Rotavirus double-layered particles (DLPs) are studied in the gas phase with a high-resolution differential mobility analyzer (DMA). DLPs were transferred to 10 mM aqueous ammonium acetate, ...electrosprayed into the gas phase, converted into primarily singly charged particles, and DMA-analyzed. Up to seven slightly different conformations were resolved, whose apparently random, fast (minutes), and reversible interconversions were followed in real time. They sometimes evolved into just two distinct structures, with periods of one dominating over the other and vice versa. Differences between the DLP structures in solution and in the gas phase are clearly revealed by the smaller DLP diameter found here (60 versus 70 nm). Nevertheless, we argue that the multiple gas-phase conformers observed originate in as many conformations pre-existing in solution. We further hypothesize that these conformers correspond to incomplete DLPs having lost some of the VP6 trimer quintets surrounding each of the 12 5-fold axes. Instances of this peculiar loss have been previously documented by cryoelectron microscopy for the rotavirus Wa strain, as well as via charge detection mass spectrometry for five other rotavirus strains included in the RotaTec vaccine. Evidence of this loss systematically found for all 7 rotavirus types so far studied in aqueous ammonium acetate may be a special feature of this electrolyte.
Infection by different coronaviruses (CoVs) causes alterations in the transcriptional and translational patterns, cell cycle, cytoskeleton, and apoptosis pathways of the host cells. In addition, CoV ...infection may cause inflammation, alter immune and stress responses, and modify the coagulation pathways. The balance between the up- and downregulated genes could explain the pathogenesis caused by these viruses. We review specific aspects of CoV-host interactions. CoV genome replication takes place in the cytoplasm in a membrane-protected microenvironment and may control the cell machinery by locating some of their proteins in the host cell nucleus. CoVs initiate translation by cap-dependent and cap-independent mechanisms. CoV transcription involves a discontinuous RNA synthesis (template switching) during the extension of a negative copy of the subgenomic mRNAs. The requirement for base-pairing during transcription has been formally demonstrated in arteriviruses and CoVs. CoV N proteins have RNA chaperone activity that may help initiate template switching. Both viral and cellular proteins are required for replication and transcription, and the role of selected proteins is addressed.
Zika virus (ZIKV) is a mosquito-borne member of the Flaviviridae family that has been known to circulate for decades causing mild febrile illness. The more recent ZIKV outbreaks in the Americas and ...the Caribbean associated with congenital malformations and Guillain-Barré syndrome in adults have placed public health officials in high alert and highlight the significant impact of ZIKV on human health. New technologies to study the biology of ZIKV and to develop more effective prevention options are highly desired. In this study we demonstrate that direct delivery in mice of an infectious ZIKV cDNA clone allows the rescue of recombinant (r)ZIKV in vivo. A bacterial artificial chromosome containing the sequence of ZIKV strain Paraiba/2015 under the control of the cytomegalovirus promoter was complexed with a commercial transfection reagent and administrated using different routes in type-I interferon receptor deficient A129 mice. Clinical signs and death associated with ZIKV viremia were observed in mice. The rZIKV recovered from these mice remained fully virulent in a second passage in mice. Interestingly, infectious rZIKV was also recovered after intraperitoneal inoculation of the rZIKV cDNA in the absence of transfection reagent. Further expanding these studies, we demonstrate that a single intraperitoneal inoculation of a cDNA clone encoding an attenuated rZIKV was safe, highly immunogenic, and provided full protection against lethal ZIKV challenge. This novel in vivo reverse genetics method is a potentially suitable delivery platform for the study of wild-type and live-attenuated ZIKV devoid of confounding factors typical associated with in vitro systems. Moreover, our results open the possibility of employing similar in vivo reverse genetic approaches for the generation of other viruses and, therefore, change the way we will use reverse genetics in the future.
Replication of the coronavirus genome requires continuous RNA synthesis, whereas transcription is a discontinuous process unique among RNA viruses. Transcription includes a template switch during the ...synthesis of subgenomic negative-strand RNAs to add a copy of the leader sequence. Coronavirus transcription is regulated by multiple factors, including the extent of base-pairing between transcription-regulating sequences of positive and negative polarity, viral and cell protein-RNA binding, and high-order RNA-RNA interactions. Coronavirus RNA synthesis is performed by a replication-transcription complex that includes viral and cell proteins that recognize
cis
-acting RNA elements mainly located in the highly structured 5′ and 3′ untranslated regions. In addition to many viral nonstructural proteins, the presence of cell nuclear proteins and the viral nucleocapsid protein increases virus amplification efficacy. Coronavirus RNA synthesis is connected with the formation of double-membrane vesicles and convoluted membranes. Coronaviruses encode proofreading machinery, unique in the RNA virus world, to ensure the maintenance of their large genome size.
A previously unknown coronavirus, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), was isolated in Wuhan, China in December 2019, from a patient with a respiratory disease linked to ...potential contact with wild animals ....
Fractional killing is the main cause of tumour resistance to chemotherapy. This phenomenon is observed even in genetically identical cancer cells in homogeneous microenvironments. To understand this ...variable resistance, here we investigate the individual responses to TRAIL in a clonal population of HeLa cells using live-cell microscopy and computational modelling. We show that the cellular mitochondrial content determines the apoptotic fate and modulates the time to death, cells with higher mitochondrial content are more prone to die. We find that all apoptotic protein levels are modulated by the mitochondrial content. Modelling the apoptotic network, we demonstrate that these correlations, and especially the differential control of anti- and pro-apoptotic protein pairs, confer mitochondria a powerful discriminatory capacity of apoptotic fate. We find a similar correlation between the mitochondria and apoptotic proteins in colon cancer biopsies. Our results reveal a different role of mitochondria in apoptosis as the global regulator of apoptotic protein expression.
Middle East respiratory syndrome coronavirus (MERS-CoV) is an emerging coronavirus infecting humans that is associated with acute pneumonia, occasional renal failure, and a high mortality rate and is ...considered a threat to public health. The construction of a full-length infectious cDNA clone of the MERS-CoV genome in a bacterial artificial chromosome is reported here, providing a reverse genetics system to study the molecular biology of the virus and to develop attenuated viruses as vaccine candidates. Following transfection with the cDNA clone, infectious virus was rescued in both Vero A66 and Huh-7 cells. Recombinant MERS-CoVs (rMERS-CoVs) lacking the accessory genes 3, 4a, 4b, and 5 were successfully rescued from cDNA clones with these genes deleted. The mutant viruses presented growth kinetics similar to those of the wild-type virus, indicating that accessory genes were not essential for MERS-CoV replication in cell cultures. In contrast, an engineered mutant virus lacking the structural E protein (rMERS-CoV-ΔE) was not successfully rescued, since viral infectivity was lost at early passages. Interestingly, the rMERS-CoV-ΔE genome replicated after cDNA clone was transfected into cells. The infectious virus was rescued and propagated in cells expressing the E protein in trans, indicating that this virus was replication competent and propagation defective. Therefore, the rMERS-CoV-ΔE mutant virus is potentially a safe and promising vaccine candidate to prevent MERS-CoV infection.
Since the emergence of MERS-CoV in the Arabian Peninsula during the summer of 2012, it has already spread to 10 different countries, infecting around 94 persons and showing a mortality rate higher than 50%. This article describes the development of the first reverse genetics system for MERS-CoV, based on the construction of an infectious cDNA clone inserted into a bacterial artificial chromosome. Using this system, a collection of rMERS-CoV deletion mutants has been generated. Interestingly, one of the mutants with the E gene deleted was a replication-competent, propagation-defective virus that could only be grown in the laboratory by providing E protein in trans, whereas it would only survive a single virus infection cycle in vivo. This virus constitutes a vaccine candidate that may represent a balance between safety and efficacy for the induction of mucosal immunity, which is needed to prevent MERS-CoV infection.
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