infected with CHIKV (MOI = 50) for 16 h and then stained with anti-dsRNA J2 monoclonal antibody (left). The statistical results of positive cells are shown on the right. Scale bars, 10 µm. (E) Effects of YBX1 on CHIKV translation and replication. The Con or YBX1-KO HEK293T cells were transfected with in vitro transcribed wild-type (GDD, CHIKV-nsP3-Fluc) or replication-deficient (GAA, CHIKV-nsP3-Fluc-nsP4^GAA) RNA. The firefly luciferase activity was assessed at the indicated times. (F) Effects of YBX1 on CHIKV-induced formation of plasma membrane (PM)-associated spherules (S). The Con or YBX1-KO HEK293T cells were incubated with CHIKV-181/25 (MOI of 50) for 18 h before transmission electron microscopy analyses. Replication S, together with viral particles (V) at the PM. Scale bars, 500 nm. (G) YBX1 efficiently accumulates in large cytoplasmic CHIKV nsP3-containing complexes. The Con or YBX1-KO HEK293T cells were incubated with CHIKV-181/25-nsP3-GFP (MOI of 50) for the indicated times, then fixed with 4% paraformaldehyde and stained with indicated antibodies before confocal microscopy. The areas in the dashed boxes on the left are enlarged on the right. Images are representative of three experiments. Scale bars, 5 µm. Data are represented as mean ± SD. *P < 0.05, ***P < 0.001. ns, not significant.

Fig 4

YBX1 directly interacts with CHIKV nsP3 through CSD and C-terminal domain (CTD) of YBX1 and HVD of CHIKV nsP3. (A) YBX1 interacts with CHIKV nsPs in the mammalian overexpression system. HEK293T cells were transfected with indicated plasmids for 24 h. Co-immunoprecipitation and immunoblot analysis were performed with the indicated antibodies. (B) Direct interaction between YBX1 and CHIKV nsP3. Purified His-YBX1 and GST-nsP3 were incubated as indicated. Pull-down was performed with Ni magnetic beads, and the proteins present in the pellet were denatured and subjected to SDS-PAGE and immunoblotting with the indicated antibodies. (C and D) Domain mapping of YBX1-nsP3 interaction. HEK293T cells were transfected with the indicated plasmids for 24 h before co-immunoprecipitation and immunoblot analysis with the indicated antibodies. The schematic representations of YBX1 and nsP3 truncations are shown on the left.

Fig 5

Mutations in the YBX1-binding site of nsP3 HVD attenuate CHIKV replication. (A) A schematic diagram of Flag-HVD cassettes applied for mapping of YBX1-binding sites. (B) YBX1-binding sites are located in the D fragment of the CHIKV HVD. HEK293T cells were transfected with the indicated plasmids for 24 h. Co-immunoprecipitation and immunoblot analysis were performed with the indicated antibodies. The schematic representations of YBX1 mutations are shown at the top. (C) The peptide located between aa 487 and 492 of CHIKV HVD is a potential YBX1-binding site. The experiments were similarly performed as in panel B except that different YBX1 mutants were used. (D) The four conserved motifs of CHIKV nsP3 HVD are not responsible for interaction with YBX1. The experiments were similarly performed as in panel B except that different YBX1 mutants were used. (E and F) Deletion of the YBX1-binding site in the CHIKV genome attenuates CHIKV replication. BHK-21 cells were transfected with CHIKV and CHIKV-Δ6 RNA, respectively. The supernatants were harvested at the indicated times. Production of progeny viruses was measured by plaque assay (E). The representative plaques were photographed and recorded (F).

Fig 6

RNA-binding activity of YBX1 is required for efficient virus replication. (A) The CSD and CTD domains of YBX1 are important for CHIKV replication. YBX1-deficient (YBX1-KO) HeLa cells reconstituted with empty vector, YBX1-Flag, or the indicated YBX1 truncations were inoculated with CHIKV (MOI = 1) for 12 h before RT-qPCR analysis. The structural schematic representations of YBX1 are shown at the top. (B) RNA-binding ability of YBX1 is required for infection of CHIKV and SINV. YBX1-KO HeLa cells reconstituted with empty vector, YBX1-Flag, or the indicated YBX1 mutations were inoculated with CHIKV (MOI = 1) or SINV (MOI = 1) for 12 h before RT-qPCR analysis. 4A, Y72A/F74A/F85A/ H87A. For bar graphs, data are normalized to that of the vector-reconstituted cells. (C) RNA-binding ability of YBX1 is required for interaction with CHIKV nsP3. HEK293T cells were transfected with the indicated plasmids for 24 h. Co-immunoprecipitation and immunoblot analysis were performed with the indicated antibodies. (D) YBX1 promotes the binding of CHIKV nsP3 to CHIKV RNA. Purified His-YBX1, GST-nsP3, and biotin-labeled CHIKV RNA were incubated as indicated. Pull-down was performed with streptavidin magnetic beads, and the proteins present in the pellet were denatured and subjected to SDS-PAGE and immunoblotting with the indicated antibodies. Data are represented as mean ± SD. ***P < 0.001. ns, not significant.

Fig 7

The replication of CHIKV is independent of m5C machinery. (A and B) HFF cells were transfected with the indicated siRNAs for 48 h and then infected with CHIKV-181/25 (MOI = 1) for 12 h before RT-qPCR analysis. Data are normalized to that of control siRNA-transfected cells. Data are represented as mean ± SD. *P < 0.05, ***P < 0.001. ns, not significant.