The M, E, and N structural proteins of the severe acute respiratory syndrome coronavirus are required for efficient assembly, trafficking, and release of virus-like particles

Copyright @ 2008 American Society for Microbiology.The production of virus-like particles (VLPs) constitutes a relevant and safe model to study molecular determinants of virion egress. The minimal requirement for the assembly of VLPs for the coronavirus responsible for severe acute respiratory syndrome in humans (SARS-CoV) is still controversial. Recent studies have shown that SARS-CoV VLP formation depends on either M and E proteins or M and N proteins. Here we show that both E and N proteins must be coexpressed with M protein for the efficient production and release of VLPs by transfected Vero E6 cells. This suggests that the mechanism of SARS-CoV assembly differs from that of other studied coronaviruses, which only require M and E proteins for VLP formation. When coexpressed, the native envelope trimeric S glycoprotein is incorporated onto VLPs. Interestingly, when a fluorescent protein tag is added to the C-terminal end of N or S protein, but not M protein, the chimeric viral proteins can be assembled within VLPs and allow visualization of VLP production and trafficking in living cells by state-of-the-art imaging technologies. Fluorescent VLPs will be used further to investigate the role of cellular machineries during SARS-CoV egress.The University of Hong Kong and the French Ministry of Health

Supertropical semirings and supervaluations

We interpret a valuation $v$ on a ring $R$ as a map $v: R \to M$ into a so called bipotent semiring $M$ (the usual max-plus setting), and then define a \textbf{supervaluation} $\phi$ as a suitable map into a supertropical semiring $U$ with ghost ideal $M$ (cf. [IR1], [IR2]) covering $v$ via the ghost map $U \to M$. The set \Cov(v) of all supervaluations covering $v$ has a natural ordering which makes it a complete lattice. In the case that $R$ is a field, hence for $v$ a Krull valuation, we give a complete explicit description of \Cov(v). The theory of supertropical semirings and supervaluations aims for an algebra fitting the needs of tropical geometry better than the usual max-plus setting. We illustrate this by giving a supertropical version of Kapranov's lemma.Comment: 47 page

Crystal Structure of SARS-CoV-2 Main Protease in Complex with the Non-Covalent Inhibitor ML188

Viral proteases are critical enzymes for the maturation of many human pathogenic viruses and thus are key targets for direct acting antivirals (DAAs). The current viral pandemic caused by SARS-CoV-2 is in dire need of DAAs. The Main protease (M(pro)) is the focus of extensive structure-based drug design efforts which are mostly covalent inhibitors targeting the catalytic cysteine. ML188 is a non-covalent inhibitor designed to target SARS-CoV-1 M(pro), and provides an initial scaffold for the creation of effective pan-coronavirus inhibitors. In the current study, we found that ML188 inhibits SARS-CoV-2 M(pro) at 2.5 microM, which is more potent than against SAR-CoV-1 M(pro). We determined the crystal structure of ML188 in complex with SARS-CoV-2 M(pro) to 2.39 A resolution. Sharing 96% sequence identity, structural comparison of the two complexes only shows subtle differences. Non-covalent protease inhibitors complement the design of covalent inhibitors against SARS-CoV-2 main protease and are critical initial steps in the design of DAAs to treat CoVID 19

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

International audienceCoronavirus M proteins represent the major protein component of the viral envelope. They play an essential role during viral assembly by interacting with all 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, since substituting Asp-211 and Glu-213 with alanine induced retention of the MERS-CoV M in the ER. The second motif, 199 KxGxYR 204 , 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 199 KxGxYR 204 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

Generic trees

We continue the investigation of the Laver ideal ℓ0 and Miller ideal m 0 started in [GJSp] and [GRShSp]; these are the ideals on the Baire space associated with Laver forcing and Miller forcing. We solve several open problems from these papers. The main result is the construction of models for t < add(ℓ0), < add(m 0), where add denotes the additivity coefficient of an ideal. For this we construct amoeba forcings for these forcings which do not add Cohen reals. We show that = ω 2 implies add(m 0) ≤ . We show that , implies cov(ℓ0) ≤ +, cov(m 0) ≤ + respectively. Here cov denotes the covering coefficient. We also show that in the Cohen model cov(m 0) < holds. Finally we prove that Cohen forcing does not add a superperfect tree of Cohen real