TRIM5 is an innate immune sensor for the retrovirus capsid lattice.

TRIM5 is a RING domain-E3 ubiquitin ligase that restricts infection by human immunodeficiency virus (HIV)-1 and other retroviruses immediately following virus invasion of the target cell cytoplasm. Antiviral potency correlates with TRIM5 avidity for the retrovirion capsid lattice and several reports indicate that TRIM5 has a role in signal transduction, but the precise mechanism of restriction is unknown. Here we demonstrate that TRIM5 promotes innate immune signalling and that this activity is amplified by retroviral infection and interaction with the capsid lattice. Acting with the heterodimeric, ubiquitin-conjugating enzyme UBC13-UEV1A (also known as UBE2N-UBE2V1), TRIM5 catalyses the synthesis of unattached K63-linked ubiquitin chains that activate the TAK1 (also known as MAP3K7) kinase complex and stimulate AP-1 and NFκB signalling. Interaction with the HIV-1 capsid lattice greatly enhances the UBC13-UEV1A-dependent E3 activity of TRIM5 and challenge with retroviruses induces the transcription of AP-1 and NF-κB-dependent factors with a magnitude that tracks with TRIM5 avidity for the invading capsid. Finally, TAK1 and UBC13-UEV1A contribute to capsid-specific restriction by TRIM5. Thus, the retroviral restriction factor TRIM5 has two additional activities that are linked to restriction: it constitutively promotes innate immune signalling and it acts as a pattern recognition receptor specific for the retrovirus capsid lattice.

Vpx rescue of HIV-1 from the antiviral state in mature dendritic cells is independent of the intracellular deoxynucleotide concentration.

BACKGROUND: SIVMAC/HIV-2 Vpx recruits the CUL4A-DCAF1 E3 ubiquitin ligase complex to degrade the deoxynucleotide hydrolase SAMHD1. This increases the concentration of deoxynucleotides available for reverse transcription in myeloid cells and resting T cells. Accordingly, transduction of these cells by SIVMAC requires Vpx. Virus-like particles containing SIVMAC Vpx (Vpx-VLPs) also increase the efficiency of HIV-1 transduction in these cells, and rescue transduction by HIV-1, but not SIVMAC, in mature monocyte-derived dendritic cells (MDDCs). Differences in Vpx mechanism noted at that time, along with recent data suggesting that SAMHD1 gains additional restriction capabilities in the presence of type I IFN prompted further examination of the role of Vpx and SAMHD1 in HIV-1 transduction of mature MDDCs. RESULTS: When challenged with Vpx-VLPs, SAMHD1 was degraded in MDDCs even after cells had been matured with LPS, though there was no increase in deoxynucleotide levels. Steady-state levels of HIV-1 late reverse transcription products in mature MDDCs were increased to the same extent by either Vpx-VLPs or exogenous nucleosides. In contrast, only Vpx-VLPs increased the levels of 2-LTR circles and proviral DNA in myeloid cells. These results demonstrate that exogenous nucleosides and Vpx-VLPs both increase the levels of HIV-1 cDNA in myeloid cells, but only Vpx-VLPs rescue 2-LTR circles and proviral DNA in myeloid cells with a previously established antiviral state. Finally, since trans-acting Vpx-VLPs provide long-lasting rescue of HIV-1 vector transduction in the face of the antiviral state, and exogenous nucleosides do not, exogenous nucleosides were used to achieve efficient transduction of MDDCs by vectors that stably encode Vprs and Vpxs from a collection of primate lentiviruses. Vpr from SIVDEB or SIVMUS, Vpx from SIVMAC251 or HIV-2, but not SIVRCM, degraded endogenous SAMHD1, increased steady-state levels of HIV-1 cDNA, and rescued HIV-1 from the antiviral state in MDDCs. CONCLUSION: Inhibition of deoxynucleotide hydrolysis by promoting SAMHD1 degradation is not the only mechanism by which Vpx rescues HIV-1 in MDDCs from the antiviral state. Vpx has an additional effect on HIV-1 transduction of these cells that occurs after completion of reverse transcription and acts independently of deoxynucleotide levels.

Vpx rescues HIV-1 transduction of dendritic cells from the antiviral state established by type 1 interferon.

BACKGROUND: Vpx is a virion-associated protein encoded by SIVSM, a lentivirus endemic to the West African sooty mangabey (Cercocebus atys). HIV-2 and SIVMAC, zoonoses resulting from SIVSM transmission to humans or Asian rhesus macaques (Macaca mulatta), also encode Vpx. In myeloid cells, Vpx promotes reverse transcription and transduction by these viruses. This activity correlates with Vpx binding to DCAF1 (VPRBP) and association with the DDB1/RBX1/CUL4A E3 ubiquitin ligase complex. When delivered experimentally to myeloid cells using VSV G-pseudotyped virus-like particles (VLPs), Vpx promotes reverse transcription of retroviruses that do not normally encode Vpx. RESULTS: Here we show that Vpx has the extraordinary ability to completely rescue HIV-1 transduction of human monocyte-derived dendritic cells (MDDCs) from the potent antiviral state established by prior treatment with exogenous type 1 interferon (IFN). The magnitude of rescue was up to 1,000-fold, depending on the blood donor, and was also observed after induction of endogenous IFN and IFN-stimulated genes (ISGs) by LPS, poly(I:C), or poly(dA:dT). The effect was relatively specific in that Vpx-associated suppression of soluble IFN-ß production, of mRNA levels for ISGs, or of cell surface markers for MDDC differentiation, was not detected. Vpx did not rescue HIV-2 or SIVMAC transduction from the antiviral state, even in the presence of SIVMAC or HIV-2 VLPs bearing additional Vpx, or in the presence of HIV-1 VLPs bearing all accessory genes. In contrast to the effect of Vpx on transduction of untreated MDDCs, HIV-1 rescue from the antiviral state was not dependent upon Vpx interaction with DCAF1 or on the presence of DCAF1 within the MDDC target cells. Additionally, although Vpx increased the level of HIV-1 reverse transcripts in MDDCs to the same extent whether or not MDDCs were treated with IFN or LPS, Vpx rescued a block specific to the antiviral state that occurred after HIV-1 cDNA penetrated the nucleus. CONCLUSION: Vpx provides a tool for the characterization of a potent, new HIV-1 restriction activity, which acts in the nucleus of type 1 IFN-treated dendritic cells.

IBDoc Canadian User Performance Evaluation.

BACKGROUND: Fecal calprotectin (FC) is a stool biomarker that has been shown to be sensitive and specific for mucosal inflammation in patients with inflammatory bowel disease (IBD). The test is limited by the requirement for patients to collect and return stool samples. A home-based FC test may improve test adherence. The aim of this study is to evaluate the usability of the IBDoc, a home-based FC measuring test, and to determine the accuracy of results compared with traditional lab-based ELISA values. METHODS: Patients were prospectively enrolled from 3 tertiary sites across Canada between May and August 2017. Patients completed a questionnaire establishing ease-of-use of the IBDoc. Patients completed a FC measurement using the IBDoc, and results were compared with an ELISA-determined FC measurement on the same stool sample. RESULTS: Sixty-one participants were enrolled in the study (29 CD, 32 UC). Seventy-nine percent of patients (48 of 61) agreed that the IBDoc was easy to use, with 85% (52 of 61) of patients strongly agreeing that they were willing use the home kit in the future. The IBDoc and ELISA measurement comparison showed an 88% agreement across all values. There were no false positives or negatives using qualitative comparison. CONCLUSIONS: The home-based IBDoc FC measuring test is acceptable to patients and correlates extremely well with the standard ELISA-determined FC value. The IBDoc enables clinicians to more easily adopt a treat-to-target approach, improve long-term outcomes, and patients' quality of life with IBD. This study is registered at ClinicalTrials.gov, number NCT03408249.

Usability of a home-based test for the measurement of fecal calprotectin in asymptomatic IBD patients.

The aim of our work was to test the usability of fecal calprotectin (FC) home-based test in inflammatory bowel disease (IBD) patients. METHODS: IBD patients were prospectively recruited. They had to measure FC with a dedicated tool and smartphone application, 5 times at two weeks intervals over an 8 weeks period. They had to fill in a usability questionnaire at the first and the last FC measurement. A System Usability Scale (SUS: 0-100) and the Global Score of Usability (GSU: 0-85) were calculated. FC was also centrally measured by ELISA. RESULTS: Fifty-eight patients were recruited. Forty-two performed at least one FC measurement and 27 performed all the FC requested measurements. The median (IQR) SUS (0-100) at the first and last use were 85 (78-90) and 81 (70-88), respectively; the median (IQR) GSU (0-85) at the first and last use were 74 (69-80) and 77 (68-83), respectively. Adherence to the planned measurements and usability of the tool were higher in females and in less severe disease. The intra-class correlation coefficient between home-based and centrally measured FC was 0.88. CONCLUSION: The adherence to home-based measurement of FC was fair. Usability scores for the home-based test were high. There was a good correlation with the centrally measured FC by ELISA.