Identifying binding hot spots on protein surfaces by mixed-solvent molecular dynamics: HIV-1 protease as a test case

The host factor protein TRIM5α plays an important role in restricting the host range of HIV-1, interfering with the integrity of the HIV-1 capsid. TRIM5 triggers an antiviral innate immune response by functioning as a capsid pattern recognition receptor, although the precise mechanism by which the restriction is imposed is not completely understood. Here we used an integrated magic-angle spinning nuclear magnetic resonance and molecular dynamics simulations approach to characterize, at atomic resolution, the dynamics of the capsid’s hexameric and pentameric building blocks, and the interactions with TRIM5α in the assembled capsid. Our data indicate that assemblies in the presence of the pentameric subunits are more rigid on the microsecond to millisecond timescales than tubes containing only hexamers. This feature may be of key importance for controlling the capsid’s morphology and stability. In addition, we found that TRIM5α binding to capsid induces global rigidification and perturbs key intermolecular interfaces essential for higher-order capsid assembly, with structural and dynamic changes occurring throughout the entire CA polypeptide chain in the assembly, rather than being limited to a specific protein-protein interface. Taken together, our results suggest that TRIM5α uses several mechanisms to destabilize the capsid lattice, ultimately inducing its disassembly. Our findings add to a growing body of work indicating that dynamic allostery plays a pivotal role in capsid assembly and HIV-1 infectivity.

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Nature, Position, and Frequency of Mutations Made in a Single Cycle of HIV-1 Replication

Journal of Virology ◽

10.1128/jvi.00915-10 ◽

2010 ◽

Vol 84 (19) ◽

pp. 9864-9878 ◽

Cited By ~ 148

Author(s):

Michael E. Abram ◽

Andrea L. Ferris ◽

Wei Shao ◽

W. Gregory Alvord ◽

Stephen H. Hughes

Keyword(s):

Viral Replication ◽

Mutation Rate ◽

Hot Spots ◽

High Rate ◽

Published Data ◽

Single Cycle ◽

Efficient System ◽

Hiv 1

ABSTRACT There is considerable HIV-1 variation in patients. The extent of the variation is due to the high rate of viral replication, the high viral load, and the errors made during viral replication. Mutations can arise from errors made either by host DNA-dependent RNA polymerase II or by HIV-1 reverse transcriptase (RT), but the relative contributions of these two enzymes to the mutation rate are unknown. In addition, mutations in RT can affect its fidelity, but the effect of mutations in RT on the nature of the mutations that arise in vivo is poorly understood. We have developed an efficient system, based on existing technology, to analyze the mutations that arise in an HIV-1 vector in a single cycle of replication. A lacZα reporter gene is used to identify viral DNAs that contain mutations which are analyzed by DNA sequencing. The forward mutation rate in this system is 1.4 × 10−5 mutations/bp/cycle, equivalent to the retroviral average. This rate is about 3-fold lower than previously reported for HIV-1 in vivo and is much lower than what has been reported for purified HIV-1 RT in vitro. Although the mutation rate was not affected by the orientation of lacZα, the sites favored for mutations (hot spots) in lacZα depended on which strand of lacZα was present in the viral RNA. The pattern of hot spots seen in lacZα in vivo did not match any of the published data obtained when purified RT was used to copy lacZα in vitro.

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