The envelope proteins from SARS-CoV-2 and SARS-CoV potently reduce the infectivity of HIV-1, reports a study published in Retrovirology
forward oligonucleotide primer, MH535: 5'- AACTAGGGAACCCACTGCTTAAG-3'; b) Alu-reverse oligonucleotide primer: SB704: 5'-TGCTGGGATTACAGGCGTGAG-3'. One µl of sample from the first round PCR and subject to real-time PCR with R/U5 primers and probe [
] with each experiment, a standard curve of the amplicon being measured was run in duplicate ranging from 10 to 1 × 10copies plus a no-template control. Reactions contained 1 × premix , 0.5 µM forward primer, 0.5 µM reverse primer, 0.25 µM probe primer, 100–500 ng of template DNA, and 1X RoxDyeII in a 20 µl volume. After initial incubations of 95 °C for 30 s, 40 cycles of amplification were carried out at 5 s at 95 °C, and 34 s at 60 °C.
For analysis of the HIV-1 transcription, HEK293 cells were transfected with either the empty pcDNA3.1 vector or one expressing the SARS-CoV-2 E. At 24 h post-transfection, cells were infected with pseudotyped HIV-1ΔE/VSV-G at an MOI of 0.1. At 48 h post-transfection, cells were washed, pelleted and the RNA was extracted using the Invitrogen PureLink RNA Extraction kit. The RNA was treated with PureLink DNase I for 15 min at room temperature followed by heat inactivation.
For analysis of LC3-I lipidation, cells were solubilized in triple-detergent buffer supplemented with phosphatase inhibitors and protease inhibitor cocktail and briefly sonicated. The protein concentration was determined with the aid of the Bio-Rad protein assay . Ten to forty micrograms of total proteins per sample were subjected to further analysis. The rabbit polyclonal antibody against LC3-B was used in a 1:5,000 dilution.
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