Abstract
Nearly full-length genome sequencing of HIV-1 using peripheral blood mononuclear cells (PBMC) DNA as a template for PCR is now a relatively routine laboratory procedure. However, this has not been the case when using virion RNA as the template and this has made full genome analysis of circulating viruses difficult. Therefore, a well-developed procedure for sequencing of full-length HIV-1 RNA directly from plasma was needed. Plasma from U.S. donors representing a range of viral loads (VL) was used to develop the assay. RNA was extracted from plasma and reverse-transcribed. Two or three overlapping regions were PCR amplified to cover the entire viral genome and sequenced for verification. The success of the procedure was sensitive to VL but was routinely successful for VL greater than 105 and the rate declined in proportion to the VL. While the two-amplicon strategy had an advantage of increasing the possibility of amplifying a single species of HIV-1, the three-amplicon strategy was more successful in amplifying samples with low viral loads. This protocol provides a useful tool for molecular analysis to understand the HIV epidemic and pathogenesis, as well as diagnosis, therapy and future vaccine strategies.
Highlights
Since the first complete sequence of the HIV-1 genome was published in 1985 [1], there have been numerous reports documenting the genetic variability of the virus based on sequencing
The accuracy of direct sequencing of full-length virion RNA from HIV-1 relies on the ability to extract RNA from clinical materials, to conduct cDNA synthesis, and to amplify the cDNA successfully
An ultracentrifugation step to concentrate the viral particles before the extraction of RNA contributed to increased success especially for plasma samples with low viral loads (VL); a total of at least 250 virus copies (10,000 copies/ml6500 ml plasma460 ml RNA63 ml used) had to be present for the procedure to be successful
Summary
Since the first complete sequence of the HIV-1 genome was published in 1985 [1], there have been numerous reports documenting the genetic variability of the virus based on sequencing. Only 74 sequences from 47 individuals are identified as derived from RNA and all of them were generated using cloning; there are no full-length HIV-1 sequences that are clearly from direct PCR amplification and sequencing of virion RNA. The reason for this is that generating a single, nearly full-length PCR amplicon of HIV-1 using virion RNA as the template has been relatively difficult. It is often the case that PBMC are not available from important study populations, and studies must be conducted using plasma or serum Such studies have often been restricted to partial genome characterization because of the technical difficulty of using RNA as the template [10,11]
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