Vartanian JP, Suspene R, Guetard D, Henry M, Meyerhans A, Wain-Hobson S; IAS Conference on HIV Pathogenesis and Treatment (2nd : 2003 : Paris, France).
Antivir Ther. 2003; 8 (Suppl.1): abstract no. 146.
Unite de Retrovirologie Moleculaire, Institut Pasteur, France
By studying splenocytes from two HIV-1 infected individuals, it was possible to show by fluorescent in situ hybridization (FISH) that, on average, the proviral copy number per cell was 3-4 with a range of 1-8. Greater than 75% of infected cells harboured two or more proviruses. By laser microdissecting individual FISH+ nuclei, followed by PCR, cloning and sequencing, it was possible to show that a single cell harboured a genetically diverse collection of genomes with up to 29% amino acid variation in the V1V2 hypervariable regions of the envelope protein. Hence the work may not only provide insights into the infection process in vivo but also highlight the tempo of recombination and its impact on HIV evolution. Building on the observation that in vivo cells harbour multiple proviruses, the question we ask here is how many viruses infect a cell in vivo? It is known that proviruses are accompanied by unintegrated forms in the nucleus, either covalently closed circles with one or two LTRs or linear molecules with ends degraded by exonucleases. By extensive sequencing of cloned PCR material from microdissected nuclei from human splenocytes, the number of discrete sequences asymptote to a finite number. Since the cross sectional sequence variation in this patient was complex and extensive, these numbers are close to the absolute number of discrete DNA molecules per nucleus. The ratio of unintegrated/integrated DNA is ~10-20. How many incoming virions are destroyed in the cytoplasm? Using infected PBMCs, as opposed to established T cell lines, and the proteasome inhibitor epoxomycin we have shown by TaqMan quantitation of nascent DNA formation that the proteasome degrades up to 75% of incoming virions. Combining the two studies the fraction of HIV RNA that gets converted into a provirus could be as low as 1:100. Hence, as a splenocyte may harbour 3-4 proviruses in vivo, this means that the cell was originally infected by ~300-400 virions. The high ratio and wide range in the number of virions making it to the provirus indicates a substantial stochastic component to the infection process.
Publication Types:
Keywords:
- Acquired Immunodeficiency Syndrome
- Base Sequence
- Cell Line
- Cell Nucleus
- DNA
- DNA Primers
- DNA, Viral
- HIV Infections
- HIV Seropositivity
- HIV-1
- Humans
- Polymerase Chain Reaction
- Proviruses
- Recombination, Genetic
- Reverse Transcription
- genetics
Other ID:
UI: 102262451
From Meeting Abstracts