Cells, tissues, and viruses.
African green monkey (Vero) cells and HeLa cells were obtained from the American Type Culture Collection and maintained in Dulbecco's modified Eagle's medium, supplemented with 10% fetal bovine serum, at 37°C and 5% CO2. Cynomolgus macaque TG were obtained at necropsy from the Southwest National Primate Research Center (SNPRC), immediately frozen in liquid nitrogen, and stored at −80°C in BSL-3 containment until processed. This occurs as rapidly as 20 min following euthanasia. BV strain E2490 (NCBI reference sequence no. NC_004812) was used for the in vitro experiments in this study.
BV is classified as a Risk Group 4 agent (CDC) and may only be propagated in a maximum containment laboratory. All experiments using infectious virus were performed in the Animal Biosafety Level 4 (ABSL-4) laboratory at the Texas Biomedical Research Institute (certified by the CDC) by trained personnel wearing protective biosafety suits.
Illumina sequencing and data analysis.
cDNA libraries for deep sequencing were prepared using the small RNA sample prep kit v1.5.0 protocol from Illumina. Between 100 ng and 500 ng small enriched RNA was ligated to adapters, followed by reverse transcription (RT) and PCR amplification. The amplified cDNA libraries were resolved using a 6% polyacrylamide Tris-borate-EDTA (TBE) gel. cDNAs that corresponded to approximately 18 to 30 nucleotides (nt) were identified by comparison to an SRA ladder (Illumina) and excised from the gel. The cDNA was eluted from the gel for 4 h with 100 μl 1× gel elution buffer (Illumina). Gel debris was removed using Spin-X cellulose acetate filters (Illumina), and the cDNA was precipitated with 1 μl glycogen (Ambion), 3 M sodium acetate (Ambion), and 325 μl chilled 100% ethanol. After being washed with 70% ethanol, the cDNA pellet was resuspended in resuspension buffer (Illumina).
Raw sequence data were collapsed into a single file of nonredundant sequences with an associated abundance. Adapter sequences were removed, and the file was converted to FASTA format. The analysis was performed with Formatdb, Megablast, filter_alignments, excise_candidate, auto_blast, and miRDeep scripts from the miRDeep package (15
), together with the BV genomic sequence (NC_004812) and miRBase database (release 14). These algorithms consider pre-miRNA structure, the relative frequency at which a mature miRNA sequence and star sequence were observed, and likelihood that the mature miRNA was generated through a Dicer-dependent mechanism. However, for the very rare reads that are sometimes observed for alphaherpesvirus miRNAs, considering the relative frequency of mature sequences to star sequences can be too stringent. Therefore, reads that failed based on this criterion were further analyzed manually: a candidate sequence was considered a potential miRNA when the sequence was present in a hairpin that was predicted as the lowest free-energy structure using mfold.
Plasmids, small interfering RNAs (siRNAs), and transfections.
To generate plasmid pAMBVgB, the BV virion membrane glycoprotein B (gB; UL27) coding sequence was amplified from BV strain E2490 DNA by PCR and cloned into pCR2.1 TOPO using the TOPO TA cloning system (Invitrogen). The PCR primers were as follows: BV glycoprotein B forward primer (5′-TCCGGGCCCGGAATGCGGCCCCGCGCC-3′) (IDT) and reverse primer (5′-GCGGCCGCCTATAGCTCCTCTTCGTCTGTGTCGCCG-3′) (IDT).
A portion of the second exon of immediate early protein ICP0 coding sequence (between bases 122943 and 123375) was synthesized as a “minigene” by IDT using the published BV sequence, yielding pIDTSMART-KAN:ICP0. The minigene was digested with HindIII and XhoI (NEB) to release the ICP0 sequence insert, which was then inserted into pCR2.1 TOPO using the TOPO TA cloning system (Invitrogen) to yield pMAICP0.
To generate miRNA expression plasmids, each miRNA sequence together with ~200 nt of flanking sequence was amplified from BV strain E2490 DNA by PCR and first subcloned into pCR2.1TOPO or pCR-Blunt II TOPO using the TOPO cloning system (Invitrogen). The PCR primers, along with the TOPO subcloning vectors and enzymes, are listed in Table S1 in the supplemental material. To generate a cassette-type vector that can accommodate cloned inserts from pCR2.1 TOPO or pCR-Blunt II TOPO, regardless of initial insert orientation, a multiple cloning site was cloned into pSIREN-Retro-Q-ZsGreen1 (Clontech), using a duplex of synthetic oligonucleotides. The oligonucleotides used were as follows: 5′-GATCCGTTAACGCGGCCGCGGGCCCCCAGTGTGCTGGTCTAGAT-3′ and 5′-AATTATCTAGACCAGCACACTGGGGGCCCGCGGCCGCGTTAACG-3′ (BamHI, HpaI, NotI, ApaI, BstXI, XbaI). The recognition sequence for EcoRI was destroyed to serve as a diagnostic tool. Oligonucleotides were annealed, and the resulting duplex was ligated to BamHI- and EcoRI-digested pSIREN-Retro-Q-ZsGreen1 vector to yield pSIREN-MCS. miRNA sequences were excised from the TOPO vectors and cloned into pSIREN-MCS using the appropriate restriction sites for correct orientation of the insert.
All constructs generated by oligonucleotide cloning or PCR were confirmed to be correct by sequencing at the Nucleic Acids Core Facility at the University of Texas Health Science Center, San Antonio (UTHSCSA).
Candidate miRNA sequence-containing plasmids were transfected into HeLa cells using the Neon transfection system (Invitrogen) according to the manufacturer's recommendations. To confirm that the candidate miRNAs were synthesized in a Drosha-dependent manner, 44 nM Drosha siRNA (RNASEN siRNA identification no. s26492; Ambion) was also cotransfected; this amount of siRNA had been determined to reduce Drosha expression by approximately 90%. A second siRNA (Silencer negative control 2 siRNA; Ambion) was used as a negative control. Briefly, 2 × 107 HeLa cells were resuspended in 100 μl of buffer R (Invitrogen), containing 8 μg plasmid and 44 nM Drosha siRNA or negative control siRNA. The electroporation was performed using 2 pulses at 1,400 V for 20 ms. After electroporation, cells were plated on 60-mm dishes in 10% fetal calf serum (FCS) without antibiotics. Forty-eight hours later, total RNA was harvested using the mirVana miRNA isolation kit (Ambion) according to the manufacturer's instructions.
RNA and DNA extractions.
To harvest RNA from infected cells, 60-mm dishes of 80% confluent Vero cells were infected with BV at a multiplicity of infection (MOI) of 10. At various times postinfection, cells were scraped from the dishes and pelleted in a low-speed centrifuge; the supernatant was removed and the pellet was flash-frozen in liquid nitrogen. Dry pellets were stored at −80°C until processed. Both large (>200 nt) and small (<200 nt) RNA were harvested from infected cells using the mirVana miRNA isolation kit (Ambion) according to the manufacturer's instructions.
Nucleic acid isolation from macaque TG began in the BSL-3 laboratory. Lysis/binding solution (600 μl; from the mirVana kit) was added to each ganglion, and the tissues were homogenized on ice using an RNase-free plastic pestle and tube. The tissues were processed immediately without allowing the tissue to thaw, thereby preventing the rupture of cells by ice crystals and the subsequent release of RNases. miRNA homogenate additive (60 μl; from the mirVana kit) was added to the homogenized tissue, and the samples were incubated on ice for 10 min. Fifty microliters of the homogenized tissue was then set aside for DNA isolation, and the remainder was used for RNA isolation. An equal volume of acid phenol-chloroform (Ambion) was added to the homogenate set aside for RNA isolation, and the samples were vortexed thoroughly. Likewise, an equal volume of phenol-chloroform (Ambion) was added to the homogenate set aside for DNA isolation, and the samples were vortexed thoroughly. Samples were then transferred to the BSL-2 laboratory, where the remainder of the RNA isolation was performed using the mirVana miRNA isolation kit according to the manufacturer's instructions. RNA concentration was determined by spectrophotometry.
In the BSL-2 laboratory, the samples set aside for DNA isolation were centrifuged and the aqueous layer was removed. DNA was precipitated by adding one volume of isopropanol (Sigma-Aldrich) and centrifuged for 1 h at 4°C and 14,000 × g. The supernatant was removed, and the DNA pellet was washed with 70% ethanol, followed by resuspension in sterile water and spectrophotometry to determine concentration.
Various pharmacological agents were used to investigate whether miRNA accumulation was affected by blocking expression of genes from particular kinetic classes. To inhibit BV DNA synthesis, which prevents expression of true late-class genes, cells were pretreated with 300 μg/ml phosphonoacetic acid (PAA) (Sigma-Aldrich) for 30 min before cells were infected with BV at an MOI of 10 in the presence of the same concentration of the drug. At 18 h postinfection (hpi), cells were processed as described above. To confirm PAA activity, RNA was harvested from infected cells and subjected to Northern blot hybridization probed for the true late-class viral gene UL44 (see B).
Fig. 7. Expression kinetics of BV-encoded miRNAs. BV-infected cells were treated with phosphonoacetic acid (PAA), cycloheximide (CHX), or actinomycin D (ActD). (A) At 18 hpi in the presence of PAA, small RNA was harvested and subjected to RT-qPCR for miRNAs as (more ...)
To inhibit expression of all but immediate early class genes, Vero cells were pretreated with 100 μg/ml of the protein synthesis inhibitor cycloheximide (CHX) (Sigma-Aldrich) for 30 min before cells were infected with BV at an MOI of 10 in the presence of the same concentration of drug. At 1 hpi, cells were scraped from the dishes and pelleted in a low-speed centrifuge, the supernatant was removed, and the pellet was flash-frozen in liquid nitrogen and stored at −80°C until processed. To confirm CHX activity, uninfected Vero cells were treated with and without CHX in the presence of 0.1 mCi/ml of [35S]methionine (PerkinElmer) in parallel to the infected cells. After 1 h, the cell lysates were resolved by SDS-PAGE and processed for autoradiography (see C).
To inhibit both viral and cellular transcription, actinomycin D (act D; MP Biomedical) was used similarly to CHX, but at a concentration of 2 μg/ml. To confirm act D activity, RNA was harvested from infected cells and subjected to Northern blot hybridization probed for the immediate early class viral gene ICP4 (see D).
Purification of virions.
Virions were purified by centrifugation through a 20% sorbitol cushion as described by Stinski (34
). Briefly, Vero cells were grown to 80% confluence in 150-cm2
flasks and infected at an MOI of 10. The cells were harvested 24 hpi and centrifuged at 2,000 rpm for 5 min. The cell pellet was lysed in reticulocyte standard buffer (RSB) (10 mM Tris, pH 7.5, 10 mM KCl, 1.5 mM MgCl2
) containing 0.5% (vol/vol) NP-40. The lysate was incubated on ice for 10 min, and the nuclei was pelleted. Viral particles in the supernatant were recovered by centrifugation at 50,000 rpm for 1 h at room temperature in a SW 55 Ti rotor through 20% (wt/vol) D-sorbitol (Sigma-Aldrich) in 50 mM Tris-HCl, pH 7.5, 1 mM MgCl2
, and 100 μg/ml bacitracin (Sigma-Aldrich). The pellet was resuspended in RNase ONE buffer (Promega) containing 9.15 × 107
molecules of synthetic ath-miR-166 miRNA standard (IDT) and 50 U RNase ONE (Promega). ath-miR-166 is a plant miRNA that is absent from mammalian cells (26
), including Vero cells (M. A. Amen and A. Griffiths, unpublished observations) and was used to determine the efficiency of RNase digestion of miRNAs. The reaction mixture was incubated at 37°C for 30 min. To boost digestion, an additional 50 U RNase ONE (Promega) was added and the reaction mixture was incubated for a further 30 min at 37°C. To dissociate aggregates, 5 mM urea (Fisher) was added and the mixture was sonicated for 10 to 15 s. An equal volume of phenol-chloroform (Ambion) was added and vortexed thoroughly before the sample was transferred to the BSL-2 laboratory. Next, the aqueous phase was harvested and half was devoted to RNA isolation and half to DNA isolation. RNA isolation was performed using the mir
Vana miRNA isolation kit (Ambion) according to the manufacturer's instructions, and the final RNA concentration was determined by spectrophotometry. DNA was precipitated by adding one volume of isopropanol (Sigma-Aldrich) and centrifuged for 1 h at 4°C and 14,000 × g
. The supernatant was removed, and the DNA pellet was washed with 70% ethanol, followed by resuspension in sterile water and spectrophotometry to determine concentration.
To determine if macaque TG contained BV DNA, we used a previously reported quantitative PCR (qPCR) assay (21
). The primers and probe were designed to detect the conserved glycoprotein B (gB) sequence of BV and differentiate between BV and other alphaherpesviruses such as HSV. A standard curve was generated using DNA isolated from uninfected Vero cells spiked with a known amount of pAMBVgB.
To account for the possibility that an infected TG was supporting lytic cycle replication at the time of necropsy, a reverse transcription-qPCR (RT-qPCR) assay was developed to detect expression of the immediate early ICP0 gene. Large RNA (5 ng/μl) was subjected to reverse transcription using 500 nM ICP0 reverse transcription primer (5′-AACTGGTGCCCCTACCAC-3′) (IDT), 5× iScript select reaction mix (Bio-Rad), GSP enhancer solution (Bio-Rad), and iScript reverse transcriptase (Bio-Rad) in a total volume of 20 μl. RTs were performed at 42°C for 60 min and 85°C for 5 min. One microliter of the resulting cDNA was added to 1× iQ SYBR green Supermix (Bio-Rad), 500 nM ICP0 forward primer (5′-CAGACGTGCCTCGCGTA-3′) (IDT), and 500 nM ICP0 reverse primer (5′-TCGACAACGCGTACCCG-3′) (IDT) in a total volume of 25 μl. A standard curve was generated using large RNA isolated from uninfected Vero cells spiked with a known amount of ICP0 RNA in vitro
transcribed using an mMESSAGE mMACHINE kit (Ambion) from pMAICP0. Reverse transcription and qPCR were performed using the iScript select cDNA synthesis kit (Bio-Rad) and iQ SYBR green Supermix (Bio-Rad), respectively, according to the manufacturers' instructions. To determine if there was equivalent RNA recovery between samples of large RNA, a previously reported RT-qPCR assay was utilized to detect RPL13a, a cellular marker shown to be effective for use with rhesus macaque tissues (1
). The sequence of RPL13a for cynomolgus macaque has several base differences from the rhesus macaque sequence. To account for these differences, the following forward primer was used: 5′-CTTGGAGGAGAAGAGGAAGGAGA-3′ (IDT).
For analyses of relative expression of miRNAs, stem-loop RT-qPCR using a single RT reaction mixture containing multiple RT primers was used as previously described (13
). Stem-loop RT-qPCR of miRNAs has been shown to be highly specific for a particular miRNA, even able to differentiate between miRNAs of different sizes and miRNAs that have single-base substitutions in an miRNA sequence (11
). Briefly, 250 ng small RNA was reverse transcribed with a final concentration of 12.5 nM of each stem-loop RT primer (IDT) (see Table S2 in the supplemental material), 1× RT buffer (Applied Biosystems), 0.25 mM (each) deoxynucleoside triphosphates (dNTPs) (Applied Biosystems), 20 U/μl RNase inhibitor (Applied Biosystems), and 3.33 U/μl MultiScribe reverse transcriptase (Applied Biosystems). The volume of the RT reaction mixture was adjusted according to the number of PCRs to be performed. The RT reactions were performed at 16°C for 30 min, followed by 42°C for 30 min and 85°C for 5 min. The efficiencies of two reactions (hbv-mir-B3RC-3P and hbv-mir-B14RC-3P) were improved by increasing the RT temperature. These RTs were performed at 19°C for 30 min, followed by 48°C for 30 min and 85°C for 5 min. For real-time PCR, a 20-μl reaction mixture was made containing 1.33 μl reverse transcription reaction, 1× TaqMan universal PCR master mix (Applied Biosystems), 0.2 μM TaqMan probe, 1.5 μM forward primer, and 0.7 μM reverse primer (5′-AGTGCAGGGTCCGAGGTA-3′) (IDT). The reactions were performed in triplicate and incubated in a 96-well plate at 95°C for 10 min, followed by 40 cycles of 95°C for 15 s and 60°C for 1 min. Detection of the most commonly sequenced isoform of each miRNA was performed using forward primers (IDT) and TaqMan probes (Applied Biosystems). The primer and probe sequences are listed in Table S2 in the supplemental material.
To confirm that Drosha mRNA was knocked down in the presence of the Drosha siRNA, a Drosha qPCR assay was used (Applied Biosystems, Hs00294820_m1). An assay to detect 18S rRNA (Hs03928990_g1; Applied Biosystems) was used as a control. Briefly, 100 ng/μl total RNA was DNase treated using a Turbo DNA-free kit (Ambion) according to manufacturer's instructions. The DNase-treated samples were reverse transcribed with oligo(dT)20 primers using the SuperScript III first-strand synthesis system (Invitrogen) according to the manufacturer's instructions. For the real-time PCR, a 20-μl reaction mixture was made containing 1 μl Drosha or 18S TaqMan assay mix, 10 μl TaqMan universal PCR master mix, no AmpErase uracil N-glycosylase (UNG) (Applied Biosystems), and 1 μl cDNA. The reactions were performed in triplicate and incubated in a 96-well plate at 95°C for 10 min, followed by 40 cycles of 95°C for 15 s and 60°C for 1 min.
To estimate the number of molecules of each miRNA packaged in virions, standard curves were generated using small RNA isolated from uninfected Vero cells spiked with a known amount of synthetic RNA oligonucleotides (IDT) representing each miRNA sequence. Eighty-five nanograms of small RNA isolated from purified virions treated with and without RNase was used for RT-qPCR of the miRNAs as described above. To estimate the number of viral genomes in each sample, 293 ng DNA isolated from purified virions treated with and without RNase was used for the qPCR of gB as described above.
To determine the changes in levels of individual miRNAs between multiple samples (time course and multiple TG), we used a relative quantification strategy. To determine if the cellular miRNA Let-7a
was an appropriate normalization factor for these assays, we measured its expression during infection. Using absolute quantification we determined that it was expressed at constant levels between samples harvested at various times postinfection using RT-qPCR (see Fig. S1 in the supplemental material). This confirmed our previous observation, using Northern blot hybridization that Let-7a
expression was unaffected by virus infection (6
). Briefly, a standard curve was generated using a synthetic RNA oligonucleotide (IDT) that represents the 22-nt form of Let-7a
. Reverse transcription and qPCR was performed as described above (see Table S2 in the supplemental material).
Northern blot hybridization.
RNA species of >200 nt isolated from infected cell lysates (1 hpi and 18 hpi) were resolved using a glyoxal-agarose gel and transferred to a nylon membrane using the NorthernMax-Gly kit (Ambion) according to the manufacturer's instructions. The RNA was cross-linked to the membrane using a Stratalinker (Stratagene). The membranes were probed with DNA oligonucleotides (IDT) end-labeled with [32P]ATP (MP Biomedicals, Irvine, CA). Unincorporated nucleotides were removed using micro Bio-Spin columns, (Bio-Rad) according to the manufacturer's instructions. Only probes with specific activities exceeding 5 × 106 dpm/pmol, as measured using a liquid scintillation counter, were used. The sequences of the probes are detailed in Table S3 in the supplemental material. Hybridizations were performed in ULTRAhyb-Oligo buffer (Ambion) according to the manufacturer's instructions. Probed membranes were exposed to phosphor storage screens (GE Life Sciences) and detected on a Storm phosphorimager (GE Life Sciences).