TRIM5 alpha drives SIVsmm evolution in rhesus macaques

Abstract

The antagonistic interaction with host restriction proteins is a major driver of evolutionary change for viruses. We previously reported that polymorphisms of the TRIM5α B30.2/SPRY domain impacted the level of SIVsmm viremia in rhesus macaques. Viremia in macaques homozygous for the non-restrictive TRIM5α allele TRIM5(Q) was significantly higher than in macaques expressing two restrictive TRIM5alpha alleles TRIM5(TFP/TFP) or TRIM5(Cyp/TFP). Using this model, we observed that despite an early impact on viremia, SIVsmm overcame TRIM5α restriction at later stages of infection and that increasing viremia was associated with specific amino acid substitutions in capsid. Two amino acid substitutions (P37S and R98S) in the capsid region were associated with escape from TRIM5(TFP) restriction and substitutions in the CypA binding-loop (GPLPA87-91) in capsid were associated with escape from TRIM5(Cyp). Introduction of these mutations into the original SIVsmE543 clone not only resulted in escape from TRIM5α restriction in vitro but the P37S and R98S substitutions improved virus fitness in macaques with homozygous restrictive TRIM(TFP) alleles in vivo. Similar substitutions were observed in other SIVsmm strains following transmission and passage in macaques, collectively providing direct evidence that TRIM5α exerts selective pressure on the cross-species transmission of SIV in primates.

Figure 1. Replication of SIVsmE543-3 in rhesus macaques with different TRIM5 alleles.

Viral loads (red lines) were quantified and shown as RNA copies in plasma samples from rhesus macaques Rh447 (A, TRIM5^TFP/TFP), Rh458 (B, TRIM5^TFP/CypA), Rh063 (C, TRIM5 ^TFP/CypA) and Rh444 (D, TRIM5^Q/Q). Peripheral CD4⁺ T cells (blue lines) were quantified by FACS and shown as the absolute numbers per microliter of blood.

Figure 2. SIV clones from macaques with restrictive TRIM5 alleles overcame restriction at later stage of infection.

Single-cycle infectivity of SIV clones from Rh447 (A, TRIM5^TFP/TFP) and Rh458 (B, TRIM5^TFP/CypA) was measured on a panel of cell lines stably expressing the rhesus TRIM5^TFP allele (dark blue bars), TRIM5^Q allele (light blue bars) and TRIM5^CypA (orange bars). Infectivity was measured as percent GFP positive cells. Black bars are negative vector controls. SIV clones SIVmac239 (C) and SIVsmE543-3 (D) were used as controls.

Figure 3. Identification of amino acid substitutions associated with escape from TRIM5 restriction.

The capsid amino acids of SIV clones from Rh444 (TRIM5^Q/Q), Rh447 (TRIM5^TFP/TFP), Rh458 (TRIM5^TFP/CypA) and Rh063 (TRIM5^TFP/CypA) were aligned to parental SIVsmE543-3. Identical amino acids were shown as dot (.), deletions are shown as dash (-). Amino acid substitutions shared among SIV clones from different macaques were highlighted with yellow. Amino acid substitutions in the CypA binding loop are highlighted in red. The critical amino acid residues identified as responsible for escape from TRIM restriction are indicated by numbers above the sequence.

Figure 4. Introduction of amino acid substitutions into SIVsmE543-3 capsid conferred virus resistance to TRIM5 restriction.

Single or combinations of amino acid substitutions “P37S”, “LPA89QQ” and “R98S” were introduced into SIVsmE543-3 capsid. Single-cycle infectivity of these mutants was measured on a panel of cell lines stably expressing a TRIM5^TFP allele (Mamu-2, dark blue bars), TRIM5^Q alleles (Mamu-4, light blue bars) and TRIM5^CypA (orange bars). Infectivity was measured as percent GFP positive cells. Black bars are negative vector controls. Infectivity on this panel are shown for SIVsmE543-3 (A), SIVsmE543-3 S³⁷ (B), SIVsmE543-3 S⁹⁸ (C), SIVsmE5433-3 S³⁷ S⁹⁸ (D), SIVsmE543-3 QQ⁸⁹ (E), SIVsmE543-3 S³⁷ QQ⁸⁹ (F), SIVsmE543-3 QQ⁸⁹ S⁹⁸ (G) and SIVsmE543-3 S³⁷ QQ⁸⁹ S⁹⁸ (H).

Figure 5. Comparison of PBMC infection with SIVsmE543-3 and SIVsmE543-3 S³⁷S⁹⁸.

PBMCs were collected from 18 macaques with a TRIM5^TFP/TFP genotype and 5 macaques with a TRIM5^Q/Q genotype and activated with PHA for 3 days. Activated PBMCs were infected with SIVsmE543-3 and SIVsmE543-3 S³⁷S⁹⁸ at a M.O.I. of 0.001. Virus production was quantified and shown as RT values in supernatants collected at 3-day intervals. A and B show the replication kinetics of SIVsmE543-3 and SIVsmE543-3 S³⁷S⁹⁸ in PBMCs of representative macaques RhDBF7 (TRIM5^TFP/TFP) and RhDCCW (TRIM5^Q/Q). Area under the replication curve (AUC) of SIVsmE543-3 and the variant were calculated for each macaque separately. The AUC of SIVsmE543-3 and its variant in the TRIM5^TFP/TFP group (C) and the TRIM5^Q/Q group (D) are shown and compared by paired t test.

Figure 6. Acquisition and replication of SIVsmE543-3 and SIVsmE543-3 S³⁷S⁹⁸ in macaques with TRIM5^TFP/TFP genotype.

Each macaque was inoculated intrarectally (I.R.) with 1000 TCID₅₀ (5×10⁵ RNA copies of virus) and the infection was monitored by measuring plasma viral RNA load. Four weeks later any of the macaques that remained uninfected were inoculated intrarectally on a weekly schedule with same amount of virus until they became infected. The acquisition of infection in each group was shown as uninfected percentage after each inoculation and compared by log-rank test. Median inoculation time was 3.5 for SIVsmE543-3 challenge group and 1 for SIVsmE543-3 S³⁷S⁹⁸ challenge group (A). Plasma viral RNA copies in each macaque (B) and median plasma viral RNA copies in each group (C) are shown. Peak plasma viral loads (D, P = 0.0152), plasma viral loads at 8 w.p.i. (E, P = 0.0411) and viral load AUC before 8 w.p.i. (F, P = 0.0260) were compared by non-parametric Mann-Whitney-test.

Figure 7. Variance of SIV capsid sequences is associated with their passage history in macaques.

The capsid N-terminal domain of SIV clones, with or without passage in rhesus, stump-tailed and pigtail macaques, were aligned to a primary SIVsm clone from sooty mangabey (top). Identical amino acids were shown as a dot (.), deletions are shown as a dash (-). The sites under TRIM5 selection are highlighted in yellow and the Cyclophilin-A binding site is highlighted in light blue and the critical amino acid residues identified as responsible for escape from TRIM restriction are indicated by numbers above the sequence.