Electron cryo-tomographic structure of cystovirus phi 12

Bacteriophage phi 12 is a member of the Cystoviridae virus family and contains a genome consisting of three segments of double-stranded RNA (dsRNA). This virus family contains eight identified members, of which four have been classified in regard to their complete genomic sequence and encoded viral proteins. A phospholipid envelope that contains the integral proteins P6, P9, P10, and P13 surrounds the viral particles. In species phi 6, host infection requires binding of a multimeric P3 complex to type IV pili. In species varphi8, phi 12, and phi 13, the attachment apparatus is a heteromeric protein assembly that utilizes the rough lipopolysaccharide (rlps) as a receptor. In phi 8 the protein components are designated P3a and P3b while in species phi 12 proteins P3a and P3c have been identified in the complex. The phospholipid envelope of the cystoviruses surrounds a nucleocapsid (NC) composed of two shells. The outer shell is composed of protein P8 with a T=13 icosahedral lattice while the primary component of the inner shell is a dodecahedral frame composed of dimeric protein P1. For the current study, the 3D architecture of the intact phi 12 virus was obtained by electron cryo-tomography. The nucleocapsid appears to be centered within the membrane envelope and possibly attached to it by bridging structures. Two types of densities were observed protruding from the membrane envelope. The densities of the first type were elongated, running parallel, and closely associated to the envelope outer surface. In contrast, the second density was positioned about 12 nm above the envelope connected to it by a flexible low-density stem. This second structure formed a torroidal structure termed "the donut" and appears to inhibit BHT-induced viral envelope fusion.     

The envelope protein of severe acute respiratory syndrome coronavirus interacts with the non-structural protein 3 and is ubiquitinated

To analyze the proteins interacting with the severe acute respiratory syndrome coronavirus (SARS-CoV) envelope (E) protein, a SARS-CoV was engineered including two tags associated to the E protein. Using this virus, complexes of SARS-CoV E and other proteins were purified using a tandem affinity purification system. Several viral and cell proteins including spike, membrane, non-structural protein 3 (nsp3), dynein heavy chain, fatty acid synthase and transmembrane protein 43 bound E protein. In the present work, we focused on the binding of E protein to nsp3 in infected cells and cell-free systems. This interaction was mediated by the N-terminal acidic domain of nsp3. Moreover, nsp3 and E protein colocalized during the infection. It was shown that E protein was ubiquitinated in vitro and in cell culture, suggesting that the interaction between nsp3 and E protein may play a role in the E protein ubiquitination status and therefore on its turnover.     

Toroidal surface complexes of bacteriophage ϕ12 are responsible for host-cell attachment

Cryo-electron tomography and subtomogram averaging are utilized to determine that the bacteriophage ?12, a member of the Cystoviridae family, contains surface complexes that are toroidal in shape, are composed of six globular domains with six-fold symmetry, and have a discrete density connecting them to the virus membrane-envelope surface. The lack of this kind of spike in a reassortant of ?12 demonstrates that the gene for the hexameric spike is located in ?12's medium length genome segment, likely to the P3 open reading frames which are the proteins involved in viral-host cell attachment. Based on this and on protein mass estimates derived from the obtained averaged structure, it is suggested that each of the globular domains is most likely composed of a total of four copies of P3a and/or P3c proteins. Our findings may have implications in the study of the evolution of the cystovirus species in regard to their host specificity.     

Localization of proteins associated with the outer hair cell plasma membrane in the gerbil cochlea

There is substantial evidence that the motility of mammalian outer hair cells is generated close to or within the plasma membrane. Several analogies between the outer hair cell cortical lattice and the membrane-related cytoskeleton of erythrocytes have been noted. In erythrocytes a member of the anion exchanger protein family, AE1, also known as Band 3, is involved in membrane-cytoskeleton linkage via Protein 4.1. In the following paper, the presence of these two proteins in gerbilline outer hair cells is confirmed by western blot. Furthermore, co-localization of these two proteins was detected in the lateral wall of outer hair cells by immunofluorescence and postembedding electron immunohistochemistry. Band 3 is restricted to this region, whereas Protein 4.1 has a somewhat more dispersed distribution.

Thus, the structure of these sensory receptor cells may result from an adaptation of a strategy used by other motile cells. The proteins investigated likely have a support function and may comprise “pillars” seen between the lateral plasma membrane and the cytoskeleton in micrographs of outer hair cells. The possibility that Band 3 comprises “protein particles” seen in the lateral plasma membrane, or maybe directly involved in the voltage-dependent force generation in outer hair cells, is also discussed.     

Development of an ID-ELISA for the detection of Rice black-streaked dwarf virus in plants

The Rice black-streaked dwarf virus (RBSDV) virion is composed of two layers of capsid proteins and 10 segments of double-stranded genomic RNA (S1-S10). Due to the fragility of the RBSDV outer capsid, it is very difficult to obtain intact virus particles for preparation of antiserum needed in virus detection. In this work, the major outer capsid protein (P10) encoded by S10 of RBSDV was expressed in Escherichia coli cells as a glutathione-S-transferase (GST) fusion protein. After purification of GST-P10 through affinity chromatography, P10 was released from the fusion protein by thrombin digestion and the purified P10 protein was used to immunize rabbits. The resulting polyclonal antiserum specifically recognized RBSDV from infected plant tissue and a planthopper vector in Western blotting assays. To facilitate screening of large numbers of samples, an indirect enzyme-linked immunosorbent assay (ID-ELISA) protocol capable of detecting RBSDV in very dilute wheat leaf extracts was developed. Based on the results, we conclude that efficient and economic detection of RBSDV can be performed routinely using polyclonal antiserum against P10 expressed in prokaryotic cells.     

Assembly of five bluetongue virus proteins expressed by recombinant baculoviruses: Inclusion of the largest protein VP1 in the core and virus-like particles

Bluetongue virus (BTV) VP1 protein, a component of the viral RNA-directed RNA polymerase, but not the VP4 or VP6 proteins, was specifically incorporated into baculovirus expressed BTV core-like particles (composed of VP3 and VP7) and BTV virus-like particles (composed of VP2, VP3, VP5, and VP7). The VP1 protein has been shown to be associated with subcore particles composed of VP3. The data suggest that the VP1 protein of BTV has both enzymatic and structural roles in the virus life cycle.     

刺突蛋白与新型冠状病毒的检测和治疗

最近,一种由新型冠状病毒2019-nCoV引起的COVID-19肺炎疫情在全球暴发。为了更好地控制疫情的蔓延,亟需对2019-nCoV的来源、传播和致病机制进行深入研究。刺突(spike)蛋白是冠状病毒表面特有的结构蛋白,包含了冠状病毒自然演化的重要信息,并在病毒识别和入侵人体细胞的过程中起到关键作用。最近十几年,在对与人类密切相关的冠状病毒的研究中,spike蛋白一直是最为重要的研究对象之一。而在COVID-19肺炎疫情暴发后,2019-nCoV病毒表面的spike蛋白也迅速成为研究的焦点。本文旨在通过介绍spike蛋白相关研究,以期为当前疫情的防控以及COVID-19的诊断和治疗提供合理的研究思路。     

Nucleic acid, protein, and protein-bound lysine and arginine patterns in epidermal nuclei of the mature and senescing onion bulb

Eight macromolecular indices were determined for nuclei of outer epidermal cells in the equatorial plane from young through old leaf bases (sequential leaf senescence) and for nuclei in basal, equatorial and three apical locations within one leaf base (apical cell senescence). The eight nuclear indices were total nucleic acid. DNA, RNA, histone, non-histone, total protein, and protein-bound lysine and arginine. Cell senescence and death were accompanied by startling decreases in these eight indices. In sequential leaf senescence, nuclei in the second innermost turgid leaf base contained about four to five times the amount of nucleic acids and nuclear proteins in the youngest leaf base. The nuclei in the outermost leaf base contained about 20% less of the nucleic acids and proteins measured than the second outermost leaf base. We infer that this represents a decline in nuclear constituents due to senescence, since less than 30% of the cells in the innermost paperlike leaf base contained nuclei and those present were almost devoid of the nuclear constituents measured. Cell senescence in the apical section of a turgid leaf base showed sequential declines in all nuclear indices studied from areas composed of living cells to areas at the apex composed of dead cells. Possible changes in histone content due to physiological ages were detected in the equatorial but not in the vertical study; the protein-bound lysine to arginine ratios increased from younger to older leaf bases.     

Structural variations in human ACE2 may influence its binding with SARS-CoV-2 spike protein

The recent pandemic of COVID-19, caused by SARS-CoV-2, is unarguably the most fearsome compared with the earlier outbreaks caused by other coronaviruses, SARS-CoV and MERS-CoV. Human ACE2 is now established as a receptor for the SARS-CoV-2 spike protein. Where variations in the viral spike protein, in turn, lead to the cross-species transmission of the virus, genetic variations in the host receptor ACE2 may also contribute to the susceptibility and/or resistance against the viral infection. This study aims to explore the binding of the proteins encoded by different human ACE2 allelic variants with SARS-CoV-2 spike protein. Briefly, coding variants of ACE2 corresponding to the reported binding sites for its attachment with coronavirus spike protein were selected and molecular models of these variants were constructed by homology modeling. The models were then superimposed over the native ACE2 and ACE2-spike protein complex, to observe structural changes in the ACE2 variants and their intermolecular interactions with SARS-CoV-2 spike protein, respectively. Despite strong overall structural similarities, the spatial orientation of the key interacting residues varies in the ACE2 variants compared with the wild-type molecule. Most ACE2 variants showed a similar binding affinity for SARS-CoV-2 spike protein as observed in the complex structure of wild-type ACE2 and SARS-CoV-2 spike protein. However, ACE2 alleles, rs73635825 (S19P) and rs143936283 (E329G) showed noticeable variations in their intermolecular interactions with the viral spike protein. In summary, our data provide a structural basis of potential resistance against SARS-CoV-2 infection driven by ACE2 allelic variants.     

Association of polioviral proteins of the P2 genomic region with the viral replication complex and virus-induced membrane synthesis as visualized by electron microscopic immunocytochemistry and autoradiography

Using high resolution electron microscopic autoradiography and immunocytochemistry with monoclonal antibodies against poliovirus proteins of the P2 genomic region, the location of these proteins in respect to the virus-induced vesicle formation and the viral RNA synthesis was followed during the viral replication cycle. It was found that P2 proteins become rER associated soon after their synthesis. At the site of protein and rER interaction, electron-dense patches appear. Simultaneously, membrane protrusions grow and form vesicles which finally budd off, carrying the patches on their outer surface. As shown by autoradiography, these patches are the site of viral RNA replication and, therefore, they represent the poliovirus replication complex. The vesicles with the replication complex, including replicating and replicated viral RNA, move away from the rER to form a continuously growing vesiculated area in the center of the infected cell, where virus maturation takes place. A likely function of the 2C protein is to attach the replication complex, or some of its components, to the vesicular membranes.     

Nucleocapsid formation and/or subsequent conformational change of rabies virus nucleoprotein (N) is a prerequisite step for acquiring the phosphatase-sensitive epitope of monoclonal antibody 5-2-26.

We investigated the antigenic maturation of rabies virus N protein, for which we used some conformational epitope-specific monoclonal antibodies (MAbs) and an MAb (5-2-26) against a phosphorylation-dependent linear epitope. Infected cells were lysed with a deoxycholate-free lysis buffer and separated by ultracentrifugation into the soluble top and the nucleocapsid fractions. None of the study MAbs recognized N proteins in the top fraction, whereas nucleocapsid-associated N proteins were recognized by all of the MAbs. Immunoprecipitation with polyclonal anti-N antibodies coprecipitated the P proteins from the top fraction, indicating that soluble N proteins are mostly associated with the P protein. The N proteins dissociated from both the N-P complex and nucleocapsids were recognized by none of the study MAbs, whereas the MAb 5-2-6 recognized the SDS-denatured N proteins of the nucleocapsid but not of the top fraction. In addition, the phosphorylation-deficient mutant N proteins were shown to be similarly accumulated as the wild-type N proteins into the viral inclusion bodies, defined as the virus-specific structures composed of viral nucleocapsids, that are produced in the cytoplasm of the infected cells. Based on these results, we believe that newly synthesized N proteins are not immediately phosphorylated at serine-389 (a common phosphorylation site) but are first associated with the P protein. After being used for encapsidation of the viral RNA, the N proteins undergo conformational changes, whereby epitopes for the conformation-specific MAbs are formed and become phosphorylated at serine-389.     

Fate of the 6K membrane protein of Semliki Forest virus during virus assembly

The Semliki Forest virus directs the synthesis of three virus-specific transmembrane proteins p62, 6K, and E1, which all are made in equimolar amounts from a polyprotein precursor molecule. The p62 and E1 spike proteins form heterodimeric complexes in the endoplasmic reticulum before being transported to the cell surface where virus budding occurs. In this study we show that the 6K protein becomes associated to the p62E1 complex in the endoplasmic reticulum and transported with the complex to the cell surface. During virus budding, E1 and p62 (which has matured into the E2 protein) are incorporated into new virions whereas the 6K is mostly excluded. Virus particles released from infected BHK cells contain only about 3% of 6K in their membrane as compared to the spike protein content. The relevance of these findings for the mechanism of SFV assembly is discussed.     

Outer membrane proteins as surface display systems

Outer membrane proteins (OMPs) of gram-negative bacteria can be used as carrier proteins to present foreign peptide epitopes on the bacterial cell surface. They all have a common structural motif of a beta-barrel that is composed of a variable number of transmembrane beta-strands connected at the periplasmic side with short turns and at the outside with long surface-accessible loops. Outer membrane proteins occur as monomers like OmpA, or assemble into trimers like the porins. Foreign gene products have been fused to surface-accessible regions of several outer membrane proteins including the porins OmpC, PhoE and LamB, lipoproteins as well as the OmpA protein. Short epitopes that are inserted into outer membrane proteins induce epitope-specific antibody responses, and are thus appealing candidates for live recombinant vaccines. Also large insertions, of more than 100 amino acids, are in some cases tolerated and do not affect the overall conformation of the carrier protein. The possible applications for outer membrane display include recombinant vaccines, peptide library screening, development of biocatalysts or whole-cell adsorbents, and adhesin-receptor interaction studies. It is expected that in the near future, development of new display systems will still increase the utilization of this emerging exciting technology.     

Antibodies against Sendai virus L protein: distribution of the protein in nucleocapsids revealed by immunoelectron microscopy

Antibodies against the L protein of Sendai virus were made by immunizing rabbits with a synthetic peptide representing a carboxyl-terminal region of the protein predicted from the base sequence of its gene. These antibodies were used to localize the L protein in viral nucleocapsids by electron microscopy. Immunogold labeling revealed that L protein molecules were distributed in clusters along nucleocapsids, suggesting that L molecules act cooperatively in viral RNA synthesis. Immunogold double-labeling showed that all L clusters were associated with clusters of P molecules. We believe that this morphological association reflects the functional cooperation of the L and P proteins in viral RNA synthesis.     

Concentration and purification of feline leukaemia virus (FeLV) and its outer envelope protein gp70 by aqueous two-phase systems

The major protective antigens of retroviruses are considered to be their glycosylated envelope proteins. However, the methods commonly employed to enrich and purify virus from culture media such as pelleting and density-gradient centrifugation result in a low recovery of the viral external glycoproteins. This is an obvious drawback when the virus is intended for use in a vaccine. In search for alternative methods to concentrate and purify FeLV, we have attempted extraction in two-phase systems based on water-soluble polymers (Albertsson PA., Biochem Biophys Acta 1958; 27: 378-395). A variety of polymer systems was tested. Some of them seem attractive for a large-scale concentration of the virus and/or its glycoprotein. The distribution between the phases of two FeLV proteins, the outer envelope protein, gp70, and the gag protein, p27, was determined. With a system composed of dextran sulfate and polyvinyl alcohol both the glycoprotein and the gag protein were almost completely recovered in the lower phase which constitutes about 3% of the total system in weight. The two proteins were more than 40-fold purified as calculated on protein basis. The proteins can be extracted readily.     

Two regions of the P protein are required to be active with the L protein for human parainfluenza virus type 1 RNA polymerase activity

The paramyxovirus P protein is an essential component of the viral RNA polymerase composed of P and L proteins. In this study, we characterized the physical and functional interactions between P and L proteins using human parainfluenza virus type 1 (hPIV1) and its counterpart Sendai virus (SV). The hPIV1 P and SV L proteins or the SV P and hPIV1 L proteins formed complexes detected by anti-P antibodies. Functional analysis using the minigenome SV RNA containing CAT gene indicated that the hPIV1 P--SV L complex, but not the SV P--hPIV1 L complex, was biologically active. Mutant SV P or hPIV1 P cDNAs, which do not express C proteins, showed the same phenotype with wild-type P cDNAs, indicating that C proteins are not responsible for the dysfunction of SV P--hPIV1 L polymerase complex. Using the chimeric hPIV1/SV P cDNAs, we identified two regions (residues 387--423 and 511--568) on P protein, which are required for the functional interaction with hPIV1 L. These regions overlap with a previously identified domain for oligomer formation and binding to nucleocapsids. Our results indicate that in addition to a P--L binding domain, hPIV1 L requires a specific region on P protein to be biologically functional as a polymerase.     

Severe COVID-19: A multifaceted viral vasculopathy syndrome

The objective of this study was to elucidate the pathophysiology that underlies severe COVID-19 by assessing the histopathology and the in situ detection of infectious SARS-CoV-2 and viral capsid proteins along with the cellular target(s) and host response from twelve autopsies. There were three key findings: 1) high copy infectious virus was limited mostly to the alveolar macrophages and endothelial cells of the septal capillaries; 2) viral spike protein without viral RNA localized to ACE2+ endothelial cells in microvessels that were most abundant in the subcutaneous fat and brain; 3) although both infectious virus and docked viral spike protein was associated with complement activation, only the endocytosed pseudovirions induced a marked up-regulation of the key COVID-19 associated proteins IL6, TNF alpha, IL1 beta, p38, IL8, and caspase 3 in endothelium. Importantly, this microvasculitis was associated with characteristic findings on hematoxylin and eosin examination that included endothelial degeneration and resultant basement membrane zone disruption and reduplication. It is concluded that serious COVID-19 infection has two distinct mechanisms: 1) a microangiopathy of pulmonary capillaries associated with a high infectious viral load where endothelial cell death releases pseudovirions into the circulation, and 2) the pseudovirions dock on ACE2+ endothelial cells most prevalent in the skin/subcutaneous fat and brain that activates the complement pathway/coagulation cascade resulting in a systemic procoagulant state as well as endothelial expression of cytokines that produce the cytokine storm. The data predicts a favorable response to therapies based on either removal of circulating viral proteins and/or blunting of the endothelial-induced response.     

Cloning and expression in Escherichia coli of opc, the gene for an unusual class 5 outer membrane protein from Neisseria meningitidis (meningococci/surface antigen).

A genomic library was constructed in a lambda gt11 vector using chromosomal DNA from a meningococcal serogroup A strain and plaques expressing the class 5C protein were recognized by screening with specific monoclonal antibodies. The opc insert was subcloned into a multicopy plasmid which induced expression of that protein in Escherichia coli as a surface-exposed major outer membrane protein. The nucleotide sequence of opc is typical of an outer membrane protein with a promoter and terminator region, a leader peptide which is cleaved during expression and a complete open reading frame. Unlike other meningococcal class 5 proteins or gonococcal P.II proteins, the sequence did not contain any pentanucleotide repeats and the sequence showed little homology to these other functionally related proteins. However, the predicted amino acid sequence of the mature protein for opc showed 27% similarity to that for a second opa gene cloned from the same meningococcal strain. This is the first report of cloning and expression of a functional meningococcal gene encoding a class 5 outer membrane protein in E. coli.     

Molecular cloning and sequencing of coat protein-encoding cDNA of rice tungro spherical virus—A plant picornavirus

Rice tungro spherical virus (RTSV) was shown to have three coat protein (CP) species by high resolution NaDodSO₄-PAGE and Western blot analyses. The sequence of a coat protein-expressing cDNA clone that was identified and selected from a RTSV cDNA library showed that the insert was composed of 2823 bp with only one large open reading frame (ORF) coding for 941 amino acids. The positions of the three coat proteins were located in the putative polyprotein by N-terminal microsequencing and were shown to start at amino acids 287, 495, and 698 for CP-1, CP-2, and CP-3, respectively. The coat proteins are expressed as a polyprotein at the 5 region of the viral RNA genome, and all are cleaved at glutamine carboxy termini, presumably by picornavirus 3C-type of protease(s). Sequence comparisons of coat proteins revealed that there are high amino acid homologies between CP-2 of RTSV and VP3s of encephalomyocarditis virus (EMCV) and Theiler's murine encephalomyelitis virus (TMEV). These results indicate that RTSV is a plant picornavirus.