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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
A block to human immunodeficiency virus type 1 assembly in murine cells
R Mariani1, G Rutter, M E Harris
1Infectious Disease Laboratory, The Salk Institute for Biological Studies, La Jolla, California 92037, USA.
Insights
Murine cells block human immunodeficiency virus type 1 (HIV-1) replication, primarily due to a major defect in virion assembly and Gag protein targeting. Overcoming this block is crucial for developing a murine model for HIV-1 replication.
Area of Science:
- Virology
- Cell Biology
- Immunology
Background:
- Human immunodeficiency virus type 1 (HIV-1) cannot replicate in murine cells.
- Understanding this replication block is essential for developing animal models for HIV-1 research.
Purpose of the Study:
- To investigate the specific post-entry steps responsible for the lack of HIV-1 replication in murine cells.
- To identify the molecular mechanisms underlying the block in HIV-1 replication in a murine cell line.
Main Methods:
- Utilized a murine NIH 3T3 reporter cell line expressing human CD4, CCR5, and cyclin T1 with an HIV-1 LTR-GFP cassette.
- Analyzed post-entry replication steps including entry, reverse transcription, integration, provirus expression, and virion assembly using pseudotyped viruses.
- Employed electron microscopy to visualize virion assembly and protein localization.
Main Results:
- HIV-1 efficiently entered murine cells, formed provirus, and expressed it, with human cyclin T1 restoring expression.
- Reverse transcription and integration occurred at levels comparable to human cells.
- A major block was identified at the virion assembly stage, with aberrant Gag protein accumulation in cytoplasmic vesicles and rare virions at the cell membrane.
Conclusions:
- The primary barrier to HIV-1 replication in murine cells is a defect in virion assembly, likely due to failed Gag protein targeting to the cell membrane.
- Despite efficient early steps, the inability to assemble and release infectious virions prevents productive HIV-1 replication in these cells.
- Addressing the virion assembly block is necessary for establishing a functional murine model for HIV-1 replication.
Abstract:
Human immunodeficiency virus type 1 (HIV-1) does not replicate in murine cells. We investigated the basis of this block by infecting a murine NIH 3T3 reporter cell line that stably expressed human CD4, CCR5, and cyclin T1 and contained a transactivatable HIV-1 long terminal repeat (LTR)-green fluorescent protein (GFP) cassette. Although the virus entered efficiently, formed provirus, and was expressed at a level close to that in a highly permissive human cell line, the murine cells did not support M-tropic HIV-1 replication. To determine why the virus failed to replicate, the efficiency of each postentry step in the virus replication cycle was analyzed using vesicular stomatitis virus G pseudotypes. The murine cells supported reverse transcription and integration at levels comparable to those in the human osteosarcoma-derived cell line GHOST.R5, and human cyclin T1 restored provirus expression, consistent with earlier findings of others. The infected murine cells contained nearly as much virion protein as did the human cells but released less than 1/500 the amount of p24(gag) into the culture medium. A small amount of p24(gag) was released and was in the form of fully infectious virus. Electron microscopy suggested that aberrantly assembled virion protein had accumulated in cytoplasmic vesicular structures. Virions assembling at the cell membrane were observed but were rare. The entry of M-tropic JR.FL-pseudotyped reporter virus was moderately reduced in the murine cells, suggesting a minor reduction in coreceptor function. A small reduction in the abundance of full-length viral mRNA transcripts was also noted; however, the major block was at virion assembly. This could have been due to a failure of Gag to target to the cell membrane. This block must be overcome before a murine model for HIV-1 replication can be developed.
