UZ Gent, AIDS referentielaboratorium
An estimated 36.7 million people are infected with HIV. Although HIV infections are decreasing, the search for a cure or vaccine for HIV remains important. To aid in this search, a good understanding of the virus is necessary. HIV has a wide range of genetic variability, mostly caused by the error prone reverse transcriptase. Mutations mostly accumulate in the HIV proviral DNA. By studying the proviral DNA, more information about HIV could be revealed, contributing to the further understanding of the virus.
A protocol for amplification of full length HIV proviral DNA in two fragments was already optimized in the laboratory. The goal of this study was the optimizing of a new protocol for the full length amplification and sequencing of HIV proviral DNA in one fragment. This must improve the analysis of the proviral DNA reservoir and discriminate replication competent from defective viruses.
Starting from frozen peripheral mononuclear blood cells, HIV DNA was extracted and subsequently clonally amplified as single full length fragments using a nested PCR protocol. PCR results were checked by submarine agarose electrophoresis. When successful, the amplicons were purified for the downstream Big Dye Sanger sequencing PCR. Using several primers spread over the genome, short sequence fragments were generated. Proofreading of all chromatograms was done and sequences were aligned against a HIV reference sequence and further analyzed with bioinformatics tools. With the obtained sequences individual provirus contig sequences that overlap the full genome were constructed.
Six samples from two patients could be successfully amplified using the optimized full length amplification protocol. Two patients, patient A and patient B, were selected for further analysis. Four out of six individual proviruses tested appeared to be small fragments with very large deletions between the LTR regions. One sample had an intact gag region, with most of the pol region also intact. Only one out of six samples was a fully intact HIV proviral genome of 8,9 kb. Small deletions and insertions were less commonly seen than expected. Evidence of hypermutation was only found in one amplicon.
An optimized protocol for the amplification of proviral HIV DNA in one fragment and the accompanying primers for sequencing is now ready for use in the laboratory. More testing regarding the efficiency of these primers may be needed to ensure consistently good results. Further longitudinal study on different samples with this protocol could lead to new information being discovered regarding the proviral HIV reservoir.
The genetic diversity of HIV is wide because of the rapid replication and the error-prone reverse transcriptase. The occurrence of selective pressure and recombination also plays a role. Certain mutations may lead to resistant viruses, no longer sensitive for treatment with the existing drugs. In some cases, mutations also lead to a defective provirus that is no longer able to infect other host cells. These defective proviruses can be seen in the viral DNA that is integrated in the DNA of the host cell. Viral DNA integrated in the host cell is called the provirus.
Amplification and sequencing of viral DNA is particularly challenging because of the interference with human DNA. The aim is to optimize the sequencing procedure for full genome sequencing of the integrated DNA. A protocol for full genome sequencing of viral RNA extracted from plasma was already available at the laboratory. The design of this study was to adapt this protocol for viral DNA in blood cells. This will allow to define the variability of the proviral DNA, identify defective viruses and make a comparison between the viral RNA sequence and the proviral DNA sequence from the same patient.
The methodology of this research relies on molecular technics. After the extraction of RNA and DNA, both are amplified with a nested PCR protocol. With this protocol two large RNA or DNA fragments are amplified that together cover the full genome of the virus. After amplification, the amplicons are loaded on an agarose gel to see if the proper amplification occurred. To avoid subsequent interference with non-specific fragments the amplicons resulting from DNA amplification then undergo slicing from the agarose gel followed by a purification. The purified amplicons from proviral DNA are then subjected to a second round PCR amplification and purification. For amplicons from viral RNA no agarose gel slicing is needed and they are purified with a common purification kit. For sequencing of the purified amplicons, primers covering the whole fragment are used in a cycle sequencing PCR. The resulting short amplicons are then separated by capillary electrophoresis whereby they pass a laser to read the fluorescent terminal nucleotide. The obtained nucleotide sequences are then aligned against a reference sequence in order to define the different mutations.
Three out of six samples from DNA and all samples from RNA were successfully amplified and three paired RNA-DNA samples could be sequenced. The LTR region gave some problems but this only resulted in the missing of a small fragment of the whole HIV genome. A comparison of the indels between RNA and DNA showed that, as expected, the Gp120 region was the most variable in lenght. Altogether, RNA was less mutated than DNA. Analysis of the hypermutations that along other causes lead to defective proviruses showed that only a recent sample was not hypermutated. The two other samples showed most hypermutations in the GAG and POL region.
Further optimization such as primers and conditions of the nested PCR is needed to successfully amplify all DNA-samples. There is also need for one or more additional primers to close the full genome completely. Only then a veracious analysis of the samples can be done. This was the first outset to amplify and sequence DNA-samples relatively quick to execute a full genome mutation analysis.
De Pintelaan 185
Mevrouw Chris Verhofstede
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