TRIM5 alpha Drives SIVsmm Evolution in Rhesus Macaques

<div><p>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. <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003577#s1" target="_blank">Introduction</a> 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.</p></div

Summary of virus mutants and TRIM5 resistance.

<p>Summary of virus mutants and TRIM5 resistance.</p

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

<p>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 (<b>A</b>), SIVsmE543-3 S³⁷ (<b>B</b>), SIVsmE543-3 S⁹⁸ (<b>C</b>), SIVsmE5433-3 S³⁷ S⁹⁸ (<b>D</b>), SIVsmE543-3 QQ⁸⁹ (<b>E</b>), SIVsmE543-3 S³⁷ QQ⁸⁹ (<b>F</b>), SIVsmE543-3 QQ⁸⁹ S⁹⁸ (<b>G</b>) and SIVsmE543-3 S³⁷ QQ⁸⁹ S⁹⁸ (<b>H</b>).</p

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

<p>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.</p

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

<p>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.</p

Passage history of SIV clones.

1<p>: For SIVmac, unrecorded passages in rhesus macaques may be involved before passage in the listed rhesus macaques.</p>2<p>. RhE543 is the only macaque with known TRIM5 genotype (TRIM5^Q/Q).</p

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

<p>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.</p

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

<p>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.</p

A Genome-Wide Investigation of MicroRNA Expression Identifies Biologically-Meaningful MicroRNAs That Distinguish between High-Risk and Low-Risk Intraductal Papillary Mucinous Neoplasms of the Pancreas

<div><p>Background</p><p>Intraductal papillary mucinous neoplasms (IPMNs) are pancreatic ductal adenocarcinoma (PDAC) precursors. Differentiating between high-risk IPMNs that warrant surgical resection and low-risk IPMNs that can be monitored is a significant clinical problem, and we sought to discover a panel of mi(cro)RNAs that accurately classify IPMN risk status.</p><p>Methodology/Principal Findings</p><p>In a discovery phase, genome-wide miRNA expression profiling was performed on 28 surgically-resected, pathologically-confirmed IPMNs (19 high-risk, 9 low-risk) using Taqman MicroRNA Arrays. A validation phase was performed in 21 independent IPMNs (13 high-risk, 8 low-risk). We also explored associations between miRNA expression level and various clinical and pathological factors and examined genes and pathways regulated by the identified miRNAs by integrating data from bioinformatic analyses and microarray analysis of miRNA gene targets. Six miRNAs (miR-100, miR-99b, miR-99a, miR-342-3p, miR-126, miR-130a) were down-regulated in high-risk versus low-risk IPMNs and distinguished between groups (<i>P</i><10⁻³, area underneath the curve (AUC) = 87%). The same trend was observed in the validation phase (AUC = 74%). Low miR-99b expression was associated with main pancreatic duct involvement (<i>P</i> = 0.021), and serum albumin levels were positively correlated with miR-99a (r = 0.52, <i>P</i> = 0.004) and miR-100 expression (r = 0.49, <i>P</i> = 0.008). Literature, validated miRNA:target gene interactions, and pathway enrichment analysis supported the candidate miRNAs as tumor suppressors and regulators of PDAC development. Microarray analysis revealed that oncogenic targets of miR-130a (<i>ATG2B, MEOX2</i>), miR-342-3p (<i>DNMT1</i>), and miR-126 (<i>IRS-1</i>) were up-regulated in high- versus low-risk IPMNs (<i>P</i><0.10).</p><p>Conclusions</p><p>This pilot study highlights miRNAs that may aid in preoperative risk stratification of IPMNs and provides novel insights into miRNA-mediated progression to pancreatic malignancy. The miRNAs identified here and in other recent investigations warrant evaluation in biofluids in a well-powered prospective cohort of individuals newly-diagnosed with IPMNs and other pancreatic cysts and those at increased genetic risk for these lesions.</p></div

Laser capture microdissection (LCM) of epithelium from A) low- grade and B) high-grade IPMN tissue.

<p>Left Panel: Hematoxylin (H&E) stained slide (× 4). Middle Panel: H&E stained slide before LCM (× 4), with the red area representing cells of interest marked for capture. Right Panel: Cap showing adherent cells.</p