Identification of system-level features in HIV migration within a host
Data files
Jul 21, 2023 version files 2.03 MB
-
env.baseline.fasta
-
README.md
Abstract
Objective: Identify system-level features in HIV migration within a host across body tissues. Evaluate heterogeneity in the presence and magnitude of these features across hosts.
Method: Using HIV DNA deep sequencing data generated across multiple tissues from 8 people with HIV, we represent the complex dependencies of HIV migration among tissues as a network and model these networks using the family of exponential random graph models (ERGMs). ERGMs allow for the statistical assessment of whether network features occur more (or less) frequently in viral migration than might be expected by chance. The analysis investigates ve potential features of the viral migration network: (1) bi-directional ow between tissues; (2) preferential migration among tissues in the same biological system; (3) heterogeneity in the level of viral migration related to HIV reservoir size; (4) hierarchical structure of migration; and (5) cyclical migration among several tissues. We calculate the Cohran's Q statistic to assess heterogeneity in the magnitude of the presence of these features across hosts. The analysis adjusts for missing data on body tissues.
Results: We observe strong evidence for bi-directional ow between tissues; migration among tissues in the same biological system; and hierarchical structure of the viral migration network. This analysis shows no evidence for differential level of viral migration with respect to the HIV reservoir size of a tissue. There is evidence that cyclical migration among three tissues occurs less frequently than expected given the amount of viral migration. The analysis also provides evidence for heterogeneity in the magnitude that these features are present across hosts. Adjusting for missing tissue data identifies system-level features within a host as well as heterogeneity in the presence of these features across hosts that are not detected when the analysis only considers the observed data.
Discussion: Identification of common features in viral migration may increase the efficiency of HIV cure efforts as it enables targeting specific processes.
Methods
Blood and tissue samples were collected during a rapid autopsy procedure.
Genomic DNA was extracted from 5 million PBMCs and snap-frozen tissues using QIAamp DNA Mini Kit (Qiagen cat#51306) per manufacturer's protocol. After extraction, precipitation was performed to concentrate DNA.
RNA was extracted from blood plasma layering 500-700µl of plasma on top of 200µl of 20% sterile filtered sucrose solution. Sample was spun at 23,500xg for 1 hour at 4°C to pellet the virus. Supernatant was removed and the pellet resuspended in 140µl of PBS. RNA was extracted using Qiagen’s QIAamp Viral RNA mini kit (cat# 52904) according to the manufacturer’s recommendation. cDNA from HIV RNA was generated using Bio-Rad One-Step RT-ddPCR Advanced Kit for Probes (cat# 186-4021).
Nested PCR. To amplify single genome FL env, DNA extracted from antemortem PBMCs and post-mortem tissues was diluted using ddPCR quantification data. This limited dilution PCR reaction can prevent PCR recombination and ambiguous base calls and allow the amplification of viral single genomes. For HIV RNA in blood plasma, cDNA was generated from RNA using SuperScript III First Strand Synthesis System (cat# 18080-051). Template cDNA and HIV DNA extracted from tissues were diluted until approximately 30% of the second-round reactions were positive for the correctly-sized amplification product. Primers used for the first round were 5’FENVouter (forward) TTAGGCATCTCCTATGGCAGGAA and 3’RENVouter (reverse) TCTTAAAGGTACCTGAGGTCTGACTGG. First round PCRs were performed using the Advantage 2 PCR Kit from Takara (cat# 639206) following manufacturer’s recommendations using the 10X SA Buffer. Cycling conditions were 95C for 1 min, 35 cycles of 95C 15sec, 57C 30sec, 68C 3min with a final extension at 68C for 10min.The second round PCR was done using 5’FENVinner: GAGCAGAAGACAGTGGCAATGA (forward) and 3’RENVinner: CCACTTGCCACCCATBTTATAGCA (reverse). Cycling conditions were 95C for 1 min, 30 cycles of 95C 15sec, 64C 30sec, 68C 3min with a final extension at 68C for 10min. PCR clean ups were done on the second round reaction products using QIAquick PCR Purification Kit (cat# 28106). DNA was quantified using Qubit dsDNA HS Assay Kit (Invitrogen cat#Q32854). Quality and integrity were measured using Genomic DNA Screen Tape in combination with the 2200 TapeStation System (Genomic DNA Reagents cat#5067-5366, Genomic DNA Screen Tape Cat #5067-5365).
Nextera XT Library Preparation. Single Genome amplicons were prepared for deep sequencing using the Nextera XT DNA Library Preparation Kit (Illumina FC-131-1096) with indexing of 96-samples per run (Nextera XT index kit set A FC-131-2001) per manufacturer’s protocols.