The measles virus (MeV), a member of the genus Morbillivirus, is an established pathogen of humans. A key feature of morbilliviruses is their ability to spread by virus–cell and cell–cell fusion. The latter process, which leads to syncytia formation in vitro and in vivo, is driven by the viral fusion (F) and haemagglutinin (H) glycoproteins. In this study, we demonstrate that MeV glycoproteins are sensitive to inhibition by bone marrow stromal antigen 2 (BST2/Tetherin/CD317) proteins. BST2 overexpression causes a large reduction in MeV syncytia expansion. Using quantitative cell–cell fusion assays, immunolabeling, and biochemistry we further demonstrate that ectopically expressed BST2 directly inhibits MeV cell–cell fusion. This restriction is mediated by the targeting of the MeV H glycoprotein, but not other MeV proteins. Using truncation mutants, we further establish that the C-terminal glycosyl-phosphatidylinositol (GPI) anchor of BST2 is required for the restriction of MeV replication in vitro and cell–cell fusion. By extending our study to the ruminant morbillivirus peste des petits ruminants virus (PPRV) and its natural host, sheep, we also confirm this is a broad and cross-species specific phenotype.
ABSTRACT Orthopneumoviruses characteristically form membrane-less cytoplasmic inclusion bodies (IBs) wherein RNA replication and transcription occur. Here, we report a strategy whereby the orthopneumoviruses sequester various components of the translational pre i nitiation complex machinery into viral inclusion bodies to facilitate translation of their own mRNAs— PIC -pocketing. Electron microscopy of respiratory syncytial virus (RSV)-infected cells revealed bi-phasic organization of IBs, specifically, spherical “droplets” nested within the larger inclusion. Using correlative light and electron microscopy, combined with fluorescence in situ hybridization, we showed that the observed bi-phasic morphology represents functional compartmentalization of the inclusion body and that these domains are synonymous with the previously reported inclusion body-associated granules (IBAGs). Detailed analysis demonstrated that IBAGs concentrate nascent viral mRNA, the viral M2-1 protein as well as components of eukaryotic translation initiation factors (eIF), eIF4F and eIF3, and 40S complexes involved in translation initiation. Interestingly, although ribopuromycylation-based imaging indicates that the majority of viral mRNA translation occurs in the cytoplasm, there was some evidence for intra-IBAG translation, consistent with the likely presence of ribosomes in a subset of IBAGs imaged by electron microscopy. Mass spectrometry analysis of sub-cellular fractions from RSV-infected cells identified significant modification of the cellular translation machinery; however, interestingly, ribopuromycylation assays showed no changes to global levels of translation. The mechanistic basis for this pathway was subsequently determined to involve the viral M2-1 protein interacting with eIF4G, likely to facilitate its transport between the cytoplasm and the separate phases of the viral inclusion body. In summary, our data show that these viral organelles function to spatially regulate early steps in viral translation within a highly selective bi-phasic biomolecular condensate. IMPORTANCE Respiratory syncytial viruses (RSVs) of cows and humans are a significant cause of morbidity and mortality in their respective populations. These RNA viruses replicate in the infected cells by compartmentalizing the cell’s cytoplasm into distinct viral microdomains called inclusion bodies (IBs). In this paper, we show that these IBs are further compartmentalized into smaller structures that have significantly different density, as observed by electron microscopy. Within smaller intra-IB structures, we observed ribosomal components and evidence for active translation. These findings highlight that RSV may additionally compartmentalize translation to favor its own replication in the cell. These data contribute to our understanding of how RNA viruses hijack the cell to favor replication of their own genomes and may provide new targets for antiviral therapeutics in vivo .
Abstract Kobuviruses are an unusual and poorly characterised genus within the picornavirus family, and can cause gastrointestinal enteric disease in humans, livestock and pets. The human Kobuvirus, Aichi virus (AiV) can cause severe gastroenteritis and deaths in children below the age of five years, however this is a very rare occurrence. During the assembly of most picornaviruses (e.g. poliovirus, rhinovirus and foot-and-mouth disease virus), the capsid precursor protein VP0 is cleaved into VP4 and VP2. However, Kobuviruses retain an uncleaved VP0. From studies with other picornaviruses, it is known that VP4 performs the essential function of pore formation in membranes, which facilitates transfer of the viral genome across the endosomal membrane and into the cytoplasm for replication. Here, we employ genome exposure and membrane interaction assays to demonstrate that pH plays a critical role in AiV uncoating and membrane interactions. We demonstrate that incubation at low pH alters the exposure of hydrophobic residues within the capsid, enhances genome exposure and enhances permeabilisation of model membranes. Furthermore, using peptides we demonstrate that the N-terminus of VP0 mediates membrane pore formation in model membranes, indicating that this plays an analogous function to VP4. Importance To initiate infection, viruses must enter a host cell and deliver their genome into the appropriate location. The picornavirus family of small non-enveloped RNA viruses includes significant human and animal pathogens and are also models to understand the process of cell entry. Most picornavirus capsids contain the internal protein VP4, generated from cleavage of a VP0 precursor. During entry, VP4 is released from the capsid. In enteroviruses this forms a membrane pore, which facilitates genome release into the cytoplasm. Due to high levels of sequence similarity, it is expected to play the same role for other picornaviruses. Some picornaviruses, such as Aichi virus, retain an intact VP0, and it is unknown how these viruses re-arrange their capsids and induce membrane permeability in the absence of VP4. Here we have used Aichi virus as a model VP0 virus to test for conservation of function between VP0 and VP4. This could enhance understanding of pore function and lead to development of novel therapeutic agents that block entry.
Respiratory syncytial virus (RSV) is the major cause of childhood respiratory disease; however, currently there is no licenced vaccine available and the only therapeutic, a monoclonal antibody against the viral Fusion (F) protein, is expensive and applied sparingly. RSV particles enter cells by membrane fusion, orchestrated by F – a type I integral membrane protein. This process was recently shown to involve macropinocytosis of the particle. Separately, RSV can spread through induction of direct cell-cell fusion – again orchestrated by F. Little is currently known about the host-factors involved in regulating or inhibiting RSV F-mediated fusion. Here, using two different high-throughput screening approaches, we have identified host-factors involved in regulating RSV fusion. Using quantitative mass-spectrometry analysis of isolated cell membrane fractions from mock and RSV-infected cells we have identified membrane proteins which are differentially regulated during RSV infection. Furthermore, using lentiviral libraries expressing individual interferon stimulated genes (ISGs) from different mammalian species we have investigated ISG-mediated inhibition of RSV fusion. Our data provides important insights into host-factors involved in RSV spread, furthering our understanding of the fusion process and identifying potential targets for antiviral therapy.
Morbilliviruses infect a broad range of mammalian hosts, including ruminants, carnivores, and humans. The recent eradication of rinderpest virus (RPV) and the active campaigns for eradication of the human-specific measles virus (MeV) have raised significant concerns that the remaining morbilliviruses may emerge in so-called vacated ecological niches. Seeking to assess the zoonotic potential of nonhuman morbilliviruses within human populations, we found that peste des petits ruminants virus (PPRV)-the small-ruminant morbillivirus-is restricted at the point of entry into human cells due to deficient interactions with human SLAMF1-the immune cell receptor for morbilliviruses. Using a structure-guided approach, we characterized a single amino acid change, mapping to the receptor-binding domain in the PPRV hemagglutinin (H) protein, which overcomes this restriction. The same mutation allowed escape from some cross-protective, human patient, anti-MeV antibodies, raising concerns that PPRV is a pathogen with zoonotic potential. Analysis of natural variation within human and ovine SLAMF1 also identified polymorphisms that could correlate with disease resistance. Finally, the mechanistic nature of the PPRV restriction was also investigated, identifying charge incompatibility and steric hindrance between PPRV H and human SLAMF1 proteins. Importantly, this research was performed entirely using surrogate virus entry assays, negating the requirement for in situ derivation of a human-tropic PPRV and illustrating alternative strategies for identifying gain-of-function mutations in viral pathogens.IMPORTANCE A significant proportion of viral pandemics occur following zoonotic transmission events, where animal-associated viruses jump species into human populations. In order to provide forewarnings of the emergence of these viruses, it is necessary to develop a better understanding of what determines virus host range, often at the genetic and structural levels. In this study, we demonstrated that the small-ruminant morbillivirus, a close relative of measles, is unable to use human receptors to enter cells; however, a change of a single amino acid in the virus is sufficient to overcome this restriction. This information will be important for monitoring this virus's evolution in the field. Of note, this study was undertaken in vitro, without generation of a fully infectious virus with this phenotype.
The Coronavirus Disease 2019 (COVID-19) pandemic, caused by SARS Coronavirus 2 (SARS-CoV-2), continues to cause significant mortality in human populations worldwide. SARS-CoV-2 has high sequence similarity to SARS-CoV and other related coronaviruses circulating in bats. It is still unclear whether transmission occurred directly from bats to humans, or through an intermediate host, bringing into question the broader host range of SARS-CoV-2. Using a combination of low biocontainment entry assays as well as live virus, we explored the receptor usage of SARS-CoV-2 using angiotensin-converting enzyme 2 (ACE2) receptors from 22 different species. We demonstrated that in addition to human ACE2, the Spike of SARS-CoV-2 has broad tropism for other mammalian ACE2s, including dog, cat and cattle. However, comparison of SARS-CoV-2 receptor usage to the related SARS-CoV and bat coronavirus, RaTG13, identified distinct patterns of receptor usage, with the two human viruses being more closely aligned. Finally, using bioinformatics, structure analysis and targeted mutagenesis, we identified key residues at the Spike-ACE2 interface which may have played a pivotal role in the emergence of SARS-CoV-2 in humans, some of which are also mutated in newly circulating variants of the virus. To summarise, the broad tropism of SARS-CoV-2 at the point of viral entry identifies the potential risk of infection of a wide range of companion animals, livestock and wildlife.
Viral glycoproteins are found on the surface of all enveloped viruses, mediating binding to host receptors and the initiation of entry events. In addition, numerous vaccines employ the same viral glycoproteins as immunogens, either vectored or recombinant in nature. During infection viral glycoproteins are thought to interact with various host-factors, facilitating their trafficking to the cell surface. However, these interactions are not currently well understood or characterised. Using a human gene expression microarray, the cellular response to expression of various viral glycoproteins (Ebola, Nipah, VSV and Measles) was assessed in vitro . Specifically, we found a number of genes with a fold change greater than 2 displaying significantly altered expression across all four glycoprotein transfections. A subset of these genes were selected for validation by qPCR and extended to RSV (respiratory syncytial virus) fusion protein (F) transfection. The expression of these genes was then further investigated in Measles and RSV infected cells. Our data has identified host genes with altered expression in response to a diverse panel of viral glycoproteins, potentially elucidating a conserved set of pathways important for viral glycoprotein activity. Greater understanding of the proteins and pathways involved in glycoprotein expression has the potential to identify mechanisms underpinning host susceptibility to disease as well as improving the yield of vaccine producing cells.
Abstract Infection with rhinovirus (RV) is associated with significant morbidity and hospitalisation in people with chronic lung disease. At present there is no approved RV vaccine or antiviral. There are approximately 180 RV serotypes, classified into 3 species (RVA, RVB, RVC). There is no cross-protection between serotypes because of high diversity in immunodominant antigenic sites. This makes it impractical to create a broadly protective vaccine using traditional methods, involving whole capsids. A more feasible strategy is to direct the immune response towards conserved but less dominant epitopes. The capsid protein VP4 is highly conserved within each RV genotype and some antibodies that target VP4 are neutralising. This makes VP4 vulnerable to antibodies and a promising vaccine target. Here we investigate the ability of RV VP4 N-terminal peptides presented on different display systems to initiate a neutralising immune response in mice. We compared three different-sized VP4 peptides (spanning residues, 1-15, 1-30 and 1-45) displayed on two different display systems-SpyCatcher Virus-like particles (VLPs) or Keyhole Limpet Hemocyanin (KLH). Overlapping regions of VP4 were antigenically different when presented on different platforms and in peptides with different length. The conformation of the 1-15 region of VP4 was critical for inducing antibodies that were both neutralising and able to bind VP4 in the context of the virus particle. Therefore, correctly displayed VP4 peptides can recapitulate virus-like antigenicity. These findings improve our understanding of VP4 antigenicity and will inform the design of future RV vaccines. This work could also impact the design of other peptide vaccines, since a variable antigenic conformation is a common characteristic of pathogen-derived peptide targets. Impact statement Rhinovirus (RV) is a highly prevalent respiratory virus that is a frequent cause of common cold symptoms. For people with chronic lung diseases such as asthma, infection can cause significant worsening of disease, leading to increased suffering and hospitalisation. There are currently no vaccines or anti-viral drugs that target RV infections. Development of a RV vaccine would be hugely beneficial to suffers of chronic lung diseases. Attempts to develop a broadly protective RV vaccine have been hindered by the presence of over 180 serotypes which have little or no natural cross-reactivity. Previous studies have identified the highly conserved VP4 epitope as a potential target for broadly protective vaccines. However, there has been little progress in evaluating its effectiveness as a vaccine candidate. In this study we assessed the ability of different RV VP4 immunogens to induce neutralising antibodies in mice. We demonstrated that antigenic conformation of VP4 varies greatly between immunogens and presentation of the first 15 N-terminal residues in a virus-like conformation is important for generating neutralising antibodies. This knowledge could help to maximise the production of neutralising VP4-specific antibodies in future studies which could ultimately lead to a universal RV vaccine.
A review of published clinical reports shows that anorexia nervosa has been found in association with several genetic anomalies, principally gonosomal aneuploidy. An additional, and unique, association is described here: a case of anorexia nervosa in a patient with the yellow mutant form of oculocutaneous albinism and no other apparent chromosomal abnormalities. While the concurrence of these two disorders in a single person is apparently a chance phenomenon, our review of experimental publications shows that feeding disturbances also occur in yellow mutant mice. Such complementary findings suggest the need for continuing investigation of the genetic foundations of eating behaviour.
'%3e%3cpath fill='%23012169' d='M0 0v30h60V0z'/%3e%3cpath stroke='%23fff' stroke-width='6' d='m0 0 60 30m0-30L0 30'/%3e%3cpath stroke='%23C8102E' stroke-width='4' d='m0 0 60 30m0-30L0 30' clip-path='url(%23b)'/%3e%3cpath stroke='%23fff' stroke-width='10' d='M30 0v30M0 15h60'/%3e%3cpath stroke='%23C8102E' stroke-width='6' d='M30 0v30M0 15h60'/%3e%3c/g%3e%3c/svg%3e)
