Highly Pathogenic New World and Old World Human Arenaviruses Induce Distinct Interferon Responses in Human Cells

Abstract

The arenavirus family includes several important pathogens that cause severe and sometimes fatal diseases in humans. The highly pathogenic Old World (OW) arenavirus Lassa fever virus (LASV) is the causative agent of Lassa fever (LF) disease in humans. LASV infections in severe cases are generally immunosuppressive without stimulating interferon (IFN) induction, a proinflammatory response, or T cell activation. However, the host innate immune responses to highly pathogenic New World (NW) arenaviruses are not well understood. We have previously shown that the highly pathogenic NW arenavirus, Junin virus (JUNV), induced an IFN response in human A549 cells. Here, we report that Machupo virus (MACV), another highly pathogenic NW arenavirus, also induces an IFN response. Importantly, both pathogenic NW arenaviruses, in contrast to the OW highly pathogenic arenavirus LASV, readily elicited an IFN response in human primary dendritic cells and A549 cells. Coinfection experiments revealed that LASV could potently inhibit MACV-activated IFN responses even at 6 h after MACV infection, while the replication levels of MACV and LASV were not affected by virus coinfection. Our results clearly demonstrated that although all viruses studied herein are highly pathogenic to humans, the host IFN responses toward infections with the NW arenaviruses JUNV and MACV are quite different from responses to infections with the OW arenavirus LASV, a discovery that needs to be further investigated in relevant animal models. This finding might help us better understand various interplays between the host immune system and highly pathogenic arenaviruses as well as distinct mechanisms underlying viral pathogenesis.

Importance: Infections of humans with the highly pathogenic OW LASV are accompanied by potent suppression of interferon or proinflammatory cytokine production. In contrast, infections with the highly pathogenic NW arenavirus JUNV are associated with high levels of IFNs and cytokines in severe and fatal cases. Arenaviruses initially target macrophages and dendritic cells, which are potent IFN/cytokine-producers. In human macrophages, JUNV reportedly does not trigger IFN responses. We here demonstrated that JUNV activated IFN responses in human dendritic cells. MACV, another highly pathogenic NW arenavirus, also activated IFN responses. LASV did not induce detectable IFN responses, in spite of higher replication levels, and blocked the MACV-triggered IFN response in a coinfection assay. Although these viruses are highly pathogenic to humans, our study highlights distinct innate immune responses to infections with the NW arenaviruses JUNV and MACV and to infection with the OW arenavirus LASV and provides important insights into the virus-host interaction and pathogenesis.

FIG 1

Activation of the IFN pathway by NW arenavirus MACV infection in human A549 cells. Human A549 cells were mock infected or infected with JUNV or MACV at an MOI of 1. Cell lysates were prepared daily from day 1 to day 3 after infection and subjected to Western blotting for phosphorylated STAT1 (p-STAT1), STAT1 protein, ISG15, RIG-I, JUNV and MACV NP protein, and human β-actin. The mouse monoclonal antibody (NA05-AG12; BEI Resources) detects the NPs of JUNV and MACV.

FIG 2

Interferon expression in human dendritic cells infected by the highly pathogenic NW arenaviruses JUNV and MACV. Human monocyte-derived dendritic cells (MoDCs) were mock infected or infected with the NW JUNV, NW MACV, or OW LASV at an MOI of 1. (A) At 0, 1, 2, and 3 days postinfection (dpi), intracellular RNA was purified and analyzed by real-time RT-qPCR to quantitate IFN-β mRNA expression. C_T values of the targeting genes were normalized to the average C_T values of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and β-actin housekeeping genes. Data are expressed as fold change of normalized IFN-β mRNA levels relative to the level of the mock-infected samples at 0 dpi. (B and C) Supernatants were harvested from virus-infected MoDCs and analyzed for IFN-β protein and IFN-α protein expression by ELISAs. Data are presented as the mean values ± SEM derived from three healthy donors in three independent experiments. Student's t tests were performed for statistical analysis (*, P < 0.01; **, P < 0.05).

FIG 3

Upregulation of interferon-stimulated gene products in NW JUNV- and MACV-infected MoDCs. Human MoDCs were mock infected or infected with the NW JUNV, NW MACV, or OW LASV at an MOI of 1. (A) At 1, 2, and 3 dpi, cellular protein lysates were prepared and subjected to Western blotting for phosphorylated STAT1 (p-STAT1), STAT1 protein, ISG15, JUNV and MACV (JUNV/MACV) NP protein, LASV NP, and human β-actin. (B) Virus titers in the supernatants of infected MoDCs were determined at the indicated time points. Data are presented as the mean values ± SEM.

FIG 4

The highly pathogenic NW arenaviruses JUNV and MACV but not the OW LASV induced type I interferon expression in A549 cells. Human A549 cells were mock infected or infected with JUNV, MACV, or LASV at an MOI of 1. (A) Virus titers were determined at the indicated time points. (B and C) Real-time RT-qPCR analysis was performed to quantitate the expression levels of IFN-β mRNA and ISG15 mRNA. The mRNA expression levels of targeting genes were normalized to the levels of housekeeping genes GAPDH and β-actin, and values are expressed as fold change relative to the mRNA levels of the corresponding genes in mock-infected samples at 0 dpi. (D) IFN-α protein expression levels in the supernatants from infected cells were measured by ELISA. Data are presented as the means ± SEM of three experiments.

FIG 5

ISG expression in MACV- and LASV-infected A549 cells. Human A549 cells were mock infected or infected with the highly pathogenic NW arenavirus MACV or the OW arenavirus LASV at an MOI of 1. At 1, 2, and 3 dpi, cellular protein lysates were prepared and subjected to Western blotting for phosphorylated STAT1 (p-STAT1), STAT1 protein, ISG15, RIG-I, MACV NP protein, LASV NP, and human β-actin. As a control, A549 cells were treated with human IFN-α for 16 h.

FIG 6

OW LASV coinfection eliminated the NW MACV-activated IFN response. A549 cells were infected with the NW MACV or OW LASV at MOI of 1. (A) Coinfection studies were performed by LASV infection of MACV-infected cells at 1 h before (T₋₁) MACV infection, at the same time (T₀) as MACV infection, or at 1 h after (T₊₁) or 3 h after (T₊₃) MACV infection. At 40 h post-MACV infection, IFN-β protein levels in the supernatant were measured by ELISA. Data are presented as the means ± SEM of three experiments. (B) WB assays were performed to detect phosphorylated STAT1 (p-STAT1), STAT1 protein, ISG15, RIG-I, MACV NP protein, LASV NP, and human β-actin. (C) LASV (MOI of 1) was added to MACV-infected cells at the same time (T₀) as MACV infection or at 6 h after (T₊₆) or 16 h after (T₊₁₆) MACV infection. At 40 h post-MACV infection, IFN-β protein in supernatants was measured by ELISA. Data are presented as the means ± SEM of three experiments. (D) A WB assay was performed to detect MACV NP protein, LASV NP, and human β-actin in T₊₆ and T₊₁₆ samples. Note that the monoclonal antibody (NA05-AG12) used to detect MACV NP did not react to LASV NP, while the mouse ascitic fluid against LASV weakly cross-reacted to MACV NP.