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Assembly and composition of intracellular particles formed by Moloney murine leukemia virus
M Hansen1, L Jelinek, R S Jones
1Vollum Institute for Advanced Biomedical Research, Department of Microbiology and Immunology, Oregon Health Sciences University, Portland 97201.
Abstract:
Assembly of type C retroviruses such as Moloney murine leukemia virus (M-MuLV) ordinarily occurs at the plasma membranes of infected cells and absolutely requires the particle core precursor protein, Pr65gag. Previously we have shown that Pr65gag is membrane associated and that at least a portion of intracellular Pr65gag protein appears to be routed to the plasma membrane by a vesicular transport pathway. Here we show that intracellular particle formation can occur in M-MuLV-infected cells. M-MuLV immature particles were observed by electron microscopy budding into and within rough endoplasmic reticulum, Golgi, and vacuolar compartments. Biochemical fractionation studies indicated that intracellular Pr65gag was present in nonionic detergent-resistant complexes of greater than 150S. Additionally, viral RNA and polymerase functions appeared to be associated with intracellular particles, as were Gag-beta-galactosidase fusion proteins which have the capacity to be incorporated into virions. Immature intracellular particles in postnuclear lysates could be proteolytically processed in vitro to mature forms, while extracellular immature M-MuLV particles remained immature as long as 10 h during incubations. The occurrence of M-MuLV-derived intracellular particles demonstrates that Pr65gag can associate with intracellular membranes and indicates that if a plasma membrane Pr65gag receptor exists, it also can be found in other membrane compartments. These results support the hypothesis that intracellular particles may serve as a virus reservoir during in vivo infections.
Insights
Moloney murine leukemia virus (M-MuLV) assembly occurs intracellularly, not just at the plasma membrane. This finding suggests M-MuLV particles may form a reservoir during infections.
Area of Science:
- Virology
- Cell Biology
- Molecular Biology
Background:
- Type C retrovirus assembly, like Moloney murine leukemia virus (M-MuLV), typically occurs at the plasma membrane.
- The precursor protein Pr65gag is essential for M-MuLV particle formation and associates with cell membranes.
- Previous research indicated Pr65gag is routed to the plasma membrane via vesicular transport.
Purpose of the Study:
- To investigate the occurrence and characteristics of intracellular M-MuLV particle formation.
- To determine if M-MuLV assembly can initiate within the cell, independent of the plasma membrane.
- To explore the implications of intracellular particle formation for viral reservoirs.
Main Methods:
- Electron microscopy to visualize M-MuLV particles within cellular compartments.
- Biochemical fractionation to identify intracellular Pr65gag complexes.
- Analysis of viral RNA, polymerase activity, and Gag-beta-galactosidase fusion proteins in intracellular particles.
- In vitro processing of immature intracellular and extracellular M-MuLV particles.
Main Results:
- Immature M-MuLV particles were observed budding within the rough endoplasmic reticulum, Golgi, and vacuolar compartments.
- Intracellular Pr65gag was found in large, nonionic detergent-resistant complexes (>150S).
- Viral RNA, polymerase functions, and Gag fusion proteins were associated with intracellular particles, which could be matured in vitro.
Conclusions:
- M-MuLV particle formation can occur intracellularly, associating with membranes beyond the plasma membrane.
- The presence of intracellular particles suggests potential alternative assembly sites and a possible viral reservoir during infection.
- If a plasma membrane receptor for Pr65gag exists, it may also be present in other intracellular membrane compartments.