Zoonotic coronavirus (CoV) infections, such as those responsible for the current severe acute respiratory syndrome-CoV 2 (SARS-CoV-2) pandemic, cause grave international public health concern. In ...infected cells, the CoV RNA-synthesizing machinery associates with modified endoplasmic reticulum membranes that are transformed into the viral replication organelle (RO). Although double-membrane vesicles (DMVs) appear to be a pan-CoV RO element, studies to date describe an assortment of additional CoV-induced membrane structures. Despite much speculation, it remains unclear which RO element(s) accommodate viral RNA synthesis. Here we provide detailed 2D and 3D analyses of CoV ROs and show that diverse CoVs essentially induce the same membrane modifications, including the small open double-membrane spherules (DMSs) previously thought to be restricted to gamma- and delta-CoV infections and proposed as sites of replication. Metabolic labeling of newly synthesized viral RNA followed by quantitative electron microscopy (EM) autoradiography revealed abundant viral RNA synthesis associated with DMVs in cells infected with the beta-CoVs Middle East respiratory syndrome-CoV (MERS-CoV) and SARS-CoV and the gamma-CoV infectious bronchitis virus. RNA synthesis could not be linked to DMSs or any other cellular or virus-induced structure. Our results provide a unifying model of the CoV RO and clearly establish DMVs as the central hub for viral RNA synthesis and a potential drug target in CoV infection.
SARS Coronavirus 2 (SARS-CoV-2) emerged in late 2019, leading to the Coronavirus Disease 2019 (COVID-19) pandemic that continues to cause significant global mortality in human populations. Given its ...sequence similarity to SARS-CoV, as well as related coronaviruses circulating in bats, SARS-CoV-2 is thought to have originated in Chiroptera species in China. However, whether the virus spread directly to humans or through an intermediate host is currently unclear, as is the potential for this virus to infect companion animals, livestock, and wildlife that could act as viral reservoirs. Using a combination of surrogate entry assays and live virus, we demonstrate that, in addition to human angiotensin-converting enzyme 2 (ACE2), the Spike glycoprotein of SARS-CoV-2 has a broad host tropism for mammalian ACE2 receptors, despite divergence in the amino acids at the Spike receptor binding site on these proteins. Of the 22 different hosts we investigated, ACE2 proteins from dog, cat, and cattle were the most permissive to SARS-CoV-2, while bat and bird ACE2 proteins were the least efficiently used receptors. The absence of a significant tropism for any of the 3 genetically distinct bat ACE2 proteins we examined indicates that SARS-CoV-2 receptor usage likely shifted during zoonotic transmission from bats into people, possibly in an intermediate reservoir. Comparison of SARS-CoV-2 receptor usage to the related coronaviruses SARS-CoV and RaTG13 identified distinct tropisms, with the 2 human viruses being more closely aligned. Finally, using bioinformatics, structural data, and targeted mutagenesis, we identified amino acid residues within the Spike-ACE2 interface, which may have played a pivotal role in the emergence of SARS-CoV-2 in humans. The apparently broad tropism of SARS-CoV-2 at the point of viral entry confirms the potential risk of infection to a wide range of companion animals, livestock, and wildlife.
The spike (S) glycoprotein of the avian gammacoronavirus infectious bronchitis virus (IBV) is comprised of two subunits (S1 and S2), has a role in virulence
, and is responsible for cellular tropism
...We have previously demonstrated that replacement of the S glycoprotein ectodomain from the avirulent Beaudette strain of IBV with the corresponding region from the virulent M41-CK strain resulted in a recombinant virus, BeauR-M41(S), with the
cell tropism of M41-CK. The IBV Beaudette strain is able to replicate in both primary chick kidney cells and Vero cells, whereas the IBV M41-CK strain replicates in primary cells only. In order to investigate the region of the IBV S responsible for growth in Vero cells, we generated a series of recombinant IBVs expressing chimeric S glycoproteins, consisting of regions from the Beaudette and M41-CK S gene sequences, within the genomic background of Beaudette. The S2, but not the S1, subunit of the Beaudette S was found to confer the ability to grow in Vero cells. Various combinations of Beaudette-specific amino acids were introduced into the S2 subunit of M41 to determine the minimum requirement to confer tropism for growth in Vero cells. The ability of IBV to grow and produce infectious progeny virus in Vero cells was subsequently narrowed down to just 3 amino acids surrounding the S2' cleavage site. Conversely, swapping of the 3 Beaudette-associated amino acids with the corresponding ones from M41 was sufficient to abolish Beaudette growth in Vero cells.
Infectious bronchitis remains a major problem in the global poultry industry, despite the existence of many different vaccines. IBV vaccines, both live attenuated and inactivated, are currently grown on embryonated hen's eggs, a cumbersome and expensive process due to the fact that most IBV strains do not grow in cultured cells. The reverse genetics system for IBV creates the opportunity for generating rationally designed and more effective vaccines. The observation that IBV Beaudette has the additional tropism for growth on Vero cells also invokes the possibility of generating IBV vaccines produced from cultured cells rather than by the use of embryonated eggs. The regions of the IBV Beaudette S glycoprotein involved in the determination of extended cellular tropism were identified in this study. This information will enable the rational design of a future generation of IBV vaccines that may be grown on Vero cells.
Autophagy is a cellular response to starvation which generates autophagosomes to carry cellular organelles and long-lived proteins to lysosomes for degradation. Degradation through autophagy can ...provide an innate defence against virus infection, or conversely autophagosomes can promote infection by facilitating assembly of replicase proteins. We demonstrate that the avian coronavirus, Infectious Bronchitis Virus (IBV) activates autophagy. A screen of individual IBV non-structural proteins (nsps) showed that autophagy was activated by IBV nsp6. This property was shared with nsp6 of mammalian coronaviruses Mouse Hepatitis Virus, and Severe Acute Respiratory Syndrome Virus, and the equivalent nsp5-7 of the arterivirus Porcine Reproductive and Respiratory Syndrome Virus. These multiple-spanning transmembrane proteins located to the endoplasmic reticulum (ER) where they generated Atg5 and LC3II-positive vesicles, and vesicle formation was dependent on Atg5 and class III PI3 kinase. The vesicles recruited double FYVE-domain containing protein (DFCP) indicating localised concentration of phosphatidylinositol 3 phosphate, and therefore shared many features with omegasomes formed from the ER in response to starvation. Omegasomes induced by viral nsp6 matured into autophagosomes that delivered LC3 to lysosomes and therefore recruited and recycled the proteins needed for autophagosome nucleation, expansion, cellular trafficking and delivery of cargo to lysosomes. The coronavirus nsp6 proteins activated omegasome and autophagosome formation independently of starvation, but activation did not involve direct inhibition of mTOR signalling, activation of sirtuin1 or induction of ER stress.
Coronaviruses are single stranded, positive sense RNA viruses, which induce the rearrangement of cellular membranes upon infection of a host cell. This provides the virus with a platform for the ...assembly of viral replication complexes, improving efficiency of RNA synthesis. The membranes observed in coronavirus infected cells include double membrane vesicles. By nature of their double membrane, these vesicles resemble cellular autophagosomes, generated during the cellular autophagy pathway. In addition, coronavirus infection has been demonstrated to induce autophagy. Here we review current knowledge of coronavirus induced membrane rearrangements and the involvement of autophagy or autophagy protein microtubule associated protein 1B light chain 3 (LC3) in coronavirus replication.
Several techniques are currently available to quickly and accurately quantify the number of virus particles in a sample, taking advantage of advanced technologies improving old techniques or ...generating new ones, generally relying on partial detection methods or structural analysis. Therefore, characterization of virus infectivity in a sample is often essential, and classical virological methods are extremely powerful in providing accurate results even in an old-fashioned way. In this chapter, we describe in detail the techniques routinely used to estimate the number of viable infectious coronavirus particles in a given sample. All these techniques are serial dilution assays, also known as titrations or end-point dilution assays (EPDA).
Infectious bronchitis virus (IBV), a gammacoronavirus, is an economically important virus to the poultry industry, as well as a significant welfare issue for chickens. As for all positive strand RNA ...viruses, IBV infection causes rearrangements of the host cell intracellular membranes to form replication organelles. Replication organelle formation is a highly conserved and vital step in the viral life cycle. Here, we investigate the localization of viral RNA synthesis and the link with replication organelles in host cells. We have shown that sites of viral RNA synthesis and virus-related dsRNA are associated with one another and, significantly, that they are located within a membrane-bound compartment within the cell. We have also shown that some viral RNA produced early in infection remains within these membranes throughout infection, while a proportion is trafficked to the cytoplasm. Importantly, we demonstrate conservation across all four coronavirus genera, including SARS-CoV-2. Understanding more about the replication of these viruses is imperative in order to effectively find ways to control them.
Quantitative real-time PCR assays are widely used for the quantification of mRNA within avian experimental samples. Multiple stably-expressed reference genes, selected for the lowest variation in ...representative samples, can be used to control random technical variation. Reference gene assays must be reliable, have high amplification specificity and efficiency, and not produce signals from contaminating DNA. Whilst recent research papers identify specific genes that are stable in particular tissues and experimental treatments, here we describe a panel of ten avian gene primer and probe sets that can be used to identify suitable reference genes in many experimental contexts. The panel was tested with TaqMan and SYBR Green systems in two experimental scenarios: a tissue collection and virus infection of cultured fibroblasts. GeNorm and NormFinder algorithms were able to select appropriate reference gene sets in each case. We show the effects of using the selected genes on the detection of statistically significant differences in expression. The results are compared with those obtained using 28s ribosomal RNA, the present most widely accepted reference gene in chicken work, identifying circumstances where its use might provide misleading results. Methods for eliminating DNA contamination of RNA reduced, but did not completely remove, detectable DNA. We therefore attached special importance to testing each qPCR assay for absence of signal using DNA template. The assays and analyses developed here provide a useful resource for selecting reference genes for investigations of avian biology.
Porcine deltacoronavirus (PDCoV) was first identified in Hong Kong in 2012 from samples taken from pigs in 2009. PDCoV was subsequently identified in the USA in 2014 in pigs with a history of severe ...diarrhea. The virus has now been detected in pigs in several countries around the world. Following the development of tissue culture adapted strains of PDCoV, it is now possible to address questions regarding virus-host cell interactions for this genera of coronavirus. Here, we presented a detailed study of PDCoV-induced replication organelles. All positive-strand RNA viruses induce the rearrangement of cellular membranes during virus replication to support viral RNA synthesis, forming the replication organelle. Replication organelles for the
,
-, and
genera have been characterized. All coronavirus genera induced the formation of double-membrane vesicles (DMVs). In addition,
- and
induce the formation of convoluted membranes, while
induce the formation of zippered endoplasmic reticulum (ER) with tethered double-membrane spherules. However, the structures induced by
, particularly the presence of convoluted membranes or double-membrane spherules, are unknown. Initially, the dynamics of PDCoV strain OH-FD22 replication were assessed with the onset of viral RNA synthesis, protein synthesis, and progeny particle release determined. Subsequently, virus-induced membrane rearrangements were identified in infected cells by electron microscopy. As has been observed for all other coronaviruses studied to date, PDCoV replication was found to induce the formation of double-membrane vesicles. Significantly, however, PDCoV replication was also found to induce the formation of regions of zippered endoplasmic reticulum, small associated tethered vesicles, and double-membrane spherules. These structures strongly resemble the replication organelle induced by avian
infectious bronchitis virus.
During infection of their host cells, viruses often inhibit the production of host proteins, a process that is referred to as host shutoff. By doing this, viruses limit the production of antiviral ...proteins and increase production capacity for viral proteins. Coronaviruses from the genera Alphacoronavirus and Betacoronavirus, such as severe acute respiratory syndrome coronavirus (SARS-CoV), establish host shutoff via their nonstructural protein 1 (nsp1). The Gammacoronavirus and Deltacoronavirus genomes, however, do not encode nsp1, and it has been suggested that these viruses do not induce host shutoff. Here, we show that the Gammacoronavirus infectious bronchitis virus (IBV) does induce host shutoff, and we find that its accessory protein 5b is indispensable for this function. Importantly, we found that 5b-null viruses, unlike wild-type viruses, induce production of high concentrations of type I interferon protein in vitro, indicating that host shutoff by IBV plays an important role in antagonizing the host's innate immune response. Altogether, we demonstrate that 5b is a functional equivalent of nsp1, thereby answering the longstanding question of whether lack of nsp1 in gammacoronaviruses is compensated for by another viral protein. As such, our study is a significant step forward in the understanding of coronavirus biology and closes a gap in the understanding of some IBV virulence strategies.
Many viruses inhibit protein synthesis by their host cell to enhance virus replication and to antagonize antiviral defense mechanisms. This process is referred to as host shutoff. We studied gene expression and protein synthesis in chicken cells infected with the important poultry pathogen infectious bronchitis virus (IBV). We show that IBV inhibits synthesis of host proteins, including that of type I interferon, a key component of the antiviral response. The IBV-induced host shutoff, however, does not require degradation of host RNA. Furthermore, we demonstrate that accessory protein 5b of IBV plays a crucial role in the onset of host shutoff. Our findings suggest that inhibition of host protein synthesis is a common feature of coronaviruses and primarily serves to inhibit the antiviral response of the host.
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