The hepatitis C virus (HCV) genome codes for highly mannosylated envelope proteins, which are naturally retained in the endoplasmic reticulum. We found that the HCV envelope glycoprotein E2 binds the dendritic cell-specific intercellular adhesion molecule 3-grabbing nonintegrin (DC-SIGN) and the related liver endothelial cell lectin L-SIGN through high-mannose N-glycans. Hepatitis C virus (HCV)1 is the major causative agent of non-A, non-B hepatitis throughout the world with more than 170 million people infected (1). Contamination with infected blood by injecting drug users is the primary risk factor for acquiring HCV infection. The majority of infected patients are unable to clear the virus, and many develop chronic liver disease, cirrhosis, and hepatocellular carcinoma (2). Replication of the HCV genome could be demonstrated in vivo and in vitro in liver hepatocytes (3, 4) and hematopoietic cells including dendritic cells and B cells (5, 6). However, the molecular mechanism by which the virus targets to these sites of replication, notably in the liver, is not known.HCV is a small, enveloped, plus-strand RNA virus belonging to the family flaviviridae and genus hepacivirus. The HCV RNA genome is 9600 nucleotides in length and encodes a single polyprotein that is post-translationally cleaved into up to 10 polypeptides including three structural proteins (core, E1, and E2), located at the N terminus, and five nonstructural proteins (1,7,8). Shortly after translocation into the endoplasmic reticulum (ER), oligosaccharide transferase catalyzes addition of Glc3Man9GlcNAc2 complexes at up to 6 (E1) and 11 (E2) N-glycosylation sites (for review see Ref. 9). Glucose residues are removed by glucosidases I and II, and correctly folded proteins are released from ER chaperones calnexin and calreticulin (10 -13). The transmembrane domains of E1 and E2 are responsible for both heterodimerization (14) and retention of the glycoproteins in a high-mannose EndoH-sensitive glycoform in the ER (15)(16)(17). By analogy to other flaviviruses it is assumed that HCV capsids bud from the cytoplasm into the ER and that enveloped particles follow the secretion pathway through the Golgi. However, attempts to produce secreted HCV particles in vitro have not been successful so far (18 -20), and it is not known if E1 and E2 on mature infectious virions possess a high-mannose, complex, or mixed glycosylation.Several receptors have been proposed that could play a role in HCV entry into hepatocytes. The low density lipoprotein (LDL) receptor has been shown to mediate HCV internalization via binding to virus-associated LDL particles (21,22). A second putative HCV receptor, the tetraspanin CD81, has been identified as a high affinity binding receptor (1.8 nM) for soluble recombinant E2 from HCV genotype 1a (23, 24). CD81 and LDL receptor are expressed in most cell types and thus likely do not account for the hepatic tropism of the virus. Furthermore E2 binds to the hepatoblastoma cell line HepG2, which does not express CD81 (25). More recently tw...

During natural transmission, bunyaviruses are introduced into the skin through arthropod bites, and dermal dendritic cells (DCs) are the first to encounter incoming viruses. DC-SIGN is a C-type lectin highly expressed on the surface of dermal DCs. We found that several arthropod-borne phleboviruses (Bunyaviridae), including Rift Valley fever and Uukuniemi viruses, exploit DC-SIGN to infect DCs and other DC-SIGN-expressing cells. DC-SIGN binds the virus directly via interactions with high-mannose N-glycans on the viral glycoproteins and is required for virus internalization and infection. In live cells, virus-induced clustering of cell surface DC-SIGN could be visualized. An endocytosis-defective mutant of DC-SIGN was unable to mediate virus uptake, indicating that DC-SIGN is an authentic receptor required for both attachment and endocytosis. After internalization, viruses separated from DC-SIGN and underwent trafficking to late endosomes. Our study provides real-time visualization of virus-receptor interactions on the cell surface and establishes DC-SIGN as a phlebovirus entry receptor.

The Bunyaviridae constitute a large family of enveloped animal viruses, many members of which cause serious diseases. However, early bunyavirus-host cell interactions and entry mechanisms remain largely uncharacterized. Investigating Uukuniemi virus, a bunyavirus of the genus Phlebovirus, we found that virus attachment to the cell surface was specific but inefficient, with 25% of bound viruses being endocytosed within 10 min, mainly via noncoated vesicles. The viruses entered Rab5a+ early endosomes and, subsequently, Rab7a+ and LAMP-1+ late endosomes. Acid-activated penetration, occurring 20-40 min after internalization, required maturation of early to late endosomes. The pH threshold for viral membrane fusion was 5.4, and entry was sensitive to temperatures below 25 degrees C. Together, our results indicate that Uukuniemi virus penetrates host cells by acid-activated membrane fusion from late endosomal compartments. This study also highlights the importance of the degradative branch of the endocytic pathway in facilitating entry of late-penetrating viruses.

Dengue virus (DV) is a mosquito-borne flavivirus that causes hemorrhagic fever in humans. In the natural infection, DV is introduced into human skin by an infected mosquito vector where it is believed to target immature dendritic cells (DCs) and Langerhans cells (LCs). We found that DV productively infects DCs but not LCs. We show here that the interactions between DV E protein, the sole mannosylated glycoprotein present on DV particles, and the C-type lectin dendritic cell-specific intercellular adhesion molecule 3-grabbing non-integrin (DC-SIGN) are essential for DV infection of DCs. Binding of mannosylated N-glycans on DV E protein to DC-SIGN triggers a rapid and efficient internalization of the viral glycoprotein. However, we observed that endocytosisdefective DC-SIGN molecules allow efficient DV replication, indicating that DC-SIGN endocytosis is dispensable for the internalization step in DV entry. Together, these results argue in favor of a mechanism by which DC-SIGN enhances DV entry and infection in cis. We propose that DC-SIGN concentrates mosquito-derived DV particles at the cell surface to allow efficient interaction with an as yet unidentified entry factor that is ultimately responsible for DV internalization and pHdependent fusion into DCs.

SARS‐CoV‐2 is a newly emerged coronavirus that caused the global COVID‐19 outbreak in early 2020. COVID‐19 is primarily associated with lung injury, but many other clinical symptoms such as loss of smell and taste demonstrated broad tissue tropism of the virus. Early SARS‐CoV‐2–host cell interactions and entry mechanisms remain poorly understood. Investigating SARS‐CoV‐2 infection in tissue culture, we found that the protease TMPRSS2 determines the entry pathway used by the virus. In the presence of TMPRSS2, the proteolytic process of SARS‐CoV‐2 was completed at the plasma membrane, and the virus rapidly entered the cells within 10 min in a pH‐independent manner. When target cells lacked TMPRSS2 expression, the virus was endocytosed and sorted into endolysosomes, from which SARS‐CoV‐2 entered the cytosol via acid‐activated cathepsin L protease 40–60 min post‐infection. Overexpression of TMPRSS2 in non‐TMPRSS2 expressing cells abolished the dependence of infection on the cathepsin L pathway and restored sensitivity to the TMPRSS2 inhibitors. Together, our results indicate that SARS‐CoV‐2 infects cells through distinct, mutually exclusive entry routes and highlight the importance of TMPRSS2 for SARS‐CoV‐2 sorting into either pathway.

Dengue virus envelope protein (E) contains two N-linked glycosylation sites, at Asn-67 and Asn-153. The glycosylation site at position 153 is conserved in most flaviviruses, while the site at position 67 is thought to be unique for dengue viruses. N-linked oligosaccharide side chains on flavivirus E proteins have been associated with viral morphogenesis, infectivity, and tropism. Here, we examined the relevance of each N-linked glycan on dengue virus E protein by removing each site in the context of infectious viral particles. Dengue viruses lacking Asn-67 were able to infect mammalian cells and translate and replicate the viral genome, but production of new infectious particles was abolished. In addition, dengue viruses lacking Asn-153 in the E showed reduced infectivity. In contrast, ablation of one or both glycosylation sites yielded viruses that replicate and propagate in mosquito cells. Furthermore, we found a differential requirement of N-linked glycans for E secretion in mammalian and mosquito cells. While secretion of E lacking Asn-67 was efficient in mosquito cells, secretion of the same protein expressed in mammalian cells was dramatically impaired. Finally, we found that viruses lacking the carbohydrate at position 67 showed reduced infection of immature dendritic cells, suggesting interaction between this glycan and the lectin DC-SIGN. Overall, our data defined different roles for the two glycans present at the E protein during dengue virus infection, highlighting the involvement of distinct host functions from mammalian and mosquito cells during dengue virus propagation.Dengue virus (DV), a member of the flavivirus genus of the Flaviviridae family, causes the most prevalent arthropod-borne viral disease of humans, which is associated with a large social and economic burden. DV contains a single-stranded RNA genome of positive polarity that is translated in the cytoplasm as a single polyprotein. Processing of the polyprotein by cellular and viral proteases results in three structural and seven nonstructural mature proteins. The structural proteins are the capsid (C), which assembles with the genomic RNA, the glycosylated envelope protein (E), and the precursor of the membrane protein (prM). Flavivirus assembly takes place in the endoplasmic reticulum (ER) (23, 24). The RNA-C complex buds into the ER lumen, acquiring the lipid bilayer, E, and prM. Furin-mediated proteolysis of prM in the trans-Golgi network (40) triggers rearrangement, the homodimerization of E, and the formation of mature viral particles (1).It is postulated that the interaction of E with a host cell receptor directs DV particles to the endocytic pathway. The acidic environment in the endosome is believed to produce major conformational changes in E that induce fusion of the viral and host cell membranes, which in turn releases the viral genome into the cytoplasm. Several studies indicate that cell surface heparan sulfates (HS) are involved in the attachment of DV to mammalian but not to mosquito cells (5,9,13,14). It was demonstrate...

During viral infection the first challenge that viruses have to overcome is gaining access to the intracellular compartment. The infection process starts when the virus contacts the surface of the host cell. A complex series of events ensues, including diffusion at the host cell membrane surface, binding to receptors, signaling, internalization, and delivery of the genetic information. The focus of this review is on the very initial steps of virus entry, from receptor binding to particle uptake into the host cell. We will discuss how viruses find their receptor, move to sub-membranous regions permissive for entry, and how they hijack the receptor-mediated signaling pathway to promote their internalization.

No abstract