M mulv rt

All RNA analyzed by reverse transcriptase-quantitative polymerase chain reaction (RT-qPCR) was treated with RQ1 DNase (Promega) before the reverse transcription (RT) reaction. RT reactions were performed using SuperScript™ II RT (Invitrogen) or M-MuLV-RT (New England Biolabs) and random hexamers (Sigma) or dT₂₀ (fractionation experiments) as per the manufacturer's protocol. cDNAs were then treated with RNase A and RNase H for 30 min at 37°C and purified with phenol:chloroform:isoamyl alcohol (PCA). cDNAs were resuspended in 20 μl H₂O and diluted in an assay-dependent manner. Primer sequences and efficiencies are described in Supplementary Table S3. Relative quantities (RQ) were determined based on amplification efficiency and Ct value. To determine the relative expression of candidate transcripts after fractionation, single knockdown or knockdown over time, RQ values were normalized to RQ values of 7SK RNA.
To assess relative stability of candidate transcripts, cells transfected with REF or Control siRNAs were treated with 1 ug/ml Actinomycin D to inhibit transcription. Cells were harvested at various time points from 0 to 4 h by addition of Tri-Reagent. RNA was extracted and analyzed by RT-qPCR to determine RNA abundance after transcription shut-off. Transcription shut-off RT-qPCR was performed using a one-step reaction with MultiScribe Reverse Transcriptase (Invitrogen).

HEK293T cells were transfected with 0.1 μg of pGCH provirus along with 0.2 μg of either Tat-FLAG or Nullbasic-FLAG plasmids or both. Total RNA was extracted 24 h post-transfection using TRIzol reagent (Invitrogen) and further isolated using the PolyAtract mRNA isolation system (Promega). The purified total RNA was reverse transcribed to cDNA using random hexamer primers and M-MuLV RT (New England BioLabs) according to the manufacturer’s instructions. Viral cDNAs were quantified by quantitative PCR using Platinum SYBR Green Supermix (Invitrogen) on the Rotor-Gene Q (Qiagen). The primers used to detect total and unspliced viral mRNA has been described previously [58 (link)]. Reverse-transcribed GAPDH cDNA was used to normalize extraction efficiency.

For total RNA isolation, infection was monitored for CPE and cells were harvested at day 4 after infection. Briefly, cells were washed with PBS, lysed in TriZol, and total RNA was isolated according to the manufacturer’s instructions. Two sets of primers (targeting the RNA-dependent RNA polymerase (RdRp) and Nucleocapsid (N) genes) were used for gene-specific reverse transcription and amplification, except for SARS-CoV-2, which was analyzed using RdRp only (Supplementary Table 1 online). Complementary DNA (cDNA) was synthesized with M-MuLV RT (NEB) using 500 ng of total RNA, according to the manufacturer’s instructions. Quantitative real-time PCR amplification was carried out using SYBR Green technology with the iTaq Universal SYBR Green PCR master mix according to the manufacturer’s instructions. Genome copies were calculated using a standard curve, and fold differences in gene expression were calculated using the 2^–ΔΔCt method.¹⁴

HeLa cells were transfected by electroporation with 5 µg viral RNA transcript, added to HeLa cell monolayers and incubated at 37 °C. Upon cytopathic effect (CPE), viruses were harvested by three repeated freeze-thaw cycles, cell debris removed by centrifugation. Viral RNA was isolated with QiaAmp viral RNA purification kit (Qiagen), as recommended by the manufacturer. The 3Dpol cDNA was prepared from purified viral RNA by reverse transcription with MMuLV-RT (New England Biolabs) with Random Hexamer Primers (manufacturer). The resulting DNA product was then PCR amplified using SuperTaq DNA polymerase (Ambion; Naugtuck, CT, USA) and oligonucleotides PV-3D-BglII-for and PV-3D-EcoRI-ApaI-polyA-rev as primers. The presence of all four mutations was determined by sequencing of the nucleic acid obtained in second PCR step with oligonucleotides PV-3D-seq100-for (5′-GAA GGG GTG AAG GAA CCA G-3′) and PV-3D-seq500-for (5’-AGG TTG AGC AGG GGA AA-3′).