Journal: Cell Host & Microbe

Article Title: Mapping the neutralizing specificity of human anti-HIV serum by deep mutational scanning

doi: 10.1016/j.chom.2023.05.025

Figure Lengend Snippet: Lentivirus platform for deep mutational scanning (A) The lentivirus genome used for deep mutational scanning. The genome contains the full 5′ and 3′ LTR sequences, including the U3 sequence usually deleted in the 3′ LTR. Env is under control of an inducible TRE3G promoter and followed by a 16N random nucleotide barcode. A CMV promoter drives ZsGreen and puromycin resistance (PuR) expression. (B) Approach for generating genotype-phenotype linked variant libraries. Lentivirus genomes carrying barcoded Env mutants are transfected into 293T cells alongside plasmids expressing the lentiviral proteins necessary for creating single-cycle infectious virions and VSV-G. The resulting VSV-G pseudotyped viruses are used to infect 293T-rtTA cells at a low multiplicity of infection, such that most infected cells receive just one viral genome. Infected cells are enriched via puromycin selection, and genotype-phenotype-linked Env-expressing virus variant libraries are generated by inducing Env expression with doxycycline and transfecting plasmids encoding the lentivirus genes. The virus variant libraries are also generated separately with VSV-G, and these VSV-G pseudotyped viruses can infect cells regardless of whether or not they have a functional Env and so can be used to readout the library composition.

Article Snippet: A second round of PCR was then performed using a forward primer that annealed to the Illumina Truseq Read 1 sequence and had a P5 Illumina adapter overhang, and reverse primers from the PerkinElmer NextFlex DNA Barcode adaptor set that annealed to the Truseq Read 2 site and had the P7 Illumina adapter and i7 sample index.

Techniques: Sequencing, Control, Expressing, Variant Assay, Transfection, Infection, Selection, Virus, Generated, Functional Assay