Date Published: March 19, 2019
Publisher: Public Library of Science
Author(s): Sevnur Kömürlü, Margret Bradley, Nikolai Smolin, Sabrina Imam, Raymond F. Pauszek, Seth L. Robia, David Millar, Emi E. Nakayama, Tatsuo Shioda, Edward M. Campbell, Luis Menéndez-Arias.
TRIM5α is an interferon inducible restriction factor which contributes to intrinsic defense against HIV infection by targeting the HIV capsid protein CA. Although human TRIM5α (huTRIM5α) does not potently inhibit HIV-1 infection, the ability of huTRIM5α to exhibit some control of HIV-1 infection is evidenced by a single nucleotide polymorphism in huTRIM5α which substitutes aspartic acid to glycine at position 249 (G249D) in the L2 region and is associated with higher susceptibility to HIV-1 infection. To understand the mechanistic basis for the reduced antiviral activity, we employed biophysical and cell biological methods coupled with molecular dynamics simulations to compare WT and the G249D polymorphism of huTRIM5α. We investigated the differences in conformational dynamics of rhesus and huTRIM5α Coiled Coil–Linker 2 (CC-L2) dimers utilizing circular dichroism and single molecule-Fluorescence Energy Transfer (sm-FRET). These methods revealed that the G249D dimer exhibits secondary structure and conformational dynamics similar to WT huTRIM5α. Homology modelling revealed that G249 was present on the hairpin of the antiparallel dimer, in a position which may act to stabilize the adjacent BBox2 domain which mediates the inter-dimeric contacts required for the formation of TRIM5 assemblies. We therefore asked if the G249D mutant forms assemblies in cells with the same efficiency as WT protein by expressing these proteins as YFP fusions and quantifying the number of assemblies in cells. In cells expressing comparable amounts of protein, the G249D mutant formed fewer assemblies than WT protein, in agreement with our homology modeling predictions and molecular dynamics simulations of dimers and higher oligomers of TRIM5α, providing a mechanistic explanation of the reduced antiviral activity of the G249D polymorphism.
TRIM5α is a potent restriction factor against retroviruses that mediates a post-entry block to infection by faciltating abortive disassembly of capsid core [1–3]. Like all other TRIpartite Motif (TRIM) family members, TRIM5 alpha contains an N-terminal RING, BBox2 and coiled-coil (CC) domains which define the TRIM family [4, 5].The C-terminal of TRIM5α contains a SPRY/PRY domain connected to the RBCC motif by a linker region (L2). The RING domain mediates E3 ubiquitin ligase activity upon higher order assembly [3, 6–8], the BBox2 domain mediates the higher-order hexagonal assemblies of individual TRIM dimers comprised of the CC domain and L2 region [9–17]. The lattice spacing of TRIM5α hexagonal assemblies matches with the HIV capsid, allowing formation of these assemblies around the viral capsid [18–20]. It is also known that formation of TRIM5α assemblies around the HIV capsid results in the abortive disassembly of the capsid core through a poorly defined mechanism.
Although the magnitude of HIV-1 restriction by huTRIM5α in single cycle infectivity assays is modest, it is worth noting that numerous studies have found that TRIM5α proteins which exhibit modest restriction in single cycle infectivity assays can facilitate immunity in vivo by involving components of the immune system. Moreover, TRIM5α was identified as one of the host factors mediating interferon inhibition of HIV-1 in an unbiased CRISPR screen along with MxB, IFITM1, and Tetherin/BST2. For example, primary cells from rhesus macaques inhibit HIV-1 infection significantly, but measurable infection is observed, even at low doses of virus [64–66], yet TRIM5α is known to provide sterilizing immunity against HIV-1 in rhesus macaques. Similarly, Richardson et al. have observed that mCherry-huTRIM5α provides sterilizing immunity against HIV-1 infection in a humanized mouse model of HIV-1 infection despite observing restriction in vitro that was incomplete and less potent than rhTRIM5alpha . This enhanced potency in vivo is likely due to a combination of the ability of TRIM5α to generate K63-linked ubiquitin chains which upregulate the expression of other antiviral genes  and the cumulative effect of modest restriction amplified over many replication cycles. Similarly, the observation that the G249D mutation induces a reproducible but modest relief of HIV-1 restriction by huTRIM5α in our experiments correlates to a significant increase in higher susceptibility to HIV-1 infection and higher viral titers in HIV infected patients [39–41].
In this study, we investigated a single nucleotide polymorphism in huTRIM5α, which is associated with increased HIV-1 susceptibility and rapid disease progression. We examined the mechanism behind the reduced antiviral activity by variety of biophysical methods and eliminated other possibilities related to secondary structure and conformational dynamics except higher-order assembly formation. Molecular dynamic simulations and in vivo imaging studies revealed that defects in assembly reduce antiviral activity of G249D polymorphism in huTRIM5α.