Bushman F, Schroder A, Ecker J, Olvera JM, Li L, Yoder KE, Mitchell RS, Butler SL, Lieber M, Martin SL; Conference on Retroviruses and Opportunistic Infections.
9th Conf Retrovir Oppor Infect Feb 24 28 2002 Wash State Conv Trade Cent Seattle Wash Conf Retrovir Oppor Infect 9th 2002 Seattle Wash. 2002 Feb 24-28; 9: abstract no. S1.
The Salk Inst. for Biological Studies, La Jolla, CA
BACKGROUND: Retroviral replication involves reverse transcription of the viral RNA genome to generate a cDNA copy, and integration of that copy into a chromosome of the host cell. The steps in this process offer many opportunities for inputs from the host.METHODS: Experimental infections in culture combined with biochemical and genomic methods were used.RESULTS: In one line of studies, we have investigated the role of the host cell non-homologous DNA end joining (NHEJ) pathway. Previous studies revealed that cells mutant for NHEJ proteins -- Ku, DNA PK or XRCC4 -- showed increased apoptosis in response to infection. We find that preintegration complexes (PICs) contain the Ku protein, and that the NHEJ system is required for circularization of the viral cDNA (2-LTR circles formation). We next studied apoptosis during infections of human lymphoid cells deleted for the Ligase 4 gene, which have a particularly strong defect in NHEJ. We found that HIV derivatives mutant in integrase induced apoptosis efficiently but that a reverse transcriptase inhibitor blocked apoptosis, implicating unintegrated genomes as the origin of the pro-apoptotic signal. These studies and others support a model in which the double-strand ends present in unintegrated cDNA promote apoptosis, as is known to be the case for chromosomal double strand breaks, and circularization removes free ends. In a second set of studies, we have investigated the effect of chromatin structure on HIV cDNA integration. Human cells were infected with HIV or an HIV-based vector and over 500 integration sites were cloned, sequenced and placed on the human genome. The data reveal that genes are favored quite strongly for integration. Transcriptional profiling of the target cells revealed that the targeted genes were disproportionately active, suggesting a connection with active transcription. Hotspots for integration were also found. For the most favored hotspots, five sites of integration were found within 4 kb. These data and others indicate that proteins bound to the human chromosomes strongly influence integration target selection.CONCLUSIONS: The NHEJ system and chromatin structure influence early steps of HIV infection.
Publication Types:
Keywords:
- AIDS Vaccines
- Acquired Immunodeficiency Syndrome
- Antigens, Nuclear
- DNA
- DNA Damage
- DNA Helicases
- DNA Repair
- DNA, Complementary
- DNA-Activated Protein Kinase
- DNA-Binding Proteins
- HIV
- HIV Infections
- HIV Seropositivity
- HIV-1
- Humans
- Integration Host Factors
- Ku autoantigen
- SET protein, human
- Terminal Repeat Sequences
- Transcription Factors
- genetics
- immunology
Other ID:
UI: 102264350
From Meeting Abstracts