Supplemental Materials and Methods

Cell Lines. HeLa, HEK293, and HEK293T cell lines were obtained from American Type Culture
Collection (ATCC) were maintained in DMEM supplemented with 10% (vol/vol) FBS and penstep (Cellgro). Dicer wild-type cells (DLD1) and cells hypomorphic for Dicer activity (DLD1
DicerEx5/) cell lines (1) were maintained in RPMI 1640 supplemented with 10% (vol/vol)
FBS and pen-strep. Human B-cell line GM19240 was obtained from Coriell Institute and
maintained in RPMI 1640 supplemented with 15% (vol/vol) FBS and pen-strep.
Plasmids. Sequences encompassing the predicted miRNA genes, SFVagm-miR-S4 mutational
series, and HSUR4 were synthesized by Integrated DNA Technologies (IDT) in the context of
the pIDT-SMART-KAN vector or as gBlocks (sequences are listed in Dataset S2). SV40 miRNA
and MHV68 miRNA expression vectors have been previously described (2). RISC Activity
Assay luciferase reporters were generated with two overlapping oligonucleotides of opposing
orientations, which encoded for two complementary binding sites for the various miRNAs
separated by eight nucleotides. Oligonucleotides were annealed and elongated using Taq DNA
polymerase (New England Biolabs). Extended target sites were then cloned into the XhoI/XbaI
sites of pcDNA3.1dsRluc (3). BACH1 (pGL3-BACH1, pGL3-BACH12 4) and p300
(psiCheck2-p300, psiCheck2-p300M) 3UTR luciferase reporters have been previously
described (4, 5). Drosha cleavage assay luciferase reporters for the FV miRNAs were generated
by subcloning the respective miRNAs from the pIDT-SMART-KAN vector into the restriction
sites immediately following Renilla luciferase of pcDNA3.1dsRluc. The control plasmids
(pcDNA3.1dsRluc-SV40, pcDNA3.1dsRluc-MHV68, and pcDNA3.1dsRluc-KapB) have been
previously described (2, 6). The miR-155 expression vector was generated by PCR amplification
from DLD1 genomic DNA and subcloning into pcDNA3.1puro (primers listed in Dataset S2).
The miR-132 expression vector was generated by extension of synthetic oligos followed by
subcloning into pcDNA3.1puro (oligos listed in Dataset S2).
Generation of Lentiparticles. SFVagm-miR-S6S7 or an irrelevant miRNA (ebv-mir-BART4) was
subcloned into the pLB2 CAG P2Gm (Addgene #19752) lentivector using XhoI and XbaI sites.
The following plasmids were transfected into a T75 of HEK 293T cells using Lipofectamine
2000 (Invitrogen): 11.7 ug miR or empty expression vector, 3.5 ug pMD2.G (Addgene #12259),

2.9 ug pRSV-Rev (Addgene #12253), and 5.9 ug pMDLg/pRRE (Addgene #12251). Supernatant
was collected after 24 hours and the HEK293T cells were given fresh media. The next day, the
supernatant was collected and combined with the previous day supernatant and 0.2 micron
filtered. The stocks were titered on 10^5 HEK293T cells in 24 well plates. Twenty microliters of
serial dilutions of lentiparticle stocks were added to the cells in triplicate. After 48 hours, the
cells were washed with PBS. Lentiparticle titers were determined by counting the percent of
HEK293T cells that were GFP positive. Transducing units (TU) per mL was calculated using the
following equation: ((Percent positive GFP x number of cells)/ 100 x volume of viral dilution
added to each well) x 1 / dilution factor.
Generation of Stable Cell Lines Expressing SFVagm-miR-S6S7 and controls. 5x10^5 GM19240
B-cells (Coriell Institute) were seeded into a 6-well plate. 1 mL of lentiparticles (1x10^6 TU/mL)
and 5ul polybrene (Sigma-Aldrich) (10ug/mL) were added to each well in duplicate along with a
polybrene only control. After 24 hours, the cells were pelleted and resuspended in fresh media
along with puromycin (Sigma-Aldrich) (0.5ug/mL). Cells were passed every 3 days in media that
contained puromycin. The control B-cells did not survive past 2 passages with puromycin
selection. After 11 days, selection was removed and cells were used for subsequent experiments.
miRNA Prediction Software. Python scripts to predict RNAP III miRNA genes in viral genomic
sequence utilized the RNAfold (7) and Biopython (8) software packages. Scripts and associated
data will be made freely available at https://code.google.com/p/mirna/
Small RNA Northern Blots. Small RNA Northern blot analysis was performed as previously
described (9). Probe sequences are listed in Dataset S2. Alternate SNP probes (as determined
from RNA-seq data) were used for Northern blot analysis of SFVagm infected samples.
Preparation of FV Stocks. SFVagm (SFV-HU1, ATCC: VR-2596) was obtained from ATCC and
amplified by serial passage on BHK21 cells. SFVagm stock titers (syncytia forming unit per mL
(SFU/mL)) were determined by serial dilution and infection of BHK21 cells followed by staining
with 0.5% (w/vol) methylene blue and 50% methanol (vol/vol) solution and counting of
syncytia.
Small RNA Sequencing. Fifty-percent confluent HeLa cells in a T75 flask were infected with 5.4
x 10^4 SFU of SFVagm. Seventy-two hours post infection, total RNA was harvested with PIG-B

(10). Two hundred micrograms of total RNA was size fractionated on a 15% (vol/vol) UREAPAGE gel and used to prepare pooled small RNA libraries for Illumina small RNA sequencing.
Sequencing was performed on an Illumina HiSeq 2500 at the Genomic Sequencing and Analysis
Facility at UT Austin. Sequencing adapter sequences were trimmed from the reads using custom
Python scripts, and any sequences with ambiguous calls or less than 18 nt in length after
trimming were removed from further analysis. The preprocessed reads were then mapped to the
SFVagm (NC_10820) genomic sequences using the SHRiMP2 software package (11). 5 start site
counts and coverage were calculated using custom Python scripts and visualized using the
gnuplot software package. Genomic maps were generated from the reference sequences with the
Geneious software package (Biomatters).
Precursor miRNA Structure Prediction. miRNA stemloop secondary structures were generated
using the mfold RNA folding prediction Web server (12, 13) or the RNAfold prediction Web
server (7), as indicated.
RISC Activity Assay. Twenty-four well plates of HEK293T cells were co-transfected with five
nanograms of Renilla luciferase based 3'UTR reporter (pcDNA3.1dsRluc (3)), five nanograms of
rey reporter (pcDNA3.1dsLuc2CP (3)) and one microgram of either empty vector (pIDTSMART-KAN) or indicated miRNA expression vector in triplicate using the Turbofect reagent
(Fermentas). Twenty-four hours later, cells were harvested and assayed with the Dual-Glo
Luciferase Assay System (Promega). Luciferase activity was measured with a Luminoskan
Ascent luminometer (Thermo Electronic). Students t-test was used to assess statistical
significance of observed differences.
RNA Polymerase Activity Assay. HEK293T cells were transfected with miRNA expressions
vectors as indicated. Two hours later, cells were treated with 50 ug/mL -amanitin (SigmaAldrich). Total RNA was harvested with PIG-B (10) at 24 hours post transfection and Northern
blot analysis was performed.
Drosha siRNA Assay. HEK293T cells were transfected with 20 nM Drosha siRNA (6) or
negative control siRNA (6) using Lipofectamine RNAi-MAX (Invitrogen). Twenty-four hours
later, a second transfection of siRNA was performed followed by transfection of miRNA
expression vectors using the Turbofect reagent according to the manufacturers recommendations

(Fermentas). Twenty-four or thirty hours later, total RNA was extracted with PIG-B (10) and
Northern blot analysis was performed
Microprocessor Overexpression. Fifty-percent confluent HEK293T cells in twelve well plates
were co-transfected with 600 ng of the indicated miRNA expression vector and 300 ng each
Drosha and DGCR8 expression vectors (6) using the Turbofect reagent according to the
manufacturers recommendations (Fermentas). Twenty-four hours later, total RNA was extracted
with PIG-B (10) and Northern blot analysis was performed.
Drosha Cleavage Luciferase Assay. Fifty-percent confluent HEK293T cells in twenty-four well
plates were co-transfected with 0.5 ng of the indicated experimental Renilla luciferase reporter,
0.5 ng control firefly luciferase reporter, and 250 ng each Drosha and DGCR8 expression vectors
in triplicate using the Turbofect reagent according to the manufacturers recommendations
(Fermentas). Twenty-four hours later, cells were harvested and assayed with the Dual-Glo
Luciferase Assay System (Promega). Luciferase activity was measured with a Luminoskan
Ascent luminometer (Thermo Electronic). Students t-test was used to assess statistical
significance of observed differences.
Dicer Dependence Assay. Dicer wild-type cells (DLD1) and cells hypomorphic for Dicer activity
(DLD1 DicerEx5/) (1) were transfected with miRNA expression vectors using the Turbofect
reagent (Fermentas). Twenty-four hours post transfection, total RNA was extracted with PIG-B
and Northern blot analysis was performed.
S4 Mutation Series Analysis. Fifty-percent confluent six-well plates of HEK293T cells were cotransfected with 2 ug of the indicated miRNA expression vector and 500 ng MHV68 expression
vector. Total RNA was extracted with PIG-B at forty-eight hours post transfection and Northern
blot analysis was performed.
Host Target 3UTR Luciferase Assays. Fifty-percent confluent HEK293T cells in twenty-four
well plates were co-transfected with 0.5 ng of the indicated luciferase reporters (pGL3 based
BACH1 reporters and empty vector controls included co-transfection of pcDNA3.1dsLuc2CP
control vector; psiCheck2 based p300 reporters express both firefly and Renilla luciferase from
the same plasmid) and 0.5 ug of the indicated miRNA expression vectors using the Turbofect
reagent (Fermentas) in triplicate. Twenty-four hours post transfection, cells were harvested and

assayed with the Dual-Glo Luciferase Assay System (Promega). Luciferase activity was
measured with a Luminoskan Ascent luminometer (Thermo Electronic). Students t-test was used
to assess statistical significance of observed differences.
Western Immunoblot Analysis. Fifty percent confluent six well plates of HeLa cells were infected
with 1.35 * 10^4 SFU of SFVagm or mock infected. Twenty-four hours later, media was replaced
with fresh media. Ninety-six hours after infection, lysates were prepared with RIPA buffer (0.1%
SDS, 1% Triton X-100, 1% deoxycholate, 5 mM EDTA, 150 mM NaCl, and 10 mM Tris at pH
7.2) including Complete, Mini Protease Inhibitor tablets (Roche). Fifty micrograms of the total
lysate was separated in NuPAGE 4-12% bis-tris gels and transferred to nitrocellulose membranes
(Biorad). Membranes were blocked with Odyssey blocking buffer (LI-COR). Primary antibodies
used were p300 (C-20): sc-585 (Santa Cruz Biotechnology), BACH1 (C-20): sc-14700 (Santa
Cruz Biotechnology), and -Actin (C4): sc-47778 (Santa Cruz Biotechnology). Blots were
probed with a 1:250 dilution of either p300 or BACH1 antibody and 1:500 dilution -Actin in
Odyssey blocking buffer. Blots were washed four times with Phosphate Buffered Saline
including 0.1% Tween 20 (PBST). IRDye 800CW (LI-COR) or Alexa-Fluor 750 (Life
Technologies) and Alexa-Fluor 680 (Life Technologies) secondary antibodies (LI-COR) were
diluted 1:5,000 in Odyssey blocking buffer containing 0.1% Tween 20 and 0.05% SDS. Blots
were washed four times with PBST. Blots were scanned on an Odyssey CLx infrared imaging
system (LI-COR). Band intensities were measured with Image Studio software (LI-COR). Three
independent experiments were performed on different days for quantification and Students t-test
was used to assess statistical significance of observed differences.
Interferon Signaling Assay. HEK293T cells were seeded in 96-well plates. The next day, 1 ul
Luciferase reporter mix (5 ng/ul pcDNA3.1dsRluc (3) and 20 ng/ul pISRE-Luc (Clonetech)) was
co-transfected along with 100 ng of the indicated miRNA expression vector or control vector
using the Turbofect transfection reagent according to the manufacturer protocol (Fermentas).
Each experimental condition was transfected into 6 wells (2 sets of triplicates-3 control wells and
3 experimental wells). After 48 hours, the media was replaced with new media with or without
100 U/ml of universal type I interferon (PBL Interferon Source). 12 hours after IFN treatment, a
dual-luciferase assay was performed. Firefly luciferase was normalized to Renilla luciferase
expression in order to control for transfection differences. Fold induction of the ISRE luciferase

reporter was calculated by dividing the comparable experimental wells by the control wells.
Induction of each condition is relative to the negative control.
Interferon Growth Assay. 3x10^4 cells of the indicated pooled stable cell lines were seeded into a
24 well plate in 1 mL growth media in triplicate. Viable cells as assayed by trypan blue (Cellgro)
staining were counted (Day 0) then 250 units universal type I interferon (IFN) (PBL Interferon
Source) or 1 ug bovine serum albumin (BSA) (Sigma-Aldrich) control was added to the wells.
Viable cell numbers were counted every day and after each cell count 250 units IFN or 1ug BSA
was added to the wells. Three independent experiments were performed on different days in
triplicate.

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