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Analysis of the retrovirus capsid interdomain linker region
Brian Arvidson1, Joshua Seeds, Mike Webb
1Vollum Institute and Department of Microbiology, Oregon Health and Sciences University, 3181 SW Sam Jackson Park Road, Portland, OR 97201-3098, USA.
Abstract:
In structural studies, the retrovirus capsid interdomain linker region has been shown as a flexible connector between the CA N-terminal domain and its C-terminal domain. To analyze the function of the linker region, we have examined the effects of three Moloney murine leukemia virus (M-MuLV) capsid linker mutations/variations in vivo, in the context of the full-length M-MuLV structural precursor protein (PrGag). Two mutations, A1SP and A5SP, respectively, inserted three and seven additional codons within the linker region to test the effects of increased linker lengths. The third variant, HIV/Mo, represented a chimeric HIV-1/M-MuLV PrGag protein, fused at the linker region. When expressed in cells, the three variants reduced the efficiency of virus particle assembly, with PrGag proteins and particles accumulating at the cellular plasma membranes. Although PrGag recognition of viral RNA was not impaired, the capsid linker variant particles were abnormal, with decreased stabilities, anomalous densities, and aberrant multiple lobed and tubular morphologies. Additionally, rather than crosslinking as PrGag dimers, particle-associated A1SP, A5SP, and HIV/Mo proteins showed an increased propensity to crosslink as trimers. Our results suggest that a wild-type retrovirus capsid linker region is required for the proper alignment of capsid protein domains.
Insights
Altering the retrovirus capsid linker region impairs virus assembly and particle stability. A functional linker is crucial for correct capsid protein domain alignment during retroviral replication.
Area of Science:
- Virology
- Structural Biology
- Molecular Biology
Background:
- The retrovirus capsid protein (CA) comprises N-terminal and C-terminal domains linked by a flexible interdomain region.
- This linker's precise role in viral assembly and particle formation remains incompletely understood.
Purpose of the Study:
- To investigate the functional significance of the Moloney murine leukemia virus (M-MuLV) capsid linker region.
- To analyze the in vivo effects of altering linker length and composition on M-MuLV PrGag protein function and virus particle formation.
Main Methods:
- Introduction of three specific variations into the M-MuLV PrGag gene: A1SP (3 additional codons), A5SP (7 additional codons), and HIV/Mo (HIV-1/M-MuLV chimera).
- Expression of modified PrGag proteins in cells to assess effects on virus particle assembly, protein localization, RNA binding, and particle characteristics.
- Analysis of protein crosslinking patterns (dimers vs. trimers) in assembled particles.
Main Results:
- All three linker variants significantly reduced the efficiency of virus particle assembly.
- PrGag proteins and assembled particles accumulated at the cellular plasma membrane.
- Capsid linker variant particles exhibited abnormal morphologies, decreased stability, and anomalous densities.
- While viral RNA recognition was unaffected, particle-associated proteins showed increased trimerization instead of the typical dimerization.
Conclusions:
- The retrovirus capsid linker region plays a critical role in proper viral assembly and particle integrity.
- Alterations in linker length or composition disrupt capsid protein domain alignment, leading to aberrant particle formation.
- A wild-type capsid linker is essential for maintaining the structural fidelity and functional competence of retroviral particles.