Role of phosphorylation clusters in the biology of the coronavirus infectious bronchitis virus nucleocapsid protein

The coronavirus infectious bronchitis virus (IBV) nucleocapsid (N) protein is an RNA binding protein which is phosphorylated at two conserved clusters. Kinetic analysis of RNA binding indicated that the C-terminal phosphorylation cluster was involved in the recognition of viral RNA from non-viral RNA. The IBV N protein has been found to be essential for the successful recovery of IBV using reverse genetics systems. Rescue experiments indicated that phosphorylated N protein recovered infectious IBV more efficiently when compared to modified N proteins either partially or non-phosphorylated. Our data indicate that the phosphorylated form of the IBV N protein plays a role in virus biology.     

RNA-binding proteins of coronavirus MHV: Detection of monomeric and multimeric N protein with an RNA overlay-protein blot assay

RNA-binding proteins of coronavirus MHV-A59 were identified using an RNA overlay-protein blot assay (ROPBA). The major viral RNA-binding protein in virions and infected cells was the phosphorylated nucleocapsid protein N (50K). A new 140K virus structural protein was identified as a minor RNA-binding protein both in virions and in infected cells. The 140K protein was antigenically related to N, and upon reduction, yielded only 50K N. Thus, the 140K protein is probably a trimer of N subunits linked by intermolecular disulfide bonds. Several cellular RNA-binding proteins were also detected. RNA-binding of N was not nucleotide sequence specific. Single-stranded RNA of MHV, VSV, or cellular origin, a DNA probe of the MHV leader sequence, and double-stranded bovine rotavirus RNA could all bind to N. Binding of MHV RNA was optimal between pH 7 and 8, and the RNA could be eluted in 0.1 M NaCl. The ROPBA is a useful method for the initial identification of RNA-binding proteins, such as N and the 140K protein of murine coronavirus.     

Sendai virus RNA synthesis and nucleocapsid formation in the presence of cycloheximide

The effect of cycloheximide on viral specific RNA synthesis and viral nucleocapsid formation was studied in chick embryo cells infected with Sendai virus. When cells were infected at a high multiplicity in the presence of cycloheximide, the 18 S and 35 S viral specific RNAs but not the 57 S (virion) RNA were synthesized during a 90-min period indicating that new protein synthesis is not necessary for the initiation of synthesis of the two smaller RNAs. When cells were treated with cycloheximide 18 hr after infection at the time of maximum viral specific RNA synthesis, the rate of synthesis of the 57 S RNA decreased much more rapidly than synthesis of the 35 S and 18 S RNAs. At the same time after infection, newly synthesized 57 S RNA continued to be converted to viral nucleocapsid in the presence of cycloheximide and the rate of incorporation of uridine-5′-³H into nucleocapsid decreased in parallel with the decrease in 57 S RNA synthesis. This indicates that nucleocapsid assembly continues in the absence of active protein synthesis.     

The virion N protein of infectious bronchitis virus is more phosphorylated than the N protein from infected cell lysates

Because phosphorylation of the infectious bronchitis virus (IBV) nucleocapsid protein (N) may regulate its multiple roles in viral replication, the dynamics of N phosphorylation were examined. 32P-orthophosphate labeling and Western blot analyses confirmed that N was the only viral protein that was phosphorylated. Pulse labeling with 32P-orthophosphate indicated that the IBV N protein was phosphorylated in the virion, as well as at all times during infection in either chicken embryo kidney cells or Vero cells. Pulse-chase analyses followed by immunoprecipitation of IBV N proteins using rabbit anti-IBV N polyclonal antibody demonstrated that the phosphate on the N protein was stable for at least 1 h. Simultaneous labeling with 32P-orthophosphate and 3H-leucine identified a 3.5-fold increase in the 32P:3H counts per minute (cpm) ratio of N in the virion as compared to the 32P:3H cpm ratio of N in the cell lysates from chicken embryo kidney cells, whereas in Vero cells the 32P:3H cpm ratio of N from the virion was 10.5-fold greater than the 32P:3H cpm ratio of N from the cell lysates. These studies are consistent with the phosphorylation of the IBV N playing a role in assembly or maturation of the viral particle.     

Hantavirus nucleocapsid protein: a multifunctional molecule with both housekeeping and ambassadorial duties

Summary. In recent years important progress has been made studying the nucleocapsid (N) protein of hantaviruses. The N protein presents a good example of a multifunctional viral macromolecule. It is a major structural component of a virion that encapsidates viral RNA (vRNA). It also interacts with the virus polymerase (L protein) and one of the glycoproteins. On top of these “house keeping” duties, the N protein performs interactive “ambassadorial” functions interfering with important regulatory pathways in the infected cells.     

Identification of putative polymerase gene product in cells infected with murine coronavirus A59

The virion RNA of mouse hepatitis virus, strain A59 (MHV-A59) is believed to be the mRNA for the viral RNA-dependent RNA polymerase. The cell-free translation of virion RNA results in the synthesis of two predominant products p220 and p28 (M. R. Denison and S. Perlman, 1986, J. Virol. 60, 12-18). p28 is a basic protein and is readily detected by two-dimensional gel electrophoresis. When infected cells and isolated virions were assayed for this protein by two-dimensional gel electrophoresis, p28 could be detected in infected cells labeled at late times after infection, but not at early times or in purified virions. p28 represents the first protein product of the putative coronavirus polymerase gene to be identified in infected cells.     

Intracellular structures involved in Sendai virus replication

Viral specific RNA-containing structures released from gently disrupted Sendai virus infected chick embryo fibroblasts were separated into four rapidly sedimenting components in sucrose density gradients. Uridine-³H was rapidly incorporated into the RNA of each component. Component 1 (>350 S) contained virion RNA (57 S) and had physical characteristics of cell-associated virus. Component 2 (250 S) contained double-stranded RNA and the kinetics of uridine-³H incorporation suggested that this structure may be involved in viral RNA synthesis. Component 3 (200 S) contained 57 S RNA and was identified as viral nucleocapsid. Component 4 (150 S) contained 35 S single-stranded RNA, and within this structure the RNA was protected from RNase digestion. The exact structure of this particle and its role in virus replication must be determined. The RNA from each intracellular component annealed specifically with virion RNA. The 57 S RNAs isolated from the cell-associated virus (component 1) and from the intracellular nucleocapsid (component 3) self annealed as previously shown for virion RNA, and the annealing was RNA concentration dependent. The 57 S RNA from virions could not be separated into more than one component by equilibrium CsCl·Cs₂SO₄ centrifugation.     

Basic virology of SARS-CoV 2

BackgroundSevere acute respiratory syndrome coronavirus 2 (SARS-CoV-2), is a highly transmissible pathogenic coronavirus emerged in late 2019 causing a pandemic of acute respiratory disease, named ‘coronavirus disease 2019’ (COVID-19). It has spread fast all over the world posing an extraordinary threat to global public health. Along with SARS-CoV-2, there are seven human coronaviruses. Those causing mild diseases are the 229E, OC43, NL63 and HKU1, and the pathogenic ones are SARSCoV, MERS-CoV and SARS-CoV-2.ObjectiveThis review has highlighted the basic virology of SARS CoV-2 including its origin, structure, genomic characteristics, pathogenesis, immunological response and clinical manifestation along with the key difference of SARS CoV2 from the previous Coronaviruses.ContentCoronaviruses are spherical and enveloped with club-shaped spikes on the surface. It has a large positive sense, single stranded RNA genome within the nucleocapsid with a helical symmetry. It has been known to cause infection to innumerable mammalian hosts, like humans, cats, bats, civets, dogs, and camels. The viral genome contains four major structural proteins: the spike (S), membrane (M), envelope (E) and the nucleocapsid (N) protein encoded within the 3’ end of the genome. Virus binds to the host cell by the S protein with specific receptor. Following receptor binding, the virus enters host cell cytosol and there is fusion of the viral and cellular membranes followed by the translation of the viral genomic RNA. Following the viral replication and sub-genomic RNA synthesis, there is formation of the mature virus. The virions are then transported to the cell surface in vesicles and are released by exocytosis.     

The intracellular sites of early replication and budding of SARS-coronavirus

In this study, we analyzed the replication and budding sites of severe acute respiratory syndrome coronavirus (SARS-CoV) at early time points of infection. We detected cytoplasmic accumulations containing the viral nucleocapsid protein, viral RNA and the non-structural protein nsp3. Using EM techniques, we found that these putative viral replication sites were associated with characteristic membrane tubules and double membrane vesicles that most probably originated from ER cisternae. In addition to its presence at the replication sites, N also accumulated in the Golgi region and colocalized with the viral spike protein. Immuno-EM revealed that budding occurred at membranes of the ERGIC (ER-Golgi intermediate compartment) and the Golgi region as early as 3 h post infection, demonstrating that SARS-CoV replicates surprisingly fast. Our data suggest that SARS-CoV establishes replication complexes at ER-derived membranes. Later on, viral nucleocapsids have to be transported to the budding sites in the Golgi region where the viral glycoproteins accumulate and particle formation occurs.     

Structural maturation of rubella virus in the Golgi complex

Rubella virus is a small enveloped virus that assembles in association with Golgi membranes. Freeze-substitution electron microscopy of rubella virus-infected cells revealed a previously unrecognized virion polymorphism inside the Golgi stacks: homogeneously dense particles without a defined core coexisting with less dense, mature virions that contained assembled cores. The homogeneous particles appear to be a precursor form during the virion morphogenesis process as the forms with mature morphology were the only ones detected inside secretory vesicles and on the exterior of cells. In mature virions potential remnants of C protein membrane insertion were visualized as dense strips connecting the envelope with the internal core. In infected cells Golgi stacks were frequently seen close to cytopathic vacuoles, structures identified as the sites for viral RNA replication, along with the rough endoplasmic reticulum and mitochondria. These associations could facilitate the transfer of viral genomes from the cytopathic vacuoles to the areas of rubella assembly in Golgi membranes.     

Frog virus 3 ORF 53R, a putative myristoylated membrane protein, is essential for virus replication in vitro

Although previous work identified 12 complementation groups with possible roles in virus assembly, currently only one frog virus 3 protein, the major capsid protein (MCP), has been linked with virion formation. To identify other proteins required for assembly, we used an antisense morpholino oligonucleotide to target 53R, a putative myristoylated membrane protein, and showed that treatment resulted in marked reductions in 53R levels and a 60% drop in virus titers. Immunofluorescence assays confirmed knock down and showed that 53R was found primarily within viral assembly sites, whereas transmission electron microscopy detected fewer mature virions and, in some cells, dense granular bodies that may represent unencapsidated DNA-protein complexes. Treatment with a myristoylation inhibitor (2-hydroxymyristic acid) resulted in an 80% reduction in viral titers. Collectively, these data indicate that 53R is an essential viral protein that is required for replication in vitro and suggest it plays a critical role in virion formation.     

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.     

Phosphorylation of human respiratory syncytial virus P protein at serine 54 regulates viral uncoating

The human respiratory syncytial virus (HRSV) structural P protein, phosphorylated at serine (S) and threonine (T) residues, is a co-factor of viral RNA polymerase. The phosphorylation of S54 is controlled by the coordinated action of two cellular enzymes: a lithium-sensitive kinase, probably glycogen synthetase kinase (GSK-3) β and protein phosphatase 2A (PP2A).Inhibition of lithium-sensitive kinase, soon after infection, blocks the viral growth cycle by inhibiting synthesis and/or accumulation of viral RNAs, proteins and extracellular particles. P protein phosphorylation at S54 is required to liberate viral ribonucleoproteins (RNPs) from M protein, during the uncoating process. Kinase inhibition, late in infection, produces a decrease in genomic RNA and infectious viral particles.LiCl, intranasally applied to mice infected with HRSV A2 strain, reduces the number of mice with virus in their lungs and the virus titre. Administration of LiCl to humans via aerosol should prevent HRSV infection, without secondary effects.     

Analyses of the mRNA transcription processes of Punta Toro phlebovirus (Bunyaviridae)

The time course of the syntheses of Punta Toro (PT) phlebovirus (Bunyaviridae) small (S)-size viral RNA (S vRNA), viral complementary RNA (S vcRNA), and messenger RNA (S mRNA) species has been analyzed using single-stranded DNA probes representing the two S-coded gene products. The data obtained support the conclusion that PT S RNA has an ambisense coding strategy (T. Ihara, H. Akashi, and D. H. L. Bishop, Virology 136, 293-306, 1984) with the viral nucleocapsid protein, N, encoded in a viral-complementary, subgenomic, mRNA species and a putative nonstructural protein, NSs, encoded in a viral-sense, subgenomic, second S mRNA species. In the absence of puromycin (or cycloheximide) full-length S vRNA, S vcRNA, and subgenomic N mRNA and putative NSs mRNA species were identified in PT virus-infected cell extracts. In the presence of inhibitors of protein synthesis (puromycin or cycloheximide) newly synthesized N mRNA species were detected, but not full-length S vcRNA, nor S vRNA, nor the S coded NSs mRNA species. The mRNA species recovered from drug-treated cells have been translated in vitro to synthesize viral N protein. Analyses of the 5' ends of the N and NSs mRNA species have shown them to be heterogeneous in sequence and some 11-18 bases longer than the ends of the genomic RNA species, indicating that they represent nonviral primer sequences like those identified for bunyavirus mRNA species (D. H. L. Bishop, M. E. Gay, and Y. Matsuoka, Nucleic Acids Res. 11, 6409-6418, 1983). The presence of such additional sequences on mRNA derived from representatives of two Bunyaviridae genera appears by these analyses to be a more conserved feature than the S RNA coding arrangement of the respective viruses.     

Ultrastructure of cell trafficking pathways and coronavirus: how to recognise the wolf amongst the sheep

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic has resulted in an urgent need to understand the pathophysiology of SARS-CoV-2 infection, to assist in the identification of treatment strategies. Viral tissue tropism is an active area of investigation, one approach to which is identification of virus within tissues by electron microscopy of post-mortem and surgical specimens. Most diagnostic histopathologists have limited understanding of the ultrastructural features of normal cell trafficking pathways, which can resemble intra- and extracellular coronavirus; in addition, viral replication pathways make use of these trafficking pathways. Herein, we review these pathways and their ultrastructural appearances, with emphasis on structures which may be confused with coronavirus. In particular, we draw attention to the fact that, when using routine fixation and processing, the typical ‘crown’ that characterises a coronavirus is not readily identified on intracellular virions, which are located in membrane-bound vacuoles. In addition, the viral nucleocapsid is seen as black dots within the virion and is more discriminatory in differentiating virions from other cellular structures. The identification of the viral replication organelle, a collection of membranous structures (convoluted membranes) seen at a relatively low scanning power, may help to draw attention to infected cells, which can be sparse. © 2020 The Authors. The Journal of Pathology published by John Wiley & Sons, Ltd. on behalf of The Pathological Society of Great Britain and Ireland.