The C-terminal domain of the MERS coronavirus M protein contains a trans-Golgi network localization signal.

Coronavirus M proteins represent the major protein component of the viral envelope. They play an essential role during viral assembly by interacting with all of the other structural proteins. Coronaviruses bud into the endoplasmic reticulum (ER)-Golgi intermediate compartment (ERGIC), but the mechanisms by which M proteins are transported from their site of synthesis, the ER, to the budding site remain poorly understood. Here, we investigated the intracellular trafficking of the Middle East respiratory syndrome coronavirus (MERS-CoV) M protein. Subcellular localization analyses revealed that the MERS-CoV M protein is retained intracellularly in the trans-Golgi network (TGN), and we identified two motifs in the distal part of the C-terminal domain as being important for this specific localization. We identified the first motif as a functional diacidic DxE ER export signal, because substituting Asp-211 and Glu-213 with alanine induced retention of the MERS-CoV M in the ER. The second motif, 199KxGxYR204, was responsible for retaining the M protein in the TGN. Substitution of this motif resulted in MERS-CoV M leakage toward the plasma membrane. We further confirmed the role of 199KxGxYR204 as a TGN retention signal by using chimeras between MERS-CoV M and the M protein of infectious bronchitis virus (IBV). Our results indicated that the C-terminal domains of both proteins determine their specific localization, namely TGN and ERGIC/cis-Golgi for MERS-M and IBV-M, respectively. Our findings indicate that MERS-CoV M protein localizes to the TGN because of the combined presence of an ER export signal and a TGN retention motif.

HCoV-229E spike protein fusion activation by trypsin-like serine proteases is mediated by proteolytic processing in the S2' region.

Human coronavirus 229E (HCoV-229E) is responsible for common colds. Like other coronaviruses, HCoV-229E exploits cellular proteases to activate fusion mediated by the spike protein. We analysed the proteolytic processing of the HCoV-229E spike protein by trypsin-like serine proteases leading to activation of the fusion process. Unlike in other coronaviruses, HCoV-229E fusion activation appears to be a one-step process. Indeed, cleavage of the S1/S2 interface does not seem to be a prerequisite, and the fusion activation is highly reliant on the S2' region, with arginine residue 683 acting as the recognition site.

Aging increases circulating BH2 without modifying BH4 levels and impairs peripheral vascular function in healthy adults.

Little is known about the mechanisms of aging on vascular beds and its relationship with tetra and di-hydrobiopterin (BH4 and BH2) levels. This observational clinical study analyzed the impact of aging on plasma and platelet biopterins, cutaneous blood flow (CBF), and coronary flow reserve (CFR) in healthy adults. The study enrolled healthy adults in 3 age groups: 18-30, 50-59, and 60-70 years (n = 25/group). Biopterins were assessed by LC-MS/MS using newly defined pre-analytical conditions limiting BH4 oxidation and improving long-term stability. CBF was measured by Laser Speckle Contrast Imaging coupled with acetylcholine-iontophoresis and CFR by adenosine stress cardiac magnetic resonance. In healthy adults, aging (60-70 years vs 18-30 years) significantly increased platelet BH2 (+75%, P = 0.033) and BH2 + BH4 (+31%, P = 0.033), and to a lesser extent plasma BH2 (+29%, P = 0.009) without affecting BH4 and BH4/BH2. Simultaneously, CBF was decreased (-23%, P = 0.004) but not CFR, CBF being inversely correlated with platelet BH2 (r = -0.42, P = 0.001) and BH2 + BH4 (r = -0.41, P = 0.002). The proportion of adults with abnormal platelet BH2 increased with age (+28% in 60-70y). These abnormal BH2 levels were significantly associated with reduced CBF and CFR (-16%, P = 0.03 and -26%, P = 0.02). In conclusion, our study showed that age-related peripheral endothelial dysfunction was associated with an increase in circulating BH2 without decreasing BH4, the effect being more marked in platelets, the most relevant blood compartment to assess biopterin bioavailability. Peripheral but not coronary vascular function is progressively impaired with aging in healthy adults. All these findings support biopterins as therapeutic targets to improve vascular function.