Sindbis virus consists of a nucleocapsid core surrounded by a lipid membrane through which penetrate 80 glycoprotein trimers. The structure of the core protein comprising the coat surrounding the genomic RNA has been determined. The polypeptide fold from residue 114 to residue 264 is homologous to that of chymotrypsin-like serine proteinases with catalytic residues His 141, Asp 163 and Ser 215 of the core protein positioned as in other serine proteinases. The C-terminal tryptophan remains in the P1 substrate site subsequent to the autocatalytic cis cleavage of the capsid protein, thus rendering the proteinase inactive. Model building of the Sindbis core protein dimer shows that the nucleocapsid is likely to have T = 4 quasisymmetry.

Structural and mutational analyses of residues within the hydrophobic pocket suggest that budding results in a switch between two conformations of the capsid hydrophobic pocket. This is the first description of a viral budding mechanism in molecular detail.

Background: The coronavirus disease 2019 (COVID-19) pandemic is an emerging threat worldwide. It remains unclear how comorbidities affect the risk of infection and severity of COVID-19. Methods: This is a nationwide retrospective case-control study of 219,961 individuals, aged 18 years or older, whose medical costs for COVID-19 testing were claimed until May 15, 2020. COVID-19 diagnosis and infection severity were identified from reimbursement data using diagnosis codes and on the basis of respiratory support use, respectively. Odds ratios (ORs) were estimated using multiple logistic regression, after adjusting for age, sex, region, healthcare utilization, and insurance status. Results: The COVID-19 group (7,341 of 219,961) was young and had a high proportion of female. Overall, 13.0% (954 of 7,341) of the cases were severe. The severe COVID-19 group had older patients and a proportion of male ratio than did the non-severe group. Diabetes (odds ratio range [

Alphaviruses are enveloped, insect-borne viruses, which contains a positive-sense RNA genome. The protein capsid is surrounded by a lipid membrane, which is penetrated by glycoprotein spikes. The structure of the Sindbis virus (SINV) (the type virus) core protein (SCP) was previously determined and found to have a chymotrypsin-like structure. SCP is a serine proteinase which cleaves itself from a polyprotein. Semliki Forest virus (SFV) is among the most distantly related alphaviruses to SINV. Similar to SCP, autocatalysis is inhibited in SFCP after cleavage of the polyprotein by leaving the carboxy-terminal tryptophan in the specificity pocket. The structures of two different crystal forms (I and II) of SFV core protein (SFCP) have been determined to 3.0 A and 3.3 A resolution, respectively. The SFCP monomer backbone structure is very similar to that of SCP. The dimeric association between monomers, A and B, found in two different crystal forms of SCP is also present in both crystal forms of SFCP. However, a third monomer, C, occurs in SFCP crystal form I. While monomers A and B make a tail-to-tail dimer contact, monomers B and C make a head-to-head dimer contact. A hydrophobic pocket on the surface of the capsid protein, the proposed site of binding of the E2 glycoprotein, has large conformational differences with respect to SCP and, in contrast to SCP, is found devoid of bound peptide. In particular, Tyr184 is pointing out of the hydrophobic pocket in SFCP, whereas the equivalent tyrosine in SCP is pointing into the pocket. The conformation of Tyr184, found in SFCP, is consistent with its availability for iodination, as observed in the homologous SINV cores. This suggests, by comparison with SCP, that E2 binding to cores causes major conformational changes, including the burial of Tyr184, which would stabilize the intact virus on budding from an infected cell. The head-to-tail contacts found in the pentameric and hexameric associations within the virion utilize in the same monomer surface regions as found in the crystalline dimer interfaces.

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SummaryAlphaviruses are a group of icosahedral, positive-strand RNA, enveloped viruses. The membrane bi-layer, which surrounds the ~ 400 Å diameter nucleocapsid, is penetrated by 80 spikes arranged in a T = 4 lattice. Each spike is a trimer of heterodimers consisting of glycoproteins E1 and E2. Cryoelectron microscopy and image reconstruction of Ross River virus showed that the T = 4 quaternary structure of the nucleocapsid consists of pentamer and hexamer clusters of the capsid protein, but not dimers, as have been observed in several crystallographic studies. The El-E2 heterodimers form one-to-one associations with the nucleocapsid monomers across the lipid bilayer. Knowledge of the atomic structure of the capsid protein and our reconstruction allows us to identify capsid-protein residues that interact with the RNA, the glycoproteins, and adjacent capsid-proteins.

In this article, we investigate the application of contactless high frequency ultrasound microbeam stimulation (HFUMS) for determining the invasion potential of breast cancer cells. In breast cancer patients, the finding of tumor metastasis significantly worsens the clinical prognosis. Thus, early determination of the potential of a tumor for invasion and metastasis would significantly impact decisions about aggressiveness of cancer treatment. Recent work suggests that invasive breast cancer cells (MDA-MB-231), but not weakly invasive breast cancer cells (MCF-7, SKBR3, and BT-474), display a number of neuronal characteristics, including expression of voltage-gated sodium channels. Since sodium channels are often co-expressed with calcium channels, this prompted us to test whether single-cell stimulation by a highly focused ultrasound microbeam would trigger Ca2+ elevation, especially in highly invasive breast cancer cells. To calibrate the diameter of the microbeam ultrasound produced by a 200-MHz single element LiNbO3 transducer, we focused the beam on a wire target and performed a pulse-echo test. The width of the beam was ~17 μm, appropriate for single cell stimulation. Membrane-permeant fluorescent Ca2+ indicators were utilized to monitor Ca2+ changes in the cells due to HFUMS. The cell response index (CRI), which is a composite parameter reflecting both Ca2+ elevation and the fraction of responding cells elicited by HFUMS, was much greater in highly invasive breast cancer cells than in the weakly invasive breast cancer cells. The CRI of MDA-MB-231 cells depended on peak-to-peak amplitude of the voltage driving the transducer. These results suggest that HFUMS may serve as a novel tool to determine the invasion potential of breast cancer cells, and with further refinement may offer a rapid test for invasiveness of tumor biopsies in situ.

Structure determination of macromolecules often depends on phase improvement and phase extension by use of real-space averaging of electron density related by noncrystallographic symmetry.