A novel betacoronavirus, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), which caused a large respiratory outbreak in Wuhan, China in December 2019, is currently spreading across many countries globally. Here, we show that a TMPRSS2expressing VeroE6 cell line is highly susceptible to SARS-CoV-2 infection, making it useful for isolating and propagating SARS-CoV-2. Our results reveal that, in common with SARS-and Middle East respiratory syndrome-CoV, SARS-CoV-2 infection is enhanced by TMPRSS2.

No abstract

(RuV) causes a systemic infection and transplacental fetal infection causes congenital rubella syndrome. In this study, we showed that treatment of cells with sphingomyelinase inhibited RuV infection. Assays using inhibitors of serine palmitoyl transferase and ceramide transport protein demonstrated the contribution of sphingomyelin (SM) to RuV infection. Compelling evidence for direct binding of RuV to lipid membranes at neutral pH was obtained using liposome co-flotation assays. The absence of either SM or cholesterol (Chol) abrogated the RuV--liposome interaction. SM and Chol (SM/Chol) were also critical for RuV binding to erythrocytes and lymphoid cells. Removal of Ca from the assay buffer or mutation of RuV envelope E1 protein Ca-binding sites abrogated RuV binding to liposomes, erythrocytes, and lymphoid cells. However, RuV bound to various non-lymphoid adherent cell lines independently of extracellular Ca or SM/Chol. Even in these adherent cell lines, both the E1 protein Ca-binding sites and cellular SM/Chol were essential for the early stage of RuV infection, possibly affecting envelope-membrane fusion in acidic compartments. Myelin oligodendrocyte glycoprotein (MOG) has recently been identified as a cellular receptor for RuV. However, RuV bound to MOG-negative cells in a Ca-independent manner. Collectively, our data demonstrate that RuV has two distinct binding mechanisms: one is Ca-dependent and the other Ca-independent. Ca-dependent binding observed in lymphoid cells occurs by the direct interaction between E1 protein fusion loops and SM/Chol-enriched membranes. Clarification of the mechanism of Ca-independent RuV binding is an important next step in understanding the pathology of RuV infection. Rubella has a significant impact on public health as infection during early pregnancy can result in babies being born with congenital rubella syndrome. Despite effective rubella vaccines being available, rubella outbreaks still occur in many countries. We studied the entry mechanism of (RuV) and found that RuV binds directly to the host plasma membrane in the presence of Ca at neutral pH. This Ca-dependent binding is specifically directed to membranes enriched in sphingomyelin and cholesterol, and is critical for RuV infection. Importantly, RuV also binds to many cell lines in a Ca-independent manner. An unidentified RuV receptor(s) is involved in this Ca-independent binding. We believe that the data presented here may aid the development of the first anti-RuV drug.

Mumps virus (MuV) is an airborne virus that causes a systemic infection in patients.In vivo, the epithelium is a major replication site of MuV, and thus, the mode of MuV infection of epithelial cells is a subject of interest. Our data in the present study showed that MuV entered polarized epithelial cells via both the apical and basolateral surfaces, while progeny viruses were predominantly released from the apical surface. In polarized cells, intracellular transport of viral ribonucleoprotein (vRNP) complexes was dependent on Rab11-positive endosomes, and vRNP complexes were transported to the apical membrane. Expression of a dominant negative form of Rab11 (Rab11S25N) reduced the progeny virus release in polarized cells but not in nonpolarized cells. Although in this way these effects were correlated with cell polarity, Rab11S25N did not modulate the direction of virus release from the apical surface. Therefore, our data suggested that Rab11 is not a regulator of selective apical release of MuV, although it acts as an activator of virus release from polarized epithelial cells. In addition, our data and previous studies on Sendai virus, respiratory syncytial virus, and measles virus suggested that selective apical release from epithelial cells is used by many paramyxoviruses, even though they cause either a systemic infection or a local respiratory infection. IMPORTANCE Mumps virus (MuV) is the etiological agent of mumps and causes a systemic infection.However, the precise mechanism by which MuV breaks through the epithelial barriers and achieves a systemic infection remains unclear. In the present study, we show that the entry of MuV is bipolar, while the release is predominantly from the apical surface in polarized epithelial cells. In addition, the release of progeny virus was facilitated by a Rab11-positive recycling endosome and microtubule network. Our data provide important insights into the mechanism of transmission and pathogenesis of MuV. Mumps is a common childhood illness characterized by painful swelling of the parotid glands and is often accompanied by severe complications such as orchitis, aseptic meningitis, pancreatitis, and deafness (1). However, despite the prevalence and seriousness of the disease, the molecular basis of the pathogenesis of mumps is still poorly understood.Mumps virus (MuV), which belongs to the genus Rubulavirus within the family Paramyxoviridae, is the causative agent of mumps (2). The virus infection of cells is initiated by the binding of the hemagglutinin-neuraminidase (HN) protein to sialic acids of the cell surface (3). After receptor binding, the fusion (F) protein induces pH-independent fusion of the viral envelope with the host plasma membrane, and the viral genomic RNA is released into the cytoplasm (4). The viral genomic RNA encapsidated by the nucleocapsid (N) protein forms an active template for RNA replication and transcription, a viral ribonucleoprotein (vRNP), with viral polymerases composed of the phosphoprotein (P protein) and large (L) protein (5...

Nucleocapsid formation is a primary function of the rubella virus capsid protein, which also promotes viral RNA synthesis via an unknown mechanism. The present study demonstrates that in infected cells, the capsid protein is associated with the nonstructural p150 protein via the short self-interacting N-terminal region of the capsid protein. Mutational analyses indicated that hydrophobic amino acids in this N-terminal region are essential for its N-terminal self-interaction, which is critical for the capsid-p150 association. An analysis based on a subgenomic replicon system demonstrated that the self-interacting N-terminal region of the capsid protein plays a key role in promoting viral gene expression. Analyses using a virus-like particle (VLP) system also showed that the self-interacting N-terminal region of the capsid protein is not essential for VLP production but is critical for VLP infectivity. These results demonstrate that the close cooperative actions of the capsid protein and p150 require the short self-interacting N-terminal region of the capsid protein during the life cycle of the rubella virus. IMPORTANCEThe capsid protein of rubella virus promotes viral RNA replication via an unknown mechanism. This protein interacts with the nonstructural protein p150, but the importance of this interaction is unclear. In this study, we demonstrate that the short N-terminal region of the capsid protein forms a homo-oligomer that is critical for the capsid-p150 interaction. These interactions are required for the viral-gene-expression-promoting activity of the capsid protein, allowing efficient viral growth. These findings provide information about the mechanisms underlying the regulation of rubella virus RNA replication via the cooperative actions of the capsid protein and p150. R ubella virus (RV) is the sole member of the genus Rubivirus in the family Togaviridae. RV is an enveloped, single-stranded, positive-sense RNA virus with a genome of approximately 10 kb. The genome acts as an mRNA and contains three untranslated regions and two open reading frames (ORFs). The ORF at the 5= end encodes two nonstructural proteins (NSPs), p150 and p90, which function in viral RNA replication. The other ORF, at the 3= end, encodes three structural proteins (SPs), capsid, E2, and E1, which are integral components of the virion (1). NSP p150 contains several domains that are conserved among other RNA viruses (2, 3). A putative methyltransferase domain and a protease domain are located at the amino (N) and carboxyl (C) termini, respectively (2, 4, 5). There are also two domains of unknown function, Y and X, between the methyltransferase and protease domains. The putative methyltransferase domain is considered to play a role in capping viral RNA (4). The protease domain cleaves the precursor polyprotein p200 into p150 and p90, and this step is critical in the regulation of viral RNA replication (6-8). NSP p90 has two functional domains, helicase and RNA-dependent RNA polymerase (RdRp) domains, in the N-and C-terminal reg...