Cytoplasmic domains of members of the p24 family of putative cargo receptors were shown to bind to coatomer, the coat protein of COPI-coated transport vesicles. Domains that contained dilysine endoplasmic reticulum retrieval signals bound the alpha-, beta'-, and epsilon-COP subunits of coatomer, whereas other p24 domains bound the beta-, gamma-, and zeta-COP subunits and required a phenylalanine-containing motif. Transit of a CD8-p24 chimera from the endoplasmic reticulum through the Golgi complex was slowed when the phenylalanine motif was mutated, suggesting that this motif may function as an anterograde transport signal. The either-or bimodal binding of coatomer to p24 tails suggests models for how coatomer can potentially package retrograde-directed and anterograde-directed cargo into distinct COPI-coated vesicles.
Synaptotagmin, a likely calcium sensor for synaptic transmission, and SNAP-25, a t-SNARE of the presynaptic plasma membrane, are key proteins for the docking and fusion of synaptic and other vesicles. We report that both synaptotagmin and SNAP-25 are palmitoylated with their fatty acids attached in a labile thioester-type bond. A SNAP-25 mutant with deleted palmitoylation sites is found exclusively in the cytosol after cell fractionation, whereas the palmitoylated form of SNAP-25 is membrane-bound, establishing that SNAP-25 is membraneanchored via covalently linked palmitate.
17The emergence of a novel coronavirus, SARS-CoV-2, resulted in a pandemic. Here, we used X-ray 18 structures of human ACE2 bound to the receptor-binding domain (RBD) of the spike protein (S) from 19 SARS-CoV-2 to predict its binding to ACE2 proteins from different animals, including pets, farm animals, 20 and putative intermediate hosts of SARS-CoV-2. Comparing the interaction sites of ACE2 proteins 21 known to serve or not serve as receptor allows to define residues important for binding. From the 20 22 amino acids in ACE2 that contact S up to seven can be replaced and ACE2 can still function as the SARS-23CoV-2 receptor. These variable amino acids are clustered at certain positions, mostly at the periphery 24 of the binding site, while changes of the invariable residues prevent S-binding or infection of the 25 respective animal. Some ACE2 proteins even tolerate the loss or the acquisition of N-glycosylation sites 26 located near the S-interface. Of note, Pigs and dogs, which are not or not effectively infected and have 27 only a few changes in the binding site, exhibit relatively low levels of ACE2 in the respiratory tract. 28Comparison of the RBD of S of SARS-CoV-2 with viruses from Bat-CoV-RaTG13 and Pangolin-CoV 29 revealed that the latter contains only one substitution, whereas the Bat-CoV-RaTG13 exhibits five. 30However, ACE2 of pangolin exhibit seven changes relative to human ACE2, a similar number of 31 substitutions is present in ACE2 of bats, raccoon, and civet suggesting that SARS-CoV-2 may not 32 especially adapted to ACE2 of any of its putative intermediate hosts. These analyses provide new 33 insight into the receptor usage and animal source/origin of SARS-CoV-2. 34 IMPORTANCE 35 SARS-CoV-2 is threatening people worldwide and there are no drugs or vaccines available to mitigate 36 its spread. The origin of the virus is still unclear and whether pets and livestock can be infected and 37 transmit SARS-CoV-2 are important and unknown scientific questions. Effective binding to the host 38 receptor ACE2 is the first prerequisite for infection of cells and determines the host range. Our analysis 39 provides a framework for the prediction of potential hosts of SARS-CoV-2. We found that ACE2 from 40 species known to support SARS-CoV-2 infection tolerate many amino acid changes indicating that the 41 species barrier might be low. An exception are dogs and especially pigs, which, however, revealed 42
In 2011, a new virus was isolated from pigs with influenza-like symptoms and subsequently also from cattle, which are the main reservoir of the virus. It is similar to Influenza C virus (ICV), a (predominantly) human pathogen, causing respiratory disease in children. Since the virus is unable to reassort with ICV (and based on several other criteria as discussed in the text) it is now officially named as Influenzavirus D (IDV), a new genus of the Orthomyxoviridae. We summarize the epidemiology, pathology and evolution of IDV and its biological characteristics with emphasis on the only glycoprotein HEF. Based on the limited data available we finally consider whether IDV represent a public health threat.
Influenza virus hemagglutinin (HA) has been suggested to be enriched in liquid-ordered lipid domains named rafts, which represent an important step in virus assembly. We employed
It has been supposed that the HA (haemagglutinin) of influenza virus must be recruited to membrane rafts to perform its function in membrane fusion and virus budding. In the present study, we aimed at substantiating this association in living cells by biophysical methods. To this end, we fused the cyan fluorescent protein Cer (Cerulean) to the cytoplasmic tail of HA. Upon expression in CHO (Chinese-hamster ovary) cells HA-Cer was glycosylated and transported to the plasma membrane in a similar manner to authentic HA. We measured FLIM-FRET (Förster resonance energy transfer by fluorescence lifetime imaging microscopy) and showed strong association of HA-Cer with Myr-Pal-YFP (myristoylated and palmitoylated peptide fused to yellow fluorescent protein), an established marker for rafts of the inner leaflet of the plasma membrane. Clustering was significantly reduced when rafts were disintegrated by cholesterol extraction and when the known raft-targeting signals of HA, the palmitoylation sites and amino acids in its transmembrane region, were removed. FRAP (fluorescence recovery after photobleaching) showed that removal of raft-targeting signals moderately increased the mobility of HA in the plasma membrane, indicating that the signals influence access of HA to slowly diffusing rafts. However, Myr-Pal-YFP exhibited a much faster mobility compared with HA-Cer, demonstrating that HA and the raft marker do not diffuse together in a stable raft complex for long periods of time.
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