Table 1.
Oligonucleotides used for cloning.
Table 2.
Oligonucleotides used for cloning.
Fig 1.
Morphology of rhesus macaque cell lines.
Cells were seeded in 6-well plates and bright field images taken at 10x magnification. White scale bars indicate 50 μm.
Fig 2.
Characterization of marker expression in rhesus macaque cell lines.
(A) Lysates of the indicated cell lines were analyzed by immunoblot for expression of the immortalization gene large T. Human cell lines 293T and A549 served as positive and negative controls, respectively, for large T expression. Detection of β-actin (ACTB) in the same lysates on a separate blot served as expression control. Similar results were obtained in a separate experiment. (B) Cell lines transduced with a mix of immortalization genes were analyzed by PCR for the presence of integrated transducing vector by a combination of vector specific and gene specific primers. Detection of a transduced gene is indicated by black boxes, while white boxes indicate absence.
Fig 3.
Transcriptome sequencing and cell type deconvolution.
(A) Workflow used for transcriptome sequencing and deconvolution analysis. Bulk sequencing profiles were processed to a gene expression matrix, and relevant cell types were deconvoluted by reference to a scRNAseq dataset from rhesus macaque brain tissue (GSE127774). (Created with BioRender.com). (B) Reanalysis of reference scRNAseq data recognized major CNS cell populations and was used for downstream deconvolution analysis. (C) and (D) Stack barplots show putative proportions of different cell types in deconvoluted bulk RNA sequencing experiments for Co2880-gr (C) and Co2880-lT (D) cells.
Fig 4.
Expression of the astrocyte marker GFAP in rhesus macaque cell lines.
(A) Lysates of immortalized cell lines were analyzed by immunoblot for expression of glial fibrillary acidic protein (GFAP). Detection of β-actin (ACTB) in the same lysates on a separate blot served as expression control. Similar results were obtained in a separate experiment. (B) Immunofluorescence staining of immortalized cell lines with an antibody against GFAP (green). Nuclei were stained with DAPI (blue). Scale bar indicates 100 μm. Similar results were obtained in a separate experiment. (C) Lysates of immortalized cell lines were analyzed by immunoblot for expression of oligodendrocyte (Olig1) or inhibitory neuron (GAD2, GABBR2) markers. As control, rhesus genes fused to scarlet fluorescent protein were expressed in transfected 293T cells. Expected molecular weights for the respective proteins are indicated by arrows. Detection of β-actin (ACTB) in the same lysates on a separate blot served as expression control. Similar results were obtained in a separate experiment.
Fig 5.
Evidence that the rhesus macaque cell lines have a functional interferon system.
Cells seeded in 12-well plates were either treated with 100 U/mL pan-IFN or infected with VSV ncp* (MOI 0.1). Untreated cells served as control. Cells were harvested after 24 h for RNA isolation. Expression of (A) interferon beta (IFNB1) or (B) MX1 was analyzed by quantitative RT-PCR. Transcript levels were normalized against 18S rRNA transcript levels and expression fold change was calculated with respect to control cells. The results of a representative experiment carried out with technical triplicates are shown. Error bars indicate standard deviation. Similar results were obtained in a separate experiment. Statistical significance was tested by two-way ANOVA: *, p≤0.05; **, p≤0.01; ***, p≤0.001.
Fig 6.
The rhesus macaque cell lines are susceptible to entry driven by several viral glycoproteins.
The cell lines were seeded in 96-well plates and transduced in triplicates with MLV pseudoparticles encoding firefly luciferase and bearing the viral glycoproteins VSV-G (B), IAV-HA/NA (C) and LCMV-GP (D). Pseudoparticles without viral glycoprotein (Control) served as negative control (A), while transduction of 293T cells was used as positive control. Cell lysates were harvested after 72 h and luciferase activities determined. The average of four independent experiments performed with technical triplicates is shown. Error bars indicate standard error of the mean. Statistical significance was tested by two-way ANOVA: *, p≤0.05; **, p≤0.01; ***, p≤0.001.
Fig 7.
The rhesus macaque cell lines support growth of Papiine alphaherpesvirus 2 and Zika virus.
For infection with (A) Papiine alphaherpesvirus 2 (PaHV2) or (B) Zika virus (ZIKV) strain MR766, cells were seeded in 24-well plates. Vero76 cells served as positive control. Cells were infected in triplicates at an MOI of 1. Supernatants were harvested at 1 h, as baseline, and at 72 h postinfection. Infectious virus titers were determined by plaque assay (PaHV2) or focus formation assay (ZIKV) on Vero76 cells and virus titers expressed as plaque forming units (pfu) or focus forming units (ffu), respectively. The average of three independent experiments performed with technical triplicates is shown. Error bars indicate standard error of the mean. Statistical significance tested by two-way ANOVA: *, p≤0.05; **, p≤0.01; ***, p≤0.001.