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A Restriction Enzyme Based Cloning Method to Assess the In vitro Replication Capacity of HIV-1 Subtype C Gag-MJ4 Chimeric Viruses
Published on: August 31, 2014
Low-level HIV replication in mixed glial cultures is associated with alterations in the processing of p55(Gag)
Andrew V Albright1, Robin M Vos, Francisco González-Scarano
1Department of Neurology, University of Pennsylvania, Philadelphia, PA 19104-6146, USA.
Abstract:
We report a novel long-lived infection model in human mixed glial cultures (microglia) whereby cells harbor replication-competent HIV-1 for up to 2.5 months after infection; a model that potentially mimics latency within the central nervous system (CNS). Infection of mixed glial cultures in the presence of serum, cytokines, and growth factors (activating conditions) resulted in a robust productive infection of microglial cells as previously described for purified microglia. In contrast, similar mixed glial cells cultured in serum-free medium without cytokines or growth factors (mirroring a nonactivated CNS) supported HIV-1 entry, reverse transcription, integration, and transcription, yet released little or no infectious virus. We found instead that nonactivated mixed glial cells expressed almost 10-fold less Gag protein, but more importantly, analysis of the intracellular Gag products in quiescent cells showed an aberrant p55/p24 Gag processing phenotype that appeared to be due to the premature activity of the viral protease. These results suggest that the cellular environment in nonactivated microglia cells in these mixed glial cultures is not conducive to proper Gag processing and virus release. This long-lived infection model will be useful in identifying factors that are key for viral maturation in cells of the macrophage lineage.
Insights
Researchers developed a novel model of human glial cells that harbor HIV-1 for months, mimicking central nervous system latency. This model reveals how nonactivated microglia hinder HIV-1 maturation and release due to aberrant Gag protein processing.
Area of Science:
- Neuroscience
- Virology
- Immunology
Background:
- Microglia play a crucial role in central nervous system (CNS) immunity and are a key target for Human Immunodeficiency Virus type 1 (HIV-1).
- Understanding HIV-1 latency in the CNS is critical for developing effective therapeutic strategies.
- Previous models have limitations in recapitulating the complex cellular environment of the CNS.
Purpose of the Study:
- To establish a novel, long-lived HIV-1 infection model in human mixed glial cultures that mimics CNS latency.
- To investigate the mechanisms underlying HIV-1 replication and maturation in nonactivated microglia.
- To identify factors influencing viral release from infected glial cells.
Main Methods:
- Development of a human mixed glial culture model infected with replication-competent HIV-1.
- Culturing cells under activating (serum, cytokines, growth factors) and nonactivating (serum-free medium) conditions.
- Analysis of HIV-1 entry, reverse transcription, integration, transcription, Gag protein expression, and processing.
Main Results:
- Nonactivated mixed glial cells supported HIV-1 entry, reverse transcription, integration, and transcription but released minimal infectious virus.
- Quiescent microglia exhibited significantly reduced Gag protein expression (approx. 10-fold less).
- Intracellular Gag processing in nonactivated cells was aberrant, suggesting premature viral protease activity and impaired virus maturation.
Conclusions:
- The cellular environment of nonactivated microglia in mixed glial cultures is unfavorable for proper HIV-1 Gag processing and virus release.
- This novel long-lived infection model provides a valuable tool for studying HIV-1 latency and maturation in macrophage lineage cells within the CNS.
- Findings highlight the importance of microglial activation state in controlling HIV-1 replication and persistence in the central nervous system.
