Interfering residues narrow the spectrum of MLV restriction by human TRIM5alpha

Abstract

TRIM5alpha is a restriction factor that limits infection of human cells by so-called N- but not B- or NB-tropic strains of murine leukemia virus (MLV). Here, we performed a mutation-based functional analysis of TRIM5alpha-mediated MLV restriction. Our results reveal that changes at tyrosine(336) of human TRIM5alpha, within the variable region 1 of its C-terminal PRYSPRY domain, can expand its activity to B-MLV and to the NB-tropic Moloney MLV. Conversely, we demonstrate that the escape of MLV from restriction by wild-type or mutant forms of huTRIM5alpha can be achieved through interdependent changes at positions 82, 109, 110, and 117 of the viral capsid. Together, our results support a model in which TRIM5alpha-mediated retroviral restriction results from the direct binding of the antiviral PRYSPRY domain to the viral capsid, and can be prevented by interferences exerted by critical residues on either one of these two partners.

Figure 1. Stable Expression of Wild-Type or PRYSPRY V1 Mutant Forms of huTRIM5α

(A) Schematic representation of the domains present in huTRIM5α. Numbers refer to the amino acid position. V1 through V4 designate the four variable regions found in the PRYSPRY domain. The V1 amino acid sequence is shown below, with mutated amino acids (334 to 339) indicated with an asterisk (*). Below is a western blot analysis of extracts from MDTF cells stably transduced with retroviral vectors expressing HA-tagged versions of these huTRIM5α derivatives, using HA (top) and PCNA (bottom)-specific antibodies. (B) Same analysis, with derivatives carrying the indicated amino acid substitutions or a deletion (Δ) at position 336.

Figure 2. HuTRIM5α_Y336_A Potently Blocks B-MLV at an Early Stage of Infection

(A) MDTF cells expressing wild-type (WT) or indicated mutant forms of huTRIM5α, or transduced with a control vector (vector), were challenged with serial 2-fold dilutions of MLV-based or HIV-1-based GFP vectors initially titered on permissive MDTF cells. Infections were scored at 48 hours post-infection by FACS. Curves are representatives of at least two independent experiments, and phenotype observed with huTRIM5α_Y336_A was confirmed in three independently obtained stable cell lines. (B) Cells expressing wild-type or Y³³⁶A mutated huTRIM5α, or control cells (vector), were challenged with equal doses of N-, B- or Mo-MLV. Cellular DNA extractions were performed before (0 h) or 6 h, 8 h or 5 days post-infection. Intermediate minus strand DNA reverse transcription products were amplified by PCR. For each cell line, transduction was performed in triplicate in the absence (1, 2) or presence (-) of azidothymidine (AZT), a reverse transcription inhibitor. Water (C⁻) and the plasmid pCNCG (C⁺) were used as negative and positive controls for the PCR. The mouse peripheral myelin protein (Pmp22) gene was used as a loading control. (C) In parallel of the experiment described in (B), the percentage of GFP-positive cells was determined 5 days post-infection by flow cytometry.

Figure 3. Differential Restriction Spectrum of huTRIM5α Y³³⁶ Mutants

MDTF cells expressing indicated huTRIM5α derivatives, as documented in Figure 1B, were challenged with serial dilutions of MLV- and HIV-based GFP vectors, and analyzed by flow cytometry 2 days later. The resulting infectivity curves are representatives of at least two independent experiments.

Figure 4. B-MLV and HIV Restriction Are Mutually Exclusive

(A) Single and double point mutations (*) were introduced at positions 332 and 336 in the V1 region of huTRIM5α as illustrated. (B) MDTF cell lines stably expressing these mutants were engineered by retroviral transduction, and analyzed by western blot as described in Figure 1. (C) These cells were challenged with 2-fold dilutions of either N-MLV, B-MLV, Mo-MLV or HIV-derived GFP-expressing vectors, and infections were scored by flow cytometry as described in Figure 2. Plots are representative of at least two independent experiments.

Figure 5. Context-Specific Influence of MLV Capsid Residue 110

MDTF cells stably expressing wild-type or mutant versions of huTRIM5α, or containing a control vector, were challenged with N-, B- or Mo-MLV-derived vectors packaged with wild-type or CA110-mutated capsids, as indicated. Infections were scored as described in Figure 2.

Figure 6. MLV CA82 and CA117 Can Interfere with TRIM5α-Mediated Restriction

(A) Amino acids sequence alignments of Mo-, N- and B-MLV capsids. Residues that differ between Mo-MLV and N-MLV and/or B-MLV are highlighted in grey. The residues in italics at positions 109, 110 and 159 represent the only amino acids differences between N- and B-MLV capsids. Positions targeted by site-directed mutagenesis in the present study are indicated by an arrow. (B) Infectivity assays with indicated cell lines and vectors, performed as described in Figure 2. Residues at positions 82 and 117 exert different influences whether in an N- or B-MLV context.

Figure 7. Susceptibility to huTRIM5α-Mediated Restriction of Various MLV Derivatives

^aIn addition to CA110, CA82, CA109, CA117, and CA159, Moloney MLV capsid differs from N- and B-MLV CA at eight other positions (CA4, CA46, CA147, CA202, CA214, CA229, CA242, CA244). Fold restriction: -, less than 2; (+), 2 to 5; +, 5 to 10; ++, more than 10 (calculated as described in Materials and Methods); ND: not done. The one-letter abbreviation of amino acids was used to designate residues present at CA110, CA82, CA109, CA117, and CA159 (A: alanine, D: aspartate, E: glutamate, G: glycine, H: histidine, L: leucine, N: asparagine, Q: glutamine, R: arginine, T: threonine).

Figure 8. Highlighting of Functionally Critical Positions on the Structure of the MLV Capsid

Visualization of the previously defined structure of the N-terminal domain of the N-MLV capsid in its hexameric state, each monomer being given a different color. Secondary structures in one monomer with the six α helices (α1–6) and two β-hairpins (β1 and β2) are represented in the lower right panel. The four residues at positions 82, 109, 110 and 117 are shown with their respective side chains. Atoms in the side chains are colored in grey for carbon, white for hydrogen, red for oxygen and blue for nitrogen. In the upper right panel, the molecular surface of the area framed in the red-colored monomer is enlarged and viewed in the orientation indicated by the arrow. Surfaces of the four residues at positions 82, 109, 110, and 117 are colored.