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A model system for human cytomegalovirus-mediated modulation of human immunodeficiency virus type 1 long terminal
T N Moreno1, E A Fortunato, K Hsia
1Department of Biology, University of California, San Diego, La Jolla 92093-0357, USA.
Insights
Human cytomegalovirus (HCMV) activates human immunodeficiency virus type 1 (HIV-1) long terminal repeat (LTR) activity when HIV-1 gene expression is low. However, HCMV inhibits HIV-1 LTR activity when HIV-1 gene expression reaches a threshold.
Area of Science:
- Virology
- Molecular Biology
- Neuroscience
Background:
- Previous studies demonstrated that human cytomegalovirus (HCMV) differentially affects human immunodeficiency virus type 1 (HIV-1) replication in brain cells.
- HCMV activates HIV-1 in cells with limited HIV-1 gene expression but inhibits it in cells permissive for both viruses.
- Understanding these interactions is crucial for managing co-infections, particularly in neurological contexts.
Purpose of the Study:
- To investigate the mechanisms by which HCMV modulates HIV-1 long terminal repeat (LTR) activity.
- To develop a model system that decouples HIV-1 gene expression from LTR activity.
- To determine the role of HIV-1 gene expression levels in HCMV's effect on HIV-1 LTR.
Main Methods:
- Generation of U373-MG astrocytoma/glioblastoma cell lines (LTRIG and LIGHIVDC) with integrated LTR-chloramphenicol acetyltransferase (CAT) and lacI constructs.
- LIGHIVDC cells contain an inducible HIV-1 genome regulated by a Rous sarcoma virus promoter with lac operator sequences.
- Assessment of LTR-mediated CAT activity, Tat protein detection, and effects of HCMV infection and isopropyl-beta-D-thiogalactopyranoside (IPTG) induction.
Main Results:
- Basal LTR activity was significantly higher in LIGHIVDC cells, further inducible by IPTG.
- HCMV stimulated CAT activity ~20-fold in the absence of Tat, requiring HCMV gene expression.
- HCMV infection did not affect LTR-directed transcription in LIGHIVDC but inhibited it with increased Tat levels.
Conclusions:
- HCMV can induce HIV-1 LTR activity when HIV-1 gene expression is minimal.
- A threshold level of HIV-1 gene products is necessary for HCMV to inhibit HIV-1 LTR.
- These findings elucidate the complex interplay between HCMV and HIV-1 replication in specific cellular contexts.
Abstract:
Previously, our laboratory showed that human cytomegalovirus (HCMV) activates human immunodeficiency virus type 1 (HIV-1) in brain-derived cells with limited HIV-1 gene expression but inhibits HIV-1 in cells fully permissive for replication of both viruses (F. M. Jault, S. A. Spector, and D. H. Spector, J. Virol. 68:959-973, 1994). To investigate these effects further, we developed a model system that uncouples HIV-1 gene expression from long terminal repeat (LTR) activity. Two monoclonal U373-MG astrocytoma/glioblastoma cell lines (LTRIG and LIGHIVDC) were generated, each containing an integrated copy of an LTR-chloramphenicol acetyltransferase (CAT) construct and the Escherichia coli lacI gene. LIGHIVDC also has an inducible HIV-1 genome controlled by a Rous sarcoma virus promoter with lac operator sequences. Basal LTR-mediated CAT activity is 65-fold higher in LIGHIVDC than in LTRIG, and this activity is further increased (20-fold) following incubation of LIGHIVDC with isopropyl-beta-D-thiogalactopyranoside (IPTG). Tat protein can be detected by immunostaining in LIGHIVDC. However, Rev-mediated transport and subsequent translation of the singly spliced and unspliced HIV-1 mRNAs is inefficient. In the absence of Tat, HCMV stimulated CAT activity approximately 20-fold, and this activation required HCMV gene expression but not viral DNA replication. LTR-directed transcription was unaffected by HCMV infection in LIGHIVDC but was inhibited in these cells when they contained increased Tat levels following IPTG induction. These results support the hypothesis that HCMV can induce the HIV-1 LTR when HIV-1 gene expression is minimal and that a threshold level of HIV-1 gene products is necessary for HCMV to inhibit this promoter.
