Presence of Viral RNA and Proteins in Exosomes from Cellular Clones Resistant to Rift Valley Fever Virus Infection

Abstract

Rift Valley Fever Virus (RVFV) is a RNA virus that belongs to the genus Phlebovirus, family Bunyaviridae. It infects humans and livestock and causes Rift Valley fever. RVFV is considered an agricultural pathogen by the USDA, as it can cause up to 100% abortion in cattle and extensive death of newborns. In addition, it is designated as Category A pathogen by the CDC and the NIAID. In some human cases of RVFV infection, the virus causes fever, ocular damage, liver damage, hemorrhagic fever, and death. There are currently limited options for vaccine candidates, which include the MP-12 and clone 13 versions of RVFV. Viral infections often deregulate multiple cellular pathways that contribute to replication and host pathology. We have previously shown that latent human immunodeficiency virus-1 (HIV-1) and human T-cell lymphotropic virus-1 (HTLV-1) infected cells secrete exosomes that contain short viral RNAs, limited number of genomic RNAs, and viral proteins. These exosomes largely target neighboring cells and activate the NF-κB pathway, leading to cell proliferation, and overall better viral replication. In this manuscript, we studied the effects of exosome formation from RVFV infected cells and their function on recipient cells. We initially infected cells, isolated resistant clones, and further purified using dilution cloning. We then characterized these cells as resistant to new RVFV infection, but sensitive to other viral infections, including Venezuelan Equine Encephalitis Virus (VEEV). These clones contained normal markers (i.e., CD63) for exosomes and were able to activate the TLR pathway in recipient reporter cells. Interestingly, the exosome rich preparations, much like their host cell, contained viral RNA (L, M, and S genome). The RNAs were detected using qRT-PCR in both parental and exosomal preparations as well as in CD63 immunoprecipitates. Viral proteins such as N and a modified form of NSs were present in some of these exosomes. Finally, treatment of recipient cells (T-cells and monocytic cells) showed drastic rate of apoptosis through PARP cleavage and caspase 3 activation from some but not all exosome enriched preparations. Collectively, these data suggest that exosomes from RVFV infected cells alter the dynamics of the immune cells and may contribute to pathology of the viral infection.

Keywords: Rift Valley Fever Virus; exosomes; resistant clones.

FIGURE 1

Generation of Rift infected resistant clones. (A) Vero cells were infected with RVFV at MOI 3. Approximately 1% of cells survived the infection and isolated using trypsin diluted with PBS. Clones were plated and passaged 50 times before characterization. (B) The HEK-293T based reporter cell line, HEK-Blue hTLR3 (InvivoGen), was used to detect activation of TLR3 by supernatant of RVFV resistant Vero cells. After 18 h of incubation at 37°C in 5% CO₂, the absorbance (600 nm) of each control and test condition in the 96-well plate was measured using the GloMax Multi Detection System (Promega). Readings from all positive controls (sample 2, 10 ng/mL Poly I/C) and experimental samples were normalized using the mean reading from three sterile water treated negative controls. Seven clones (as indicated) were selected for follow up experiments. Error bars on the first two samples (negative and positive controls) indicated ±1 SD of biological triplicates.

FIGURE 2

Presence of RVFV genome in resistant clones. (A) Total RNA was extracted from seven RVFV resistant Vero cells using the trizol-chloroform method. For each sample, approximately 400 ng/μl of RNA was used for cDNA synthesis with GoScript Reverse Transcription System using Random Primers. The absolute quantification of the samples was determined based on the cycle threshold (Ct) value relative to the standard curve. (B) qRT-PCR analysis of resistant clones 10 days after secondary infection with RVFV at MOI 1.

FIGURE 3

Presence of RVFV genomic RNA in exosomes. (A) Vero cells were infected with double tagged RVFV for recovery of resistant clones as described in Figure 1. Resistant clones were isolated and screened on TLR3 indicator cells. (B) qRT-PCR of second generation clones was used to detect presence of RVFV genome in cell lysates. The analysis was performed as described in Figure 2A. (C) Exosome-enriched preparations were isolated from first and second generation clones via differential ultracentrifugation method. Total RNA was extracted to test for the presence of RVFV genome. (D) Exosomes were isolated from first and second generation clones through co-immunoprecipitation of cell supernatants using CD63 Dynabeads. Total RNA was extracted to test for presence of RVFV genome.

FIGURE 4

Effects of exosomes on recipient cells. (A) Approximately 50,000–100,000 cells in 50 μl exosome free media were plated in a 96 well plate. Fifty microliters of supernatant from resistant clones (A8, C6, E5, E12, and H6) and Vero cells were centrifuged 10 min at 14,000 × g. Then 50 μl of supernatants were added to cells and allowed to incubate at 37°C for 5 days. Viability was subsequently assayed using CellTiter-Glo assay and values for exosome-free DMEM (control) were used to subtract background. Samples treated with supernatant from uninfected Vero cells were set to 100% and used to normalize the experimental values. The assay was conducted using biological triplicates and error bars indicate ±1 SD. (B) Exosomes were isolated through differential ultracentrifugation and 2 μg was added to 50,000–100,000 cells in a 96 well plate. Cells were allowed to incubate at 37°C for 5 days and viability was subsequently assayed using CellTiter-Glo assay. Again, values for exosome-free DMEM (control) were used to subtract background and samples treated with exosomes from uninfected Vero cells were set to 100% and used to normalize the experimental values. The assay was conducted using biological triplicates and error bars indicate ±1 SD. (C) Approximately 100,000 mid-log Vero cells, H6 or clone #14 were treated with either RVFV or VEEV (TC83) at MOI = 1 and incubated at 37°C for 5 days. Samples were processed for cell viability using CellTiter-Glo in a microtiter plate. Triplicate samples were treated with either of the two viruses. ^∗p < 0.05.

FIGURE 5

Presence of viral proteins in exosomes. (A) Enriched exosomes were isolated through differential centrifugation and were analyzed by Western Blotting with antibodies to N protein (generous gift from Dr. Connie Schmaljohn; 1:500), CD63 (ab8219 1:500), Alix (sc-49268 1:150), and Actin (ab49900 1:5000). (B) Exosome-containing preparations obtained from resistant clones and corresponding WCEs were analyzed by Western Blots with antibodies to NSs Flag (sc-807 1:200) and Ubiquitin (1:500). (C) Three hundred and fifty microgram of purified exosomes was used for lysis, using Freeze-Thaw and TNE 50 + 0.1% NP-40 and immunoprecipitation using either IgG or anti-flag antibody (10 μg each). Samples were IPed overnight; protein A + G beads added (30% slurry) the next day for 2 h, centrifuged and washed. Bound samples were run on a gel for western blot using anti-ubiquitin antibody (1:500). Black circles indicate potential Ubiquitinated NSs proteins present in the exosomes.

FIGURE 6

Effect of exosomes on the recipient cell apoptosis machinery. (A) Jurkat cells treated with exosome-enriched preparations from resistant clones were analyzed by Western blotting with antibodies to markers of apoptosis, which included Caspase 3 (sc-7148 1:200), PARP (sc-7150 1:200), and PKR (sc-707 1:200), or Actin (ab49900 1:5000). (B) U937 cells were treated similar to (A) and extracts were used for western blot analysis probing for the same apoptosis markers.

FIGURE 7

Effect of resistant clone supernatants on Vero cells. (A) Supernatants (1 ml) from clones H6 and #14 were added to Vero cells (2.5 × 10⁶/ml in complete media in a 24 well plate; ∼35% confluency) overnight, washed 24 h later and supplemented with complete media. Each week, supernatants (1 ml) were collected and concentrated using mixture of nanoparticles NT080 + NT082 (to concentrate exosomes; 50 μl of 30% slurry) and NT086 (to concentrate potential virus; 50 μl of 30% slurry) overnight at 4°C. Samples were pelleted the next day, washed, and total RNA was isolated for qRT/PCR. “S0” denotes the starting material and “S1–3” was supernatants from treated Vero cells in weeks 1–3. (B) Similar to (A), except that the total RNA was isolated from the cell pellets prior to qRT/PCR for Rift RNAs. GAPDH served as internal control for RNA expression. (C) Total RNA was isolated from Jurkat and U937 cells treated with exosome-containing preparations from Vero or resistant clone #14 after 2 and 4 days. qRT-PCR analysis were conducted with primers specific for NSs.

FIGURE 8

Effect of RVFV infection in immune cells. (A) Immune cells including T-cells (Jurkat) and monocytes (U937) were grown to log phase of growth in complete media (1.5 × 10⁶ cells/ml). RVFV (epitope-tagged MP12) at MOI of 0.1 and 1.0 were used for infection of immune cells and Vero cells as a positive control. Cultures were incubated for 5 days as 37°C (without removing the virus) and cell viability were assayed using CellTiter-Glo. Results in (A) are from three independent experiments. Similar results were also observed with the wild type un-tagged MP12 (data not shown). (B) Similar to (A) except cells infected at MOI of 1 after 5 days were used for cell morphology using light microscopy.