Liver-expressed Cd302 and Cr1l limit hepatitis C virus cross-species transmission to mice.

Hepatitis C virus (HCV) has no animal reservoir, infecting only humans. To investigate species barrier determinants limiting infection of rodents, murine liver complementary DNA library screening was performed, identifying transmembrane proteins Cd302 and Cr1l as potent restrictors of HCV propagation. Combined ectopic expression in human hepatoma cells impeded HCV uptake and cooperatively mediated transcriptional dysregulation of a noncanonical program of immunity genes. Murine hepatocyte expression of both factors was constitutive and not interferon inducible, while differences in liver expression and the ability to restrict HCV were observed between the murine orthologs and their human counterparts. Genetic ablation of endogenous Cd302 expression in human HCV entry factor transgenic mice increased hepatocyte permissiveness for an adapted HCV strain and dysregulated expression of metabolic process and host defense genes. These findings highlight human-mouse differences in liver-intrinsic antiviral immunity and facilitate the development of next-generation murine models for preclinical testing of HCV vaccine candidates.

Yellow Fever: Integrating Current Knowledge with Technological Innovations to Identify Strategies for Controlling a Re-Emerging Virus.

Yellow fever virus (YFV) represents a re-emerging zoonotic pathogen, transmitted by mosquito vectors to humans from primate reservoirs. Sporadic outbreaks of YFV occur in endemic tropical regions, causing a viral hemorrhagic fever (VHF) associated with high mortality rates. Despite a highly effective vaccine, no antiviral treatments currently exist. Therefore, YFV represents a neglected tropical disease and is chronically understudied, with many aspects of YFV biology incompletely defined including host range, host-virus interactions and correlates of host immunity and pathogenicity. In this article, we review the current state of YFV research, focusing on the viral lifecycle, host responses to infection, species tropism and the success and associated limitations of the YFV-17D vaccine. In addition, we highlight the current lack of available treatments and use publicly available sequence and structural data to assess global patterns of YFV sequence diversity and identify potential drug targets. Finally, we discuss how technological advances, including real-time epidemiological monitoring of outbreaks using next-generation sequencing and CRISPR/Cas9 modification of vector species, could be utilized in future battles against this re-emerging pathogen which continues to cause devastating disease.

Complex and Dynamic Interactions between Parvovirus Capsids, Transferrin Receptors, and Antibodies Control Cell Infection and Host Range.

Antibody and receptor binding are key virus-host interactions that control host range and determine the success of infection. Canine and feline parvovirus capsids bind the transferrin receptor type 1 (TfR) to enter host cells, and specific structural interactions appear necessary to prepare the stable capsids for infection. Here, we define the details of binding, competition, and occupancy of wild-type and mutant parvovirus capsids with purified receptors and antibodies. TfR-capsid binding interactions depended on the TfR species and varied widely, with no direct relationship between binding affinity and infection. Capsids bound feline, raccoon, and black-backed jackal TfRs at high affinity but barely bound canine TfRs, which mediated infection efficiently. TfRs from different species also occupied capsids to different levels, with an estimated 1 to 2 feline TfRs but 12 black-backed jackal TfRs binding each capsid. Multiple alanine substitutions within loop 1 on the capsid surface reduced TfR binding but substitutions within loop 3 did not, suggesting that loop 1 directly engaged the TfR and loop 3 sterically affected that interaction. Binding and competition between different TfRs and/or antibodies showed complex relationships. Both antibodies 14 and E competed capsids off TfRs, but antibody E could also compete capsids off itself and antibody 14, likely by inducing capsid structural changes. In some cases, the initial TfR or antibody binding event affected subsequent TfR binding, suggesting that capsid structure changes occur after TfR or antibody binding and may impact infection. This shows that precise, host-specific TfR-capsid interactions, beyond simple attachment, are important for successful infection.IMPORTANCE Host receptor binding is a key step during viral infection and may control both infection and host range. In addition to binding, some viruses require specific interactions with host receptors in order to infect, and anti-capsid antibodies can potentially disrupt these interactions, leading to neutralization. Here, we examine the interactions between parvovirus capsids, the receptors from different hosts, and anti-capsid antibodies. We show that interactions between parvovirus capsids and host-specific TfRs vary in both affinity and in the numbers of receptors bound, with complex effects on infection. In addition, antibodies binding to two sites on the capsids had different effects on TfR-capsid binding. These experiments confirm that receptor and antibody binding to parvovirus capsids are complex processes, and the infection outcome is not determined simply by the affinity of attachment.

Frequent occurrence of nonprimate hepacivirus infections in Thoroughbred breeding horses - A cross-sectional study for the occurrence of infections and potential risk factors.

Recently, several new hepaciviruses have been identified of which the nonprimate hepacivirus (NPHV) - the closest relative to hepatitis C virus (HCV) discovered to date - is highly prevalent in horses. However, potential risk factors for the transmission of NPHV among horses remain still unknown. Therefore, the objective of this study was to investigate the occurrence of NPHV infections in Thoroughbreds in northern and western Germany and to identify potential risk factors associated with NPHV infections. Using a cross-sectional study design, a total of 733 serum samples from Thoroughbred broodmares and stallions from northern and western Germany were analyzed for the presence of anti-NPHV nonstructural protein 3 (NS3) antibodies and NPHV RNA using the luciferase immunoprecipitation system (LIPS) and a quantitative real-time PCR, respectively. Information regarding signalment, stud farm, breeding history and international transportation history of each horse were collected and evaluated. A frequent occurrence of NPHV was found in the study population with 453 seropositive horses (61.8%) and 134 horses (18.3%) carrying NPHV RNA. Furthermore, statistical analysis revealed that the probability of being infected decreased for horses with a transportation history with increasing age by 20% each year. For horses that stayed in Germany no association between age and infection could be observed. In conclusion, the high occurrence of NPHV infections in breeding Thoroughbreds suggests circulating NPHV infections, endemic herds or persistent shedding in these animals and revealed the association of age and international transportation as risk factor for NPHV infections.

ABHD5/CGI-58, the Chanarin-Dorfman Syndrome Protein, Mobilises Lipid Stores for Hepatitis C Virus Production.

Hepatitis C virus (HCV) particles closely mimic human very-low-density lipoproteins (VLDL) to evade humoral immunity and to facilitate cell entry. However, the principles that govern HCV association with VLDL components are poorly defined. Using an siRNA screen, we identified ABHD5 (α/ß hydrolase domain containing protein 5, also known as CGI-58) as a new host factor promoting both virus assembly and release. ABHD5 associated with lipid droplets and triggered their hydrolysis. Importantly, ABHD5 Chanarin-Dorfman syndrome mutants responsible for a rare lipid storage disorder in humans were mislocalised, and unable to consume lipid droplets or support HCV production. Additional ABHD5 mutagenesis revealed a novel tribasic motif that does not influence subcellular localization but determines both ABHD5 lipolytic and proviral properties. These results indicate that HCV taps into the lipid droplet triglyceride reservoir usurping ABHD5 lipase cofactor function. They also suggest that the resulting lipid flux, normally devoted to VLDL synthesis, also participates in the assembly and release of the HCV lipo-viro-particle. Altogether, our study provides the first association between the Chanarin-Dorfman syndrome protein and an infectious disease and sheds light on the hepatic manifestations of this rare genetic disorder as well as on HCV morphogenesis.

Quantitative Proteomics Identifies Serum Response Factor Binding Protein 1 as a Host Factor for Hepatitis C Virus Entry.

Hepatitis C virus (HCV) enters human hepatocytes through a multistep mechanism involving, among other host proteins, the virus receptor CD81. How CD81 governs HCV entry is poorly characterized, and CD81 protein interactions after virus binding remain elusive. We have developed a quantitative proteomics protocol to identify HCV-triggered CD81 interactions and found 26 dynamic binding partners. At least six of these proteins promote HCV infection, as indicated by RNAi. We further characterized serum response factor binding protein 1 (SRFBP1), which is recruited to CD81 during HCV uptake and supports HCV infection in hepatoma cells and primary human hepatocytes. SRFBP1 facilitates host cell penetration by all seven HCV genotypes, but not of vesicular stomatitis virus and human coronavirus. Thus, SRFBP1 is an HCV-specific, pan-genotypic host entry factor. These results demonstrate the use of quantitative proteomics to elucidate pathogen entry and underscore the importance of host protein-protein interactions during HCV invasion.

DESC1 and MSPL activate influenza A viruses and emerging coronaviruses for host cell entry.

The type II transmembrane serine protease (TTSP) TMPRSS2 cleaves and activates the influenza virus and coronavirus surface proteins. Expression of TMPRSS2 is essential for the spread and pathogenesis of H1N1 influenza viruses in mice. In contrast, H3N2 viruses are less dependent on TMPRSS2 for viral amplification, suggesting that these viruses might employ other TTSPs for their activation. Here, we analyzed TTSPs, reported to be expressed in the respiratory system, for the ability to activate influenza viruses and coronaviruses. We found that MSPL and, to a lesser degree, DESC1 are expressed in human lung tissue and cleave and activate the spike proteins of the Middle East respiratory syndrome and severe acute respiratory syndrome coronaviruses for cell-cell and virus-cell fusion. In addition, we show that these proteases support the spread of all influenza virus subtypes previously pandemic in humans. In sum, we identified two host cell proteases that could promote the amplification of influenza viruses and emerging coronaviruses in humans and might constitute targets for antiviral intervention. Importance: Activation of influenza viruses by host cell proteases is essential for viral infectivity and the enzymes responsible are potential targets for antiviral intervention. The present study demonstrates that two cellular serine proteases, DESC1 and MSPL, activate influenza viruses and emerging coronaviruses in cell culture and, because of their expression in human lung tissue, might promote viral spread in the infected host. Antiviral strategies aiming to prevent viral activation might thus need to encompass inhibitors targeting MSPL and DESC1.

The spike protein of the emerging betacoronavirus EMC uses a novel coronavirus receptor for entry, can be activated by TMPRSS2, and is targeted by neutralizing antibodies.

The novel human coronavirus EMC (hCoV-EMC), which recently emerged in Saudi Arabia, is highly pathogenic and could pose a significant threat to public health. The elucidation of hCoV-EMC interactions with host cells is critical to our understanding of the pathogenesis of this virus and to the identification of targets for antiviral intervention. Here we investigated the viral and cellular determinants governing hCoV-EMC entry into host cells. We found that the spike protein of hCoV-EMC (EMC-S) is incorporated into lentiviral particles and mediates transduction of human cell lines derived from different organs, including the lungs, kidneys, and colon, as well as primary human macrophages. Expression of the known coronavirus receptors ACE2, CD13, and CEACAM1 did not facilitate EMC-S-driven transduction, suggesting that hCoV-EMC uses a novel receptor for entry. Directed protease expression and inhibition analyses revealed that TMPRSS2 and endosomal cathepsins activate EMC-S for virus-cell fusion and constitute potential targets for antiviral intervention. Finally, EMC-S-driven transduction was abrogated by serum from an hCoV-EMC-infected patient, indicating that EMC-S-specific neutralizing antibodies can be generated in patients. Collectively, our results indicate that hCoV-EMC uses a novel receptor for protease-activated entry into human cells and might be capable of extrapulmonary spread. In addition, they define TMPRSS2 and cathepsins B and L as potential targets for intervention and suggest that neutralizing antibodies contribute to the control of hCoV-EMC infection.

TMPRSS2 activates the human coronavirus 229E for cathepsin-independent host cell entry and is expressed in viral target cells in the respiratory epithelium.

Infection with human coronavirus 229E (HCoV-229E) is associated with the common cold and may result in pneumonia in immunocompromised patients. The viral spike (S) protein is incorporated into the viral envelope and mediates infectious entry of HCoV-229E into host cells, a process that depends on the activation of the S-protein by host cell proteases. However, the proteases responsible for HCoV-229E activation are incompletely defined. Here we show that the type II transmembrane serine proteases TMPRSS2 and HAT cleave the HCoV-229E S-protein (229E-S) and augment 229E-S-driven cell-cell fusion, suggesting that TMPRSS2 and HAT can activate 229E-S. Indeed, engineered expression of TMPRSS2 and HAT rendered 229E-S-driven virus-cell fusion insensitive to an inhibitor of cathepsin L, a protease previously shown to facilitate HCoV-229E infection. Inhibition of endogenous cathepsin L or TMPRSS2 demonstrated that both proteases can activate 229E-S for entry into cells that are naturally susceptible to infection. In addition, evidence was obtained that activation by TMPRSS2 rescues 229E-S-dependent cell entry from inhibition by IFITM proteins. Finally, immunohistochemistry revealed that TMPRSS2 is coexpressed with CD13, the HCoV-229E receptor, in human airway epithelial (HAE) cells, and that CD13(+) TMPRSS2(+) cells are preferentially targeted by HCoV-229E, suggesting that TMPRSS2 can activate HCoV-229E in infected humans. In sum, our results indicate that HCoV-229E can employ redundant proteolytic pathways to ensure its activation in host cells. In addition, our observations and previous work suggest that diverse human respiratory viruses are activated by TMPRSS2, which may constitute a target for antiviral intervention.