Evaluating the Biological Role of Lassa Viral Z Protein-Mediated RIG-I Inhibition Using a Replication-Competent Trisegmented Pichinde Virus System in an Inducible RIG-IN Expression Cell Line

Abstract

Lassa virus (LASV) is a mammarenavirus that can cause lethal Lassa fever disease with no FDA-approved vaccine and limited treatment options. Fatal LASV infections are associated with innate immune suppression. We have previously shown that the small matrix Z protein of LASV, but not of a nonpathogenic arenavirus Pichinde virus (PICV), can inhibit the cellular RIG-I-like receptors (RLRs), but its biological significance has not been evaluated in an infectious virus due to the multiple essential functions of the Z protein required for the viral life cycle. In this study, we developed a stable HeLa cell line (HeLa-iRIGN) that could be rapidly and robustly induced by doxycycline (Dox) treatment to express RIG-I N-terminal effector, with concomitant production of type I interferons (IFN-Is). We also generated recombinant tri-segmented PICVs, rP18tri-LZ, and rP18tri-PZ, which encode LASV Z and PICV Z, respectively, as an extra mScarlet fusion protein that is nonessential for the viral life cycle. Upon infection, rP18tri-LZ consistently expressed viral genes at a higher level than rP18tri-PZ. rP18tri-LZ also showed a higher level of a viral infection than rP18tri-PZ did in HeLa-iRIGN cells, especially upon Dox induction. The heterologous Z gene did not alter viral growth in Vero and A549 cells by growth curve analysis, while LASV Z strongly increased and prolonged viral gene expression, especially in IFN-competent A549 cells. Our study provides important insights into the biological role of LASV Z-mediated RIG-I inhibition and implicates LASV Z as a potential virulence factor. IMPORTANCE Lassa virus (LASV) can cause lethal hemorrhagic fever disease in humans but other arenaviruses, such as Pichinde virus (PICV), do not cause obvious disease. We have previously shown that the Z protein of LASV but not of PICV can inhibit RIG-I, a cytosolic innate immune receptor. In this study, we developed a stable HeLa cell line that can be induced to express the RIG-I N-terminal effector domain, which allows for timely control of RIG-I activation. We also generated recombinant PICVs encoding LASV Z or PICV Z as an extra gene that is nonessential for the viral life cycle. Compared to PICV Z, LASV Z could increase viral gene expression and viral infection in an infectious arenavirus system, especially when RIG-I signaling is activated. Our study presented a convenient cell system to characterize RIG-I signaling and its antagonists and revealed LASV Z as a possible virulence factor and a potential antiviral target.

Keywords: Lassa; Lassa virus; Pichinde; Pichinde virus; RIG-I; RIG-I signaling; Z protein; arenavirus; inducible expression; innate immune evasion; innate immunity; recombinant viruses; tri-segmented arenavirus; viral virulence.

FIG 1

Construction of an inducible stable cell line HeLa-iRIGN. (A) A schematic diagram of the PiggyBac system used to generate HeLa-iRIGN stable cells inducibly expressing RIG-IN-GFP fusion protein. The cargo plasmid contains 5′ and 3′ ITRs, HS4 insulator sequences, the rtTA gene, a puromycin resistance gene, and the RIG-IN-GFP gene under an inducible promoter TRE that is transcriptionally activated by Dox. ITR, inverted terminal repeats. rtTA, reverse tetracycline-controlled transactivator. TRE, tetracycline responsive elements. Dox, doxycycline. Puro R, puromycin resistance gene. Pol II, DNA-dependent RNA polymerase II. (B) Expression of the RIG-IN-GFP fusion protein in HeLa-iRIGN cells induced with Dox at different concentrations (0 to 2 μg/mL) for various periods (0 to 12 h) was shown by fluorescence microscopy. (C) HeLa and HeLa-iRIGN cells after Dox (2 μg/mL) induction for various periods were analyzed by Western blotting with anti-actin and anti-GFP antibodies.

FIG 2

Secretion of IFNs from Dox-induced HeLa-iRIGN cells. (A) A schematic diagram of the NDV-GFP-based biological assay to quantify the level of IFNs in supernatants collected from Dox-induced HeLa-iRIGN cells. Vero cells were pretreated with supernatants and infected with the NDV-GFP virus. Because IFN-I effectively inhibited NDV-GFP infection, the level of GFP expression in the infected Vero cells was reversely correlated with the level of IFNs in the supernatants. (B) The levels of IFNs secreted from HeLa-iRIGN cells induced by various concentrations of Dox for various periods were evaluated by an NDV-GFP-based biological assay. The GFP images of the NDV-GFP-infected Vero cells are shown.

FIG 3

LASV but not PICV Z protein can inhibit RIG-IN expressed in Dox-induced HeLa-iRIGN cells. HeLa-iRIGN cells were transfected with 100 ng of β-gal plasmid, 200 ng of the IFN-β-promoter-directed LUC reporter plasmid (IFN-β-LUC), together with different amounts (0 to 300 ng) of pCAGGS-LASV-Z-HA (A and B) or pCAGGS-PICV-Z-HA (C and D) followed by Dox induction at the indicated concentrations. Cell lysates were subjected to β-gal and firefly LUC assays. The relative LUC activity, normalized by β-gal activity, is shown for LASV Z (A) and PICV Z (C). Statistical analysis was conducted with Student's t test. ns, not significant; *, P < 0.05; **, P < 0.01; ***, P < 0.001. The same cell lysates from LASV Z (B) and PICV Z (D) transfections were analyzed by Western blotting with anti-GFP, anti-HA, and anti-actin antibodies, respectively.

FIG 4

Genome organization of tri-segmented rP18tri viruses rP18tri-LZ (A) and rP18tri-PZ (B) encoding LASV and PICV Z gene in fusion with mScarlet. Each viral genome consists of three viral RNA segments. The P18 L segment encodes the Z gene and the L gene of PICV (P18 strain) in opposite orientations. The P18 S1 segment encodes the PICV GPC gene in positive orientation and a multiple-cloning-site (MCS) sequence in place of the NP gene. The P18 S2 segment encodes the PICV NP gene in negative orientation and a Z-mScarlet fusion gene in positive orientation.

FIG 5

rP18tri-LZ virus showed a higher level of viral infection and protein expression than a rP18tri-PZ virus in HeLa-iRIGN cells. (A) HeLa and HeLa-iRIGN cells were infected with rP18tri-LZ or rP18tri-PZ, with or without Dox induction. Representative red fluorescence (Z-mScarlet) images at 48 hpi are shown (top three rows). The overlay of red (Z-mScarlet) and green (RIG-IN-GFP) fluorescence images are shown for the virus-infected Dox-treated HeLa-iRIGN cells (bottom row). (B) Expression of RIG-IN-GFP in HeLa-iRIGN cells with or without Dox induction was analyzed by flow cytometry. FITC-positive cells are gated (left), with the average MFI (FITC) of triplicates shown (right). (C) Expression of Z-mScarlet in virus-infected HeLa and HeLa-iRIGN cells with or without Dox induction was analyzed by flow cytometry. PE-positive cells are gated. (D) The PE-positive cells were quantified by the percentage (%) of parental cells (left) and MFI (right) from triplicates. ns, not significant; *, P < 0.05; **, P < 0.01; ***, P < 0.001.

FIG 6

rP18tri-LZ showed a higher level of viral RNA synthesis than rP18tri-PZ in the infected cells by qRT-PCR. (A) BHK-21, HeLa, and Dox-induced HeLa-iRIGN cells were infected with rP18tri-LZ or rP18tri-PZ at an MOI of 0.01 for 72 h. The level of PICV (P18 strain) NP gene copy numbers at different times postinfection were quantified by qRT-PCR and shown as the average of triplicates. (B) HeLa-iRIGN cells were mock-infected or infected with rP18tri vector, rP18tri-LZ, or rP18tri-PZ at an MOI of 0.01, and induced with Dox. Total RNAs were extracted at 48 hpi and analyzed by qRT-PCR with primers specific for mScarlet and PICV NP. The gene copy number of mScarlet and PICV NP is shown as the average of triplicates. M, mock infection. V, rP18tri vector. LZ, rP18tri-LZ. PZ, rP18tri-PZ. ns, not significant; *, P < 0.05; **, P < 0.01; ***, P < 0.001.

FIG 7

Additional Z gene in the context of infectious PICV system did not alter viral growth while LASV Z protein can increase and prolong viral gene expression. Vero and A549 cells were infected with respective rP18tri viruses, vector alone (V), rP18tri-LZ (LZ), or rP18tri-PZ (PZ), at MOI of 0.01 for up to 96 h. (A) The viral growth curve in Vero and A549 cells. Viral titers in the supernatants at different time points were quantified by plaque assay and compared among the groups by one-way ANOVA. ns, not statistically significant (P > 0.05). (B) Z-mScarlet expression in virus-infected Vero and A549 cells at different time points for respective viruses.