Glycomics and Proteomics Approaches to Investigate Early Adenovirus-Host Cell Interactions.

Adenoviruses as most viruses rely on glycan and protein interactions to attach to and enter susceptible host cells. The Adenoviridae family comprises more than 80 human types and they differ in their attachment factor and receptor usage, which likely contributes to the diverse tropism of the different types. In the past years, methods to systematically identify glycan and protein interactions have advanced. In particular sensitivity, speed and coverage of mass spectrometric analyses allow for high-throughput identification of glycans and peptides separated by liquid chromatography. Also, developments in glycan microarray technologies have led to targeted, high-throughput screening and identification of glycan-based receptors. The mapping of cell surface interactions of the diverse adenovirus types has implications for cell, tissue, and species tropism as well as drug development. Here we review known adenovirus interactions with glycan- and protein-based receptors, as well as glycomics and proteomics strategies to identify yet elusive virus receptors and attachment factors. We finally discuss challenges, bottlenecks, and future research directions in the field of non-enveloped virus entry into host cells

CD81 Receptor Regions outside the Large Extracellular Loop Determine Hepatitis C Virus Entry into Hepatoma Cells

Hepatitis C virus (HCV) enters human hepatocytes using four essential entry factors, one of which is human CD81 (hCD81). The tetraspanin hCD81 contains a large extracellular loop (LEL), which interacts with the E2 glycoprotein of HCV. The role of the non-LEL regions of hCD81 (intracellular tails, four transmembrane domains, small extracellular loop and intracellular loop) is poorly understood. Here, we studied the contribution of these domains to HCV susceptibility of hepatoma cells by generating chimeras of related tetraspanins with the hCD81 LEL. Our results show that non-LEL regions in addition to the LEL determine susceptibility of cells to HCV. While closely related tetraspanins (X. tropicalis CD81 and D. rerio CD81) functionally complement hCD81 non-LEL regions, distantly related tetraspanins (C. elegans TSP9 amd D. melanogaster TSP96F) do not and tetraspanins with intermediate homology (hCD9) show an intermediate phenotype. Tetraspanin homology and susceptibility to HCV correlate positively. For some chimeras, infectivity correlates with surface expression. In contrast, the hCD9 chimera is fully surface expressed, binds HCV E2 glycoprotein but is impaired in HCV receptor function. We demonstrate that a cholesterol-coordinating glutamate residue in CD81, which hCD9 lacks, promotes HCV infection. This work highlights the hCD81 non-LEL regions as additional HCV susceptibility-determining factors

The Phosphatidylserine Receptor TIM-1 Enhances Authentic Chikungunya Virus Cell Entry

Chikungunya virus (CHIKV) is a re-emerging, mosquito-transmitted, enveloped positive stranded RNA virus. Chikungunya fever is characterized by acute and chronic debilitating arthritis. Although multiple host factors have been shown to enhance CHIKV infection, the molecular mechanisms of cell entry and entry factors remain poorly understood. The phosphatidylserine-dependent receptors, T-cell immunoglobulin and mucin domain 1 (TIM-1) and Axl receptor tyrosine kinase (Axl), are transmembrane proteins that can serve as entry factors for enveloped viruses. Previous studies used pseudoviruses to delineate the role of TIM-1 and Axl in CHIKV entry. Conversely, here, we use the authentic CHIKV and cells ectopically expressing TIM-1 or Axl and demonstrate a role for TIM-1 in CHIKV infection. To further characterize TIM-1-dependent CHIKV infection, we generated cells expressing domain mutants of TIM-1. We show that point mutations in the phosphatidylserine binding site of TIM-1 lead to reduced cell binding, entry, and infection of CHIKV. Ectopic expression of TIM-1 renders immortalized keratinocytes permissive to CHIKV, whereas silencing of endogenously expressed TIM-1 in human hepatoma cells reduces CHIKV infection. Altogether, our findings indicate that, unlike Axl, TIM-1 readily promotes the productive entry of authentic CHIKV into target cells

Hepatitis C virus enters liver cells using the CD81 receptor complex proteins calpain-5 and CBLB.

Hepatitis C virus (HCV) and the malaria parasite Plasmodium use the membrane protein CD81 to invade human liver cells. Here we mapped 33 host protein interactions of CD81 in primary human liver and hepatoma cells using high-resolution quantitative proteomics. In the CD81 protein network, we identified five proteins which are HCV entry factors or facilitators including epidermal growth factor receptor (EGFR). Notably, we discovered calpain-5 (CAPN5) and the ubiquitin ligase Casitas B-lineage lymphoma proto-oncogene B (CBLB) to form a complex with CD81 and support HCV entry. CAPN5 and CBLB were required for a post-binding and pre-replication step in the HCV life cycle. Knockout of CAPN5 and CBLB reduced susceptibility to all tested HCV genotypes, but not to other enveloped viruses such as vesicular stomatitis virus and human coronavirus. Furthermore, Plasmodium sporozoites relied on a distinct set of CD81 interaction partners for liver cell entry. Our findings reveal a comprehensive CD81 network in human liver cells and show that HCV and Plasmodium highjack selective CD81 interactions, including CAPN5 and CBLB for HCV, to invade cells

The Tetraspanin CD81 Is a Host Factor for Chikungunya Virus Replication

Chikungunya virus (CHIKV) is an arthritogenic reemerging virus replicating in plasma membrane-derived compartments termed "spherules." Here, we identify the human transmembrane protein CD81 as host factor required for CHIKV replication. Ablation of CD81 results in decreased CHIKV permissiveness, while overexpression enhances infection. CD81 is dispensable for virus uptake but critically required for viral genome replication. Likewise, murine CD81 is crucial for CHIKV permissiveness and is expressed in target cells such as dermal fibroblasts, muscle and liver cells. Whereas related alphaviruses, including Ross River virus (RRV), Semliki Forest virus (SFV), Sindbis virus (SINV) and Venezuelan equine encephalitis virus (VEEV), also depend on CD81 for infection, RNA viruses from other families, such as coronaviruses, replicate independently of CD81. Strikingly, the replication-enhancing function of CD81 is linked to cholesterol binding. These results define a mechanism exploited by alphaviruses to hijack the membrane microdomain-modeling protein CD81 for virus replication through interaction with cholesterol. IMPORTANCE: In this study, we discover the tetraspanin CD81 as a host factor for the globally emerging chikungunya virus and related alphaviruses. We show that CD81 promotes replication of viral genomes in human and mouse cells, while virus entry into cells is independent of CD81. This provides novel insights into how alphaviruses hijack host proteins to complete their life cycle. Alphaviruses replicate at distinct sites of the plasma membrane, which are enriched in cholesterol. We found that the cholesterol-binding ability of CD81 is important for its function as an alphavirus host factor. This discovery thus broadens our understanding of the alphavirus replication process and the use of host factors to reprogram cells into virus replication factories

Proteins in the human liver cell CD81 complex.

<p>Proteins in the human liver cell CD81 complex.</p

CAPN5 and CBLB support a postbinding step during HCV lipoviroparticle entry.

<p>(A) Schematic overview of the experimental setup used to analyze different steps of the HCV life cycle in the CRISPR/Cas9 knockout cell lines. (B) Infection of CAPN5 (red) and CBLB (blue) knockout and parental cell lines with HCV genotype 2 reporter virus. 72 hpi infection rates were quantified as luciferase activity and normalized to infection rates in cells transduced with a non-targeting scrambled sgRNA. CD81 knockout cells served as positive control. Data from 3 independent experiments shown as mean +SEM. (C) Flow cytometric surface staining of CD81, SCARB1, CLDN1 and OCLN in cells knocked out for CAPN5 (red) or CBLB (blue). Parental cells (black) served as positive control. Isotype control stainings or stainings with secondary antibody only (white) as negative controls. (D) Entry of lentiviral particles pseudotyped with glycoproteins from HCV GT1a (strain H77) or GT1b (strain Con1). Infectivity normalized to particles without envelope protein (negative control), to particles with VSV-G envelope (positive control) and to infection of cells transduced with non-targeting scrambled sgRNA. (E) Quantification of HCV fusion activity at the plasma membrane. Cells were pretreated with concanamycin A to inhibit endosomal acidification, cold-bound with HCV luciferase reporter virus (JcR-2A; 4°C, 2 h), shifted to 37°C (1 h) and washed with a pH 5 buffer to induce artificial plasma membrane fusion. A pH 7 buffer wash served to determine the background infection rate. 48 hpi infection rate was quantified as luciferase activity. Inh: flunarizine; scr: scrambled sgRNA (F) Immunofluorescence staining of cell lines electroporated with a HCV subgenomic replicon RNA (JFH1) at 48 hpt. Green: NS5A. Blue: DAPI. 10x magnification. (G) Cell lines were electroporated with wildtype HCV subgenomic replicon RNA (JFH1) or a polymerase active site mutant JFH1-ΔGDD (dotted lines), both encoding a luciferase reporter. Replication quantified as luciferase activity at the indicated time point post electroporation. Results normalized to the 4 h time point to account for electroporation efficiency. Data from at least three independent experiments shown as representative results (C, F) or as mean ± SEM (B, D, E, G). Significance according to unpaired t-test (B, E) or to MANOVA (G) indicated by * (p≤ 0.05), ** (p≤ 0.01), *** (p≤ 0.001). See also <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1007111#ppat.1007111.s006" target="_blank">S6 Fig</a>.</p

CAPN5 and CBLB are cytoplasmic proteins enriched in the CD81 complex.

<p>(A) Whole cell proteome quantification for Lunet N hCD81 cells. Expression level as iBAQ value indicated for the CD81 interactor CAPN5 (red) and the HCV entry factors CD81 (green), SCARB1 (black square), CLDN1 (black hexagon) and OCLN (black diamond). Albumin (black dot) shown as additional positive control. (B) Comparison of protein abundance in whole cell lysates and protein enrichment in CD81 co-IPs from Lunet N hCD81 cells. CAPN5 (red) and CD81 (green) are highlighted. Dotted lines indicate median values of all detected proteins. (C, D) Flow cytometric staining of CAPN5 and CBLB on the surface of naïve Lunet N hCD81 cells or after membrane permeabilization reveals intracellular localization of CAPN5 and CBLB (E) A subfraction of CAPN5 and CBLB colocalizes with the membrane marker ZO-1. Lunet N CRISPR scrambled cells were stained with anti-ZO-1 and anti-CAPN5 (upper panel) or anti-CBLB (lower panel). Nuclei were stained with DAPI. Arrowheads indicate colocalization of ZO-1 and CAPN5 or CBLB. Representative confocal images; scale bars 10 μm. (F) Pearson’s correlation coefficient for ZO-1 and CAPN5 or CBLB calculated by intensity correlation analysis. Each symbol represents an individual frame; horizontal lines indicate the mean ± SEM.</p

Quantitative proteomics of Uukuniemi virus - host cell interactions reveals GBF1 as proviral host factor for phleboviruses.

Novel tick-borne phleboviruses in the Phenuiviridae family, which are highly pathogenic in humans and all closely related to Uukuniemi virus (UUKV), have recently emerged on different continents. How phleboviruses assemble, bud, and exit cells remains largely elusive. Here, we performed high-resolution, label-free mass spectrometry analysis of UUKV immuno-precipitated from cell lysates and identified 39 cellular partners interacting with the viral envelope glycoproteins. The importance of these host factors for UUKV infection was validated by silencing each host factor by RNA interference. This revealed Golgi-specific brefeldin A-resistance guanine nucleotide exchange factor 1 (GBF1), a guanine nucleotide exchange factor resident in the Golgi, as a critical host factor required for the UUKV life cycle. An inhibitor of GBF1, Golgicide A, confirmed the role of the cellular factor in UUKV infection. We could pinpoint the GBF1 requirement to UUKV replication and particle assembly. When the investigation was extended to viruses from various positive and negative RNA viral families, we found that not only phleboviruses rely on GBF1 for infection, but also Flavi-, Corona-, Rhabdo-, and Togaviridae In contrast, silencing or blocking GBF1 did not abrogate infection by the human adenovirus serotype 5 and immunodeficiency retrovirus type 1, the replication of both occurs in the nucleus. Together our results indicate that UUKV relies on GBF1 for viral replication, assembly and egress. This study also highlights the proviral activity of GBF1 in the infection by a broad range of important zoonotic RNA viruses

CAPN5 and CBLB have a scaffolding function in HCV infection.

<p>(A) CAPN5 (red) or CBLB (blue) knockout cells were complemented with sgRNA resistant CAPN5 (CAPN5; orange) / CBLB (CBLB; purple) or catalytically inactive CAPN5 (CAPN5 dead; light orange) / CBLB (CBLB dead; light purple), respectively. Infection of knockout and complemented cell lines with HCV genotype 2 reporter virus (upper panel). 72 hpi infection rates were quantified as luciferase activity and normalized to infection rates in knockout cells. Data from 3 independent experiments shown as mean +SEM. The protein expression level in knockout and complemented cell lines was analyzed by immunoblot (lower panel). Representative of 3 independent experiments. (B) HCV non-reporter infection in CAPN5 and CBLB knockout cells with and without complementation. Lunet N hCD81 cells with the indicated CRISPR and complementation construct were infected with non-reporter genotype 2 HCV (strain Jc1) and release of infectious particles measured at 72 h post infection by TCID50 assay. LOQ: limit of quantification. Shown are three (two for CAPN5 dead) independent experiments with technical duplicates each. (C) Schematic representation of CAPN5 and CBLB domain mutants. (D) The indicated truncated variants of CAPN5 and CBLB were overexpressed in Lunet N hCD81 cells and cells infected with <i>Renilla</i> reporter HCV. Infection was quantified 72 hpi by luciferase assay. PC: protease core; C2L: C2-like, TKB: tyrosine kinase binding; UBA: Ubiquitin associating; ctrl: empty vector control; scr: scrambled sgRNA. Bar graph shows mean + SD of one representative biological replicate (of four in total) with three technical replicates. Statistical analysis performed by ANOVA; ****p value<0.0001. (E) Model for the role of CAPN5 (red) and CBLB (blue) in HCV entry. Ub: ubiquitin, P: phosphate group. AP2: adaptor protein complex 2.</p