Impact of HIV genetic variation on Tat length, function and ability to be targeted by gene-editing

Anthony R. Mele

doi:10.17918/00001029

Human immunodeficiency virus type 1 (HIV-1) encodes for the transactivator of transcription (Tat), a 101 amino acid multi-functional protein that is involved in transcriptional enhancement of the virus and in causing neurotoxicity and central nervous system (CNS) dysfunction. Tat is also susceptible to extensive genetic diversification that alters the functionality of the protein. A meta-analysis of over 900 publications to assess Tat length usage in the previous ten years of HIV-1 literature showed that the truncated Tat 86 was used statistically more frequently than Tat 101. This analysis also highlighted that the truncated version affected certain functions associated with Tat. While assessing Tat genetic variation in the Drexel University College of Medicine CNS AIDS Research and Eradication Study (CARES) Cohort, unique Tat length variants were observed. In order to examine their functionality and to determine if these truncations were present elsewhere world-wide, subtype B HIV-1 sequences from the Los Alamos National Laboratory (LANL) database were used as a comparator. From this study, full-length Tat 101 was found to be the most frequent for patient samples world-wide. In addition, unique previously unreported Tat length variants were observed, and their functionality characterized in regard to transactivation. Given the extensive genetic variation database that was created from these studies and others, the nucleotide sequence of tat was examined to understand its impact on guide RNAs utilized in CRISPR/Cas9 gene editing systems. It was found that previously published gRNAs for Tat were already the highest rated gRNAs to account for tat genetic variation. Of the gRNAs that were designed, only one targeted the basic domain, gTatRev. However, what was not known was how CRISPR/Cas9 edits would affect the other genes in other reading frames and what types of edits would occur in an area of the HIV-1 genome not known to be bound by nucleosomes (tat) versus those that are known (the TAR region of the LTR). This is crucial to understand as the CRISPR/Cas9 system is most likely to be used in a cellular environment where there is limited transcriptional activity known as a latently infected cell. While preparing to examine these questions, the integrated HIV-1 provirus in a common latently infected model system, the J-Lat 10.6 cell line, was found to not be known. The J-Lat cells contain a replication-incompetent provirus that expresses GFP in replacement of the nef gene. Given this, Sanger sequencing and a MinION next generation sequencing approaches were used to obtain this data. With this data the frequency of insertion and deletion (InDel) events was assessed in an unstimulated environment. This was accomplished through transduction of a lentiviral vector encoding gRNAs of interest, Cas9, and an RFP reporter to target the now sequenced integrated proviral genome in J-Lat 10.6 cells. Unstimulated J-Lat 10.6 cells were assessed for InDels using TatA, a gRNA from the literature previously utilized. PCR amplification Sanger sequencing, and the publicly available assessment tool, Inference of CRISPR Edits (ICE), was used to interpret the edits, laying the groundwork for a high-throughput low sensitivity assay to determine the efficiency of gRNAs. This research will assist the field in developing more effective gRNAs.

Impact of HIV genetic variation on Tat length, function and ability to be targeted by gene-editing

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Impact of HIV genetic variation on Tat length, function and ability to be targeted by gene-editing

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