Yellow Fever Virus, but Not Zika Virus or Dengue Virus, Inhibits T-Cell Receptor-Mediated T-Cell Function by an RNA-Based Mechanism

Abstract

The Flavivirus genus within the Flaviviridae family is comprised of many important human pathogens including yellow fever virus (YFV), dengue virus (DENV), and Zika virus (ZKV), all of which are global public health concerns. Although the related flaviviruses hepatitis C virus and human pegivirus (formerly named GBV-C) interfere with T-cell receptor (TCR) signaling by novel RNA and protein-based mechanisms, the effect of other flaviviruses on TCR signaling is unknown. Here, we studied the effect of YFV, DENV, and ZKV on TCR signaling. Both YFV and ZKV replicated in human T cells in vitro; however, only YFV inhibited TCR signaling. This effect was mediated at least in part by the YFV envelope (env) protein coding RNA. Deletion mutagenesis studies demonstrated that expression of a short, YFV env RNA motif (vsRNA) was required and sufficient to inhibit TCR signaling. Expression of this vsRNA and YFV infection of T cells reduced the expression of a Src-kinase regulatory phosphatase (PTPRE), while ZKV infection did not. YFV infection in mice resulted in impaired TCR signaling and PTPRE expression, with associated reduction in murine response to experimental ovalbumin vaccination. Together, these data suggest that viruses within the flavivirus genus inhibit TCR signaling in a species-dependent manner.

Keywords: PTPRE; T-cell receptor; flavivirus; virus-derived short RNA; yellow fever virus.

Figure 1.

Flavivirus replication and envelope (env) expression in resting and activated T cells. Yellow fever virus (YFV) replication was greater in Jurkat cells lacking Lck, whereas Zika virus (ZKV) and mumps virus replication was similar in Jurkat cells with and without Lck (A) (P < .01 analysis of variance; *P < .01 t test on individual days postinfection). Inhibition of Lck function with Lck inhibitor II prior to infection enhanced YFV replication in Jurkat cells and primary human peripheral blood mononuclear cells (PBMCs) (B) in a dose-dependent manner. Infection of Jurkat cells and PBMCs with YFV reduced interleukin 2 (IL-2) release compared to uninfected cells (mock) following anti-CD3/CD28 whereas ZKV and mumps virus infection did not reduce IL-2 release (C). YFV and mumps virus multiplicity of infection was 0.5 and data represent IL-2 levels in culture supernatant fluids 5 days postinfection. IL-2 release 24 h post–TCR stimulation in Jurkat cell lines stably expressing the plasmid vector control (VC), hepatitis C virus envelope 2 protein (HCV E2), dengue virus serotype 2 envelope (DENV env), Zika virus envelope (ZKV env), and the YFV envelope (YFV env) proteins and frameshift expressing YFV env RNA without protein (D). GE indicates genomic equivalents. *P < .01 compared to control. All data represent averages (± standard error of the mean) of 3 independent experiments.

Figure 2.

Yellow fever virus (YFV) env RNA expression specifically regulates protein tyrosine phosphatase receptor epsilon (PTPRE) expression. YFV Y274 peptide coding sequence aligned with 2 sites on the 3ʹ-UTR of PTPRE (A). PTPRE expression in Jurkat cells expressing YFV env, YFV env with Y274F or Y274A substitutions, or the YFV env coding RNA with a frameshift to abolish protein translation (YFV-FS) (B). PTPRE expression was normalized using actin. The mutations introduced into the Y274 env RNA to generate amino acid mutations are underlined (B). To determine if YFV env RNA targets PTPRE 3ʹ-UTR sequences, HEK 293T cells were transfected with plasmid DNA encoding GFP with either the YFV PTPRE 3ʹUTR target site 1 and site 2 sequence or the HCV PTPRE 3ʹ-UTR site 1 and site 2 sequence. GFP expression in cells transfected with the GFP-PTPRE plasmids alone, or co-transfected with various concentrations of plasmid DNA encoding YFV native env, YFV env frameshift (with no protein translation; YFV-FS) or the Y274A mutant env were examined 72 h posttransfection (C). Data represent the averages (± standard error of the mean) of 3 independent transfection experiments. * P < .01; †P < .05. Abbreviations: FS, frameshift; GFP, green fluorescent protein; HCV, hepatitis C virus; MFI, mean fluorescent intensity; PTPRE, protein tyrosine phosphatase receptor epsilon; VC, vector control; YFV, yellow fever virus.

Figure 3.

Protein tyrosine phosphatase receptor epsilon (PTPRE) is regulated by yellow fever virus (YFV) infection and synthetic YFV RNA sequences, and short YFV RNAs are generated during YFV infection of Jurkat cells. Replication of YFV, Zika virus (ZKV), and mumps virus in HepG2 cells (A). YFV, but not ZKV or mumps virus, regulated PTPRE expression 4 days postinfection (A). Using next-generation sequencing, YFV short RNA sequences were detected 3 days postinfection in Jurkat cells (B) and in Jurkat cells stably expressing YFV env RNA sequences (C). The level of complementarity with PTPRE 3ʹ-UTR target sites 1 and 2 are shown. HEK cells were transfected with synthetic YFV sequence duplex RNAs with the putative seed sequence (underlined) at the 3ʹ end (D) (YFV RNA duplex 1) or the 5ʹ end of the YFV sequence (YFV RNA duplex 2). PTPRE expression was reduced in cells transfected with RNA duplex 1 on days 4 and 5 posttransfection, but not by RNA duplex 2 (D). PTPRE expression was normalized to actin measured on the day of transfection (PTPRE/Actin). Abbreviations: PTPRE, protein tyrosine phosphatase receptor epsilon; TCID₅₀, median tissue culture infective dose; YFV, yellow fever virus; ZKV, Zika virus.

Figure 4.

Protein tyrosine phosphatase receptor epsilon (PTPRE) levels correlate with reduced yellow fever virus (YFV) and increased Zika and mumps virus replication. Huh7 cells express significantly more PTPRE than Huh7D cells (A), and YFV replication was significantly higher in Huh7D cells (B), whereas Zika virus (ZKV) and mumps virus replicated better in Huh7 cells (C). PTPRE expression in HEK 293T cells transfected with PTPRE variant 1 lacking the native 3ʹ-UTR (YFV ΔUTR) compared to the vector control (VC), and YFV replication in the VC and YFV ΔUTR transfected cells (D). Data represent 3 independent infections, and cell viability was equivalent between the different cell types on each day postinfection. P < .01 analysis of variance, Huh7 vs Huh7D for each virus. *P < .01 by t test at each day postinfection compared to Huh7 cells. †P < .01 greater viral replication compared to Huh7D cells. Abbreviations: GE, genomic equivalent; PTPRE, protein tyrosine phosphatase receptor epsilon; VC, vector control; ZKV, Zika virus.

Figure 5.

Yellow fever virus (YFV), but not mumps virus, infection reduces T-cell receptor signaling in murine splenocytes and reduces protein tyrosine phosphatase receptor epsilon (PTPRE) protein expression in splenocytes, draining lymph nodes, and liver tissues. Mice (C57BL/6; n = 5 per group) were infected with 1 × 10⁶ median tissue culture infective dose (TCID₅₀) YFV or mumps virus intraperitoneally (IP), and sacrificed on the indicated days. Splenocytes were stimulated with anti-CD3 and interleukin 2 (IL-2) release measured (A). IL-2 measurements represent the average of 3 technical replicates and each experiment was independently repeated with consistent results. PTPRE levels relative to actin were measured in splenocytes 7 days postinfection (B), draining lymph nodes (C), and liver tissues (D) by immune blot analyses. *P < .01. Abbreviations: IL-2, interleukin 2; PTPRE, protein tyrosine phosphatase receptor epsilon; YFV, yellow fever virus.

Figure 6.

Yellow fever virus (YFV) infection reduces ovalbumin (ova)–specific T-cell cytokines in lymph nodes following ova immunization in vivo. Mice (C57BL/6; n = 5 per group) were either mock-infected, or infected with 1 × 10⁶ median tissue culture infective dose (TCID₅₀) YFV or mumps virus intraperitoneally (IP) and sacrificed on the indicated days. Subsequently these mice were immunized IP with ova in alum 7 and 14 days after YFV or mumps virus infection (n = 5 per group). Animals were killed and lymph node cells prepared and stimulated with ova at the concentrations shown. The concentrations of interferon-γ, interleukin (IL) 2, IL-5, and IL-13 released into culture supernatants were measured 3 days later (A–D). *P < .01 compared to mumps- or mock-infected animals. Abbreviations: IFN, interferon; IL, interleukin; ns, not significant; YFV, yellow fever virus.

Figure 7.

Yellow fever virus (YFV) infection reduces ovalbumin (ova)–specific T-cell cytokines in splenocytes following ova immunization in vivo. Mice (C57BL/6; n = 5 per group) were either mock-infected, or infected with 1 × 10⁶ median tissue culture infective dose (TCID₅₀) YFV or mumps virus intraperitoneally (IP) and sacrificed on the indicated days. Subsequently, these mice were immunized IP with ova in alum 7 and 14 days post-YFV or mumps virus infection (n = 5 per group). Animals were killed and splenocytes prepared and stimulated with ova at the concentrations shown. The concentrations of interferon-γ, interleukin (IL) 2, IL-5, and IL-13 released into culture supernatants were measured 3 days later (A–D). *P < .01 compared to mumps- or mock-infected animals. Abbreviations: IFN, interferon; IL, interleukin; ns, not significant; YFV, yellow fever virus.