Laboratoire de Virologie Moléculaire et Structurale

Hepatitis C virus (HCV) causes acute and chronic liver disease in humans, including chronic hepatitis, cirrhosis, and hepatocellular carcinoma. Studies of this virus have been hampered by the lack of a productive cell culture system; most information thus has been obtained from analysis of the HCV genome, heterologous expression systems, in vitro and in vivo models, and structural analyses. Structural analyses of HCV components provide an essential framework for understanding of the molecular mechanisms of HCV polyprotein processing, RNA replication, and virion assembly and may contribute to a better understanding of the pathogenesis of hepatitis C. Moreover, these analyses should allow the identification of novel targets for antiviral intervention and development of new strategies to prevent and combat viral hepatitis. This article reviews the current knowledge of HCV structural biology.

We report the crystal structure of the RNA-dependent RNA polymerase of hepatitis C virus, a major human pathogen, to 2.8-A resolution. This enzyme is a key target for developing specific antiviral therapy. The structure of the catalytic domain contains 531 residues folded in the characteristic fingers, palm, and thumb subdomains. The fingers subdomain contains a region, the "fingertips," that shares the same fold with reverse transcriptases. Superposition to the available structures of the latter shows that residues from the palm and fingertips are structurally equivalent. In addition, it shows that the hepatitis C virus polymerase was crystallized in a closed fingers conformation, similar to HIV-1 reverse transcriptase in ternary complex with DNA and dTTP [Huang H., Chopra, R., Verdine, G. L. & Harrison, S. C. (1998) Science 282, 1669-1675]. This superposition reveals the majority of the amino acid residues of the hepatitis C virus enzyme that are likely to be implicated in binding to the replicating RNA molecule and to the incoming NTP. It also suggests a rearrangement of the thumb domain as well as a possible concerted movement of thumb and fingertips during translocation of the RNA template-primer in successive polymerization rounds.

The vesicular stomatitis virus has an atypical membrane fusion glycoprotein (G) exhibiting a pH-dependent equilibrium between two forms at the virus surface. Membrane fusion is triggered during the transition from the high- to low-pH form. The structure of G in its low-pH form shows the classic hairpin conformation observed in all other fusion proteins in their postfusion conformation, in spite of a novel fold combining features of fusion proteins from classes I and II. The structure provides a framework for understanding the reversibility of the G conformational change. Unexpectedly, G is homologous to gB of herpesviruses, which raises important questions on viral evolution.

The coronavirus SARS-CoV is the primary cause of the life-threatening severe acute respiratory syndrome (SARS). With the aim of developing therapeutic agents, we have tested peptides derived from the membrane-proximal (HR2) and membrane-distal (HR1) heptad repeat region of the spike protein as inhibitors of SARS-CoV infection of Vero cells. It appeared that HR2 peptides, but not HR1 peptides, were inhibitory. Their efficacy was, however, significantly lower than that of corresponding HR2 peptides of the murine coronavirus mouse hepatitis virus (MHV) in inhibiting MHV infection. Biochemical and electron microscopical analyses showed that, when mixed, SARS-CoV HR1 and HR2 peptides assemble into a six-helix bundle consisting of HR1 as a central triple-stranded coiled coil in association with three HR2 alpha-helices oriented in an antiparallel manner. The stability of this complex, as measured by its resistance to heat dissociation, appeared to be much lower than that of the corresponding MHV complex, which may explain the different inhibitory potencies of the HR2 peptides. Analogous to other class I viral fusion proteins, the six-helix complex supposedly represents a postfusion conformation that is formed after insertion of the fusion peptide, proposed here for coronaviruses to be located immediately upstream of HR1, into the target membrane. The resulting close apposition of fusion peptide and spike transmembrane domain facilitates membrane fusion. The inhibitory potency of the SARS-CoV HR2-peptides provides an attractive basis for the development of a therapeutic drug for SARS.

Glycoprotein G of the vesicular stomatitis virus triggers membrane fusion via a low pH-induced structural rearrangement. Despite the equilibrium between the pre- and postfusion states, the structure of the prefusion form, determined to 3.0 angstrom resolution, shows that the fusogenic transition entails an extensive structural reorganization of G. Comparison with the structure of the postfusion form suggests a pathway for the conformational change. In the prefusion form, G has the shape of a tripod with the fusion loops exposed, which point toward the viral membrane, and with the antigenic sites located at the distal end of the molecule. A large number of G glycoproteins, perhaps organized as in the crystals, act cooperatively to induce membrane merging.

We report here the results of a systematic high-resolution X-ray crystallographic analysis of complexes of the hepatitis C virus (HCV) RNA polymerase with ribonucleoside triphosphates (rNTPs) and divalent metal ions. An unexpected observation revealed by this study is the existence of a specific rGTP binding site in a shallow pocket at the molecular surface of the enzyme, 30 A away from the catalytic site. This previously unidentified rGTP pocket, which lies at the interface between fingers and thumb, may be an allosteric regulatory site and could play a role in allowing alternative interactions between the two domains during a possible conformational change of the enzyme required for efficient initiation. The electron density map at 1.7-A resolution clearly shows the mode of binding of the guanosine moiety to the enzyme. In the catalytic site, density corresponding to the triphosphates of nucleotides bound to the catalytic metals was apparent in each complex with nucleotides. Moreover, a network of triphosphate densities was detected; these densities superpose to the corresponding moieties of the nucleotides observed in the initiation complex reported for the polymerase of bacteriophage phi6, strengthening the proposal that the two enzymes initiate replication de novo by similar mechanisms. No equivalent of the protein stacking platform observed for the priming nucleotide in the phi6 enzyme is present in HCV polymerase, however, again suggesting that a change in conformation of the thumb domain takes place upon template binding to allow for efficient de novo initiation of RNA synthesis.

Dengue virus (DENV) causes the major arboviral disease of the tropics, characterized in its severe forms by signs of hemorrhage and plasma leakage. DENV encodes a nonstructural glycoprotein, NS1, that associates with intracellular membranes and the cell surface. NS1 is eventually secreted as a soluble hexamer from DENV-infected cells and circulates in the bloodstream of infected patients. Extracellular NS1 has been shown to modulate the complement system and to enhance DENV infection, yet its structure and function remain essentially unknown. By combining cryoelectron microscopy analysis with a characterization of NS1 amphipathic properties, we show that the secreted NS1 hexamer forms a lipoprotein particle with an open-barrel protein shell and a prominent central channel rich in lipids. Biochemical and NMR analyses of the NS1 lipid cargo reveal the presence of triglycerides, bound at an equimolar ratio to the NS1 protomer, as well as cholesteryl esters and phospholipids, a composition evocative of the plasma lipoproteins involved in vascular homeostasis. This study suggests that DENV NS1, by mimicking or hijacking lipid metabolic pathways, contributes to endothelium dysfunction, a key feature of severe dengue disease.

Sea surface reservoir ages must be known to establish a common chronological framework for marine, continental, and cryospheric paleoproxies, and are crucial for understanding ocean-continent climatic relationships and the paleoventilation of the ocean. Radiocarbon dates of planktonic foraminifera and tephra contemporaneously deposited over Mediterranean marine and terrestrial regions reveal that the reservoir ages were similar to the modern one (approximately 400 years) during most of the past 18,000 carbon-14 years. However, reservoir ages increased by a factor of 2 at the beginning of the last deglaciation. This is attributed to changes of the North Atlantic thermohaline circulation during the massive ice discharge event Heinrich 1.

Rabies virus infection induces the formation of cytoplasmic inclusion bodies that resemble Negri bodies found in the cytoplasm of some infected nerve cells. We have studied the morphogenesis and the role of these Negri body-like structures (NBLs) during viral infection. The results indicate that these spherical structures (one or two per cell in the initial stage of infection), composed of the viral N and P proteins, grow during the virus cycle before appearing as smaller structures at late stages of infection. We have shown that the microtubule network is not necessary for the formation of these inclusion bodies but is involved in their dynamics. In contrast, the actin network does not play any detectable role in these processes. These inclusion bodies contain Hsp70 and ubiquitinylated proteins, but they are not misfolded protein aggregates. NBLs, in fact, appear to be functional structures involved in the viral life cycle. Specifically, using in situ fluorescent hybridization techniques, we show that all viral RNAs (genome, antigenome, and every mRNA) are located inside the inclusion bodies. Significantly, short-term RNA labeling in the presence of BrUTP strongly suggests that the NBLs are the sites where viral transcription and replication take place.

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 endocytosis-defective 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 pH-dependent fusion into DCs. 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 endocytosis-defective 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 pH-dependent fusion into DCs. IntroductionDengue virus (DV) 1The abbreviations used are: DV, dengue virus; Ab, antibody; BHK, baby hamster kidney; BSA, bovine serum albumin; DC, dendritic cell; DC-SIGN, dendritic cell-specific intercellular adhesion molecule 3-grabbing non-integrin; DMJ, 1-deoxymannojirimycin hydrochloride; EndoH, endoglycosidase H; FACS, fluorescence-activated cell sorting; FITC, fluorescein isothiocyanate; FCS, fetal calf serum; GM-CSF, granulocyte macrophage colony-stimulating factor; HCV, hepatitis C virus; HIV, human immunodeficiency virus; HMAF, hyperimmune mouse ascites fluids; L-SIGN, liver cell-specific intercellular adhesion molecule 3-grabbing non-integrin; LC, Langerhans cell; mAb, monoclonal antibody; m.o.i., multiplicity of infection; PBS, phosphate-buffered saline; PE, phycoerythrin; PNGase F, peptide:N-glycosydase F; sE, DV-soluble E protein; SFV, Semliki forest virus; WT, wild type. is an arthropod-borne flavivirus that belongs to the Flaviviridae family (1Chambers T.J. Monath T.P. The Flavivirus: Pathogenesis and Immunity. Elsevier Science Publishing Co., New York2003Google Scholar). The four serotypes of DV (DV-1 to DV-4) are transmitted to humans by the mosquito vector Aedes aegypti (1Chambers T.J. Monath T.P. The Flavivirus: Pathogenesis and Immunity. Elsevier Science Publishing Co., New York2003Google Scholar, 2Weaver S.C. Barrett A.D. Nat. Rev. Microbiol. 2004; 2: 789-801Crossref PubMed Scopus (447) Google Scholar). DV infection results in a spectrum of illnesses, ranging from a flu-like disease (dengue fever) to dengue hemorrhagic fever that can progress to dengue shock syndrome and death (1Chambers T.J. Monath T.P. The Flavivirus: Pathogenesis and Immunity. Elsevier Science Publishing Co., New York2003Google Scholar).DV is a lipid-enveloped virus with a single-stranded, positive sense RNA genome, which replicates in the cytoplasm of infected cells (2Weaver S.C. Barrett A.D. Nat. Rev. Microbiol. 2004; 2: 789-801Crossref PubMed Scopus (447) Google Scholar, 3Mukhopadhyay S. Kuhn R.J. Rossmann M.G. Nat. Rev. Microbiol. 2005; 3: 13-22Crossref PubMed Scopus (869) Google Scholar). The 11-kb viral RNA encodes for a large polyprotein precursor, which is processed by both host and viral proteases to yield the non-structural proteins NS1 to NS5 and three structural proteins: C (core), prM (the intracellular precursor of the M protein), and E (envelope) glycoprotein (2Weaver S.C. Barrett A.D. Nat. Rev. Microbiol. 2004; 2: 789-801Crossref PubMed Scopus (447) Google Scholar, 3Mukhopadhyay S. Kuhn R.J. Rossmann M.G. Nat. Rev. Microbiol. 2005; 3: 13-22Crossref PubMed Scopus (869) Google Scholar). The E protein is assumed to bind cellular receptors that direct DV particles to the endocytic pathway. The acidic environment in the endosome is believed to trigger major conformational changes in the E protein, which induce fusion of the viral and host cell membranes, resulting in entry of the virion into the cytoplasm (3Mukhopadhyay S. Kuhn R.J. Rossmann M.G. Nat. Rev. Microbiol. 2005; 3: 13-22Crossref PubMed Scopus (869) Google Scholar).In the natural infection, DV is introduced into human skin by an infected mosquito during a blood meal (2Weaver S.C. Barrett A.D. Nat. Rev. Microbiol. 2004; 2: 789-801Crossref PubMed Scopus (447) Google Scholar). Immature dendritic cells (DCs) and Langerhans cells (LCs), which are normally resident in the skin, have been to infected by DV and are believed to the cells by the virus Nat. PubMed Scopus Google Scholar). We have the interactions between DV and human DCs to cellular for virus entry. We and S. S. PubMed Scopus Google Scholar, PubMed Scopus Google have DC-SIGN as an essential molecule for DV infection of immature DC-SIGN molecules DV is a C-type lectin that present on the surface of viral as human immunodeficiency virus and hepatitis C virus S. PubMed Scopus Google Scholar, S. PubMed Scopus Google Scholar, Nat. Rev. 3: PubMed Scopus Google Scholar). The DV E protein, which is the glycoprotein on the surface of DV is responsible for to the host cell surface and an in viral entry (3Mukhopadhyay S. Kuhn R.J. Rossmann M.G. Nat. Rev. Microbiol. 2005; 3: 13-22Crossref PubMed Scopus (869) Google Scholar, R.J. Rossmann M.G. S. PubMed Scopus Google Scholar). present on the E glycoprotein have been to for virus to host cells PubMed Scopus Google Scholar). In to viral that bind DC-SIGN and are DV E protein at and which are used by the four DV serotypes PubMed Scopus Google Scholar). The interactions of E protein with DC-SIGN, believed to a for DV entry into are to DC-SIGN, DV is believed to and to an acidic where fusion (3Mukhopadhyay S. Kuhn R.J. Rossmann M.G. Nat. Rev. Microbiol. 2005; 3: 13-22Crossref PubMed Scopus (869) Google Scholar). of cells with which the DV infection PubMed Scopus Google Scholar). DC-SIGN is an endocytic that been to Nat. Rev. 3: PubMed Scopus Google Scholar, R.J. S. 2004; PubMed Scopus Google Scholar, Immunity. PubMed Scopus Google Scholar, PubMed Scopus Google it is DC-SIGN is responsible for DV to it the of in the of a entry we have the of DC-SIGN in the of DV entry into DCs. results the E protein as the DV responsible for to DC-SIGN and a for N-glycans in interactions between DV E protein and We that DV endocytosis is dispensable for DV infection of target cells and propose that DC-SIGN as a DV factor that interaction of viral particles with an as yet unidentified cellular which to DV of and DC-SIGN for the of prM and E proteins PubMed Scopus Google Scholar). used as a to Semliki forest virus for the DV prM and of E protein The for prM protein and the of E protein in the DV by the sense and the The encodes for the The for E with and and introduced into of the vector PubMed Scopus Google DC-SIGN wild vector Immunity. PubMed Scopus Google Scholar). DC-SIGN DC-SIGN and DC-SIGN by and into 2004; PubMed Scopus Google DC-SIGN by the protein with a DC-SIGN WT, DC-SIGN DC-SIGN DC-SIGN DC-SIGN the of the of the DC-SIGN mouse monoclonal from and from and from The and have been in and Immunity. PubMed Scopus Google Scholar, PubMed Scopus Google Scholar, 2004; PubMed Scopus Google Scholar). DV with hyperimmune mouse ascites to a from and PubMed Scopus Google Scholar). The used a is the and PubMed Scopus Google Scholar). mouse and and in with fetal calf serum and cells in with and DC-SIGN by with the vector DC-SIGN as 2004; PubMed Scopus Google Scholar). cells with of DC-SIGN by of cells by DC-SIGN vector at of infection as PubMed Scopus Google Scholar). cells by cell and Langerhans as 2004; PubMed Scopus Google Scholar). human blood cells from by with with a of and at in with FCS, human and human granulocyte macrophage colony-stimulating factor for from human blood as PubMed Scopus Google Scholar). cells in with FCS, cell factor and cells and for in with FCS, GM-CSF, and and DV and in mosquito cell in on and virus on cells by as PubMed Scopus Google infection, cells to DV for at at in with BSA, with to virus and at The cells cells with for at of DV and with the as PubMed Scopus Google viral with to cells and by and to cells in for at by with in with the for at with the to by mAb, and with in and in bovine serum from by with at a a for at and with to and with and of proteins S. PubMed Scopus Google Scholar). DV in baby hamster cells in the of and S. PubMed Scopus Google Scholar). by with endoglycosidase F; New S. PubMed Scopus Google Scholar). by at a S. PubMed Scopus Google Binding and cells in of and for at to endocytosis glycoprotein by with cells with in for at of protein used at internalization cells proteins for the at at to the cells at both and with at for to cell surface of The of the cells with at for to the glycoprotein. cells in to in to of and in a cells DC-SIGN on The cells with the in for at three with to and to for to allow DC-SIGN with for in PBS, and with for with of and for cells with and in on with the and a but by the of DV to immature DCs and we DCs as as from blood and and to DV cell the of DCs and cells for of DC-SIGN, and a C-type lectin by and in the of a the Nat. Rev. 2: PubMed Scopus Google Scholar, S. S. Immunity. PubMed Scopus Google Scholar, PubMed Scopus Google Scholar). DCs as of DC-SIGN and In not the C-type lectin which is present on of the LCs. for PubMed Scopus Google we found that in not DC-SIGN Immature DCs and infected with the in Aedes at by for NS1 protein, a non-structural protein during DV in of DCs productively infected by In of to the DV in infection of of results with DV serotypes not that DV productively infects immature DCs and not of DCs by the DV the interaction of DV with DCs in we immature DCs with mosquito-derived DV from the four serotypes at that to productively human immature DCs. that infection of DCs by is by DC-SIGN and by results with DV serotypes and not of from DCs with the four DV serotypes that infected DCs of viral of DCs with to infection virus of immature DCs to DV particles been to induce S. T.P. PubMed Scopus Google Scholar, PubMed Scopus Google Scholar). viral infection, we immature DCs with at an of in the of cell surface of and by with of immature DCs with DV in a of and cell surface a of in cells infected with We found that surface of in DC-SIGN not with Ab, of cells with as by the of and in infected cells results with the DV serotypes not In of immature DCs with DV not induce that is on viral of DV particles by DCs not these that DC-SIGN is a factor for infection of DCs by DV serotypes and for is for infection of DCs by the four DV serotypes and for DCs infected with mosquito-derived DV at of and cells and with to infection by as infection in DCs. 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S. PubMed Scopus Google Scholar, PubMed Scopus Google Scholar, PubMed Scopus Google Scholar). the interaction of E protein with DC-SIGN, the for and the of E to of polyprotein of to bovine at the C into a protein, cells infected with the precursor protein and with that protein is the major protein by as by with the natural vector of DV is the Aedes aegypti the E protein at the surface of transmitted to humans (2Weaver S.C. Barrett A.D. Nat. Rev. Microbiol. 2004; 2: 789-801Crossref PubMed Scopus (447) Google Scholar, PubMed Scopus Google Scholar). we cells with and The resulting protein, N-glycans as by to In in cells and However, both and to PNGase which both and N-glycans at the resulting in a E We found that proteins bind to cells DC-SIGN but not to cells In to we observed that sE, which to bind to indicating that N-glycans on the DV E protein are for interactions with DC-SIGN of proteins to cells is by and the we found that immature which DC-SIGN, interaction is by and by Together, these results DV E as a protein and a for N-glycans in DV E glycoprotein to glycoprotein to DC-SIGN mannosylated cells infected with for prM and and with proteins with an an an and by in cells in the of and sE, E proteins to with PNGase and by but not sE, to and for at with and three for at with and three of of to cells with in the of DC-SIGN as and an as F, DC-SIGN to dendritic with DCs in the of and and The are of three are as the of into E protein is by DC-SIGN, we cells on with to cells on to for with for to E protein from E protein to DC-SIGN at the cell We found that of the E protein to DC-SIGN by cells at In for at we observed that a large of protein to indicating that DC-SIGN internalization of DV E protein at of E protein endocytosis that DV E protein endocytosis is as of E protein is at internalization of to cells for at to and for at with to from cell surface E glycoprotein. internalization of in The are of are as the of of DC-SIGN in DV and three in that are believed to in internalization endocytic Nat. Rev. 3: PubMed Scopus Google Scholar, Nat. Rev. 2: PubMed Scopus Google Scholar). a a which are both internalization and a Nat. Rev. 3: PubMed Scopus Google Scholar, PubMed Scopus Google Scholar). the of these three in DC-SIGN and DV we DC-SIGN at the into the cells with virus vector particles, DC-SIGN with that DC-SIGN WT, and proteins at at the cell surface used to DC-SIGN endocytosis in cells with the for at and to to DC-SIGN DC-SIGN and receptors at the cell at we observed a of in DC-SIGN WT, and with an intracellular of that WT, and DC-SIGN receptors are in cells In we found that the of receptors at the surface of cells that the is for efficient DC-SIGN endocytosis and intracellular of and DC-SIGN cells with a vector for of DC-SIGN and for DC-SIGN are in of and DC-SIGN in with cells DC-SIGN WT, and proteins for at to and for at with a surface and intracellular of DC-SIGN by The are of three and proteins bind DV with DC-SIGN with efficient internalization and infection of DV, we DV entry in cells a endocytic pathway. cells and DC-SIGN molecules infected with the virus at that molecules DV infection as as DC-SIGN WT, and results in cell DC-SIGN C and a of in DV infection, we cells a DC-SIGN surface of DC-SIGN found to DC-SIGN infection show that DC-SIGN is to DV entry of cells DC-SIGN molecules with the a of Together, these that of DV entry is a pH-dependent which internalization of the However, results show that DC-SIGN endocytosis is dispensable for DV to DV entry into cells can from and internalization infection not DC-SIGN for at with and three cells infected with at for the NS1 and by infection as the of cells with a vector for WT, DC-SIGN molecules and for DC-SIGN cells WT, DC-SIGN infected with at of infection by and as the of are as the of The are of three entry by DC-SIGN cells DC-SIGN DC-SIGN infected with at a of in the of the at cells for the NS1 and viral infection by The are of and S. S. PubMed Scopus Google Scholar, PubMed Scopus Google have that the C-type lectin DC-SIGN is an essential cellular factor for DV infection of immature DCs. The of DC-SIGN on skin DCs with the of lectin to DV infection that DC-SIGN a factor the of DV In we into the by which DC-SIGN DV particles and viral of DCs in human skin, the of DV Nat. Rev. 2: PubMed Scopus Google Scholar). skin immature a found in the are believed to productively infected by DV and to the cells by the virus Nat. PubMed Scopus Google Scholar). in the skin of a human a DV have been to for DV E protein Nat. PubMed Scopus Google Scholar). cells from human skin with DV particles found to DV Nat. PubMed Scopus Google Scholar). However, these not that immature DV, the cells are a of both and DCs. progress in and DCs in as as the of and cell and DC-SIGN, of DV target cells in human DC-SIGN and are C-type that and have both been to with Nat. Rev. 2: PubMed Scopus Google Scholar, S. Nat. 3: PubMed Scopus Google Scholar). and DV particles in cells are in DV transmitted by infected DC-SIGN to target However, results show in to DCs in are by these results not the that with DV, argue a of as of DV infection and results that during DV from mosquito to that are infected by the of as HIV, and S. PubMed Scopus Google Scholar, Immunity. PubMed Scopus Google Scholar, PubMed Scopus Google Scholar). In we that DV E protein, the sole glycoprotein at the surface of DV particles, to results that the of E protein is for DV to We observed that mannosylated E and not protein with is to with In with we have that of DV particles with to immature DCs PubMed Scopus Google Scholar). Together, these results that the present at the surface of mosquito-derived are essential for DV interaction with DC-SIGN and viral entry into DCs. are found in the DV E protein PubMed Scopus Google Scholar). The is to DV, is to and E proteins are at the and E proteins are at both and PubMed Scopus Google Scholar). results show four DV serotypes DC-SIGN to productively immature in with S. S. PubMed Scopus Google Scholar). that is to DV interaction with DC-SIGN and infection of DCs. However, we the that to the interaction between DC-SIGN and to the of these in DV is at the cell surface 2004; PubMed Scopus Google and DC-SIGN molecules bind mannosylated N-glycans with S. PubMed Scopus Google Scholar, PubMed Scopus Google Scholar). that between DC-SIGN and N-glycans on viral and that the of an interaction is a of the S. PubMed Scopus Google Scholar, S. PubMed Scopus Google Scholar, PubMed Scopus Google Scholar, R.J. PubMed Scopus Google Scholar, 2005; PubMed Scopus Google Scholar). In of we found that the of DC-SIGN for DV E protein, which to that observed for the and proteins and S. PubMed Scopus Google Scholar, S. S. PubMed Scopus Google Scholar). of on cells not observed with of not are in with the of a in that a interaction between E protein and DC-SIGN 2005; PubMed Scopus Google but with the of DC-SIGN to DV of DV particles a of E protein on the virion surface R.J. Rossmann M.G. S. PubMed Scopus Google Scholar, Kuhn R.J. Rossmann M.G. PubMed Scopus Google Scholar, S. S.C. S. PubMed Scopus Google Scholar). we that a interaction between DC-SIGN and the E glycoprotein is to efficient DV we propose that the structural of N-glycans on the surface of viral particles favor the of E proteins by allow a interaction between DC-SIGN and DV particles, resulting in efficient viral and to DV is assumed to entry into target cells by to viral fusion in (3Mukhopadhyay S. Kuhn R.J. Rossmann M.G. Nat. Rev. Microbiol. 2005; 3: 13-22Crossref PubMed Scopus (869) Google Scholar). In with show that DV entry into cells is that of DV E protein to DC-SIGN triggers a rapid and efficient internalization of the viral the of the endocytic in DV However, these not to DC-SIGN as cell surface factor as an entry that virus The DC-SIGN internalization and a and a that is believed to in intracellular Nat. Rev. 3: PubMed Scopus Google Scholar, PubMed Scopus Google Scholar, Immunity. PubMed Scopus Google Scholar). of the by DC-SIGN endocytosis by In we found that both the and the are not in DC-SIGN is to that acidic in receptors are in the of to the major S. PubMed Scopus Google Scholar). to the in DC-SIGN a and is in of We observed that the endocytosis-defective DC-SIGN molecules and DV entry with to results that DV entry into cells from DC-SIGN that DC-SIGN a not as a DV cell surface factor and argue in favor of a mechanism by which DC-SIGN enhances DV entry in cis. results not the that in immature DV particles to DC-SIGN by as as for entry of PubMed Scopus Google Scholar, PubMed Scopus Google Scholar). we the in to the major a of DV immature DCs DC-SIGN and to the of entry the of a for DV internalization into DCs. We that a molecule is present at on DCs and DV entry with in the of molecules as and proteins have been to in DV entry into target cells but as cellular receptors for DV R.J. Nat. 3: PubMed Scopus Google Scholar, PubMed Google Scholar, S. 2004; PubMed Scopus Google Scholar, S. 2005; PubMed Scopus Google Scholar). In we propose that DC-SIGN with an unidentified cellular entry to infection of skin DCs during the blood DV of the to the host and the infection to these are with by DV infection The of an to the by DC-SIGN in DV in to these and to DV IntroductionDengue virus (DV) 1The abbreviations used are: DV, dengue virus; Ab, antibody; BHK, baby hamster kidney; BSA, bovine serum albumin; DC, dendritic cell; DC-SIGN, dendritic cell-specific intercellular adhesion molecule 3-grabbing non-integrin; DMJ, 1-deoxymannojirimycin hydrochloride; EndoH, endoglycosidase H; FACS, fluorescence-activated cell sorting; FITC, fluorescein isothiocyanate; FCS, fetal calf serum; GM-CSF, granulocyte macrophage colony-stimulating factor; HCV, hepatitis C virus; HIV, human immunodeficiency virus; HMAF, hyperimmune mouse ascites fluids; L-SIGN, liver cell-specific intercellular adhesion molecule 3-grabbing non-integrin; LC, Langerhans cell; mAb, monoclonal antibody; m.o.i., multiplicity of infection; PBS, phosphate-buffered saline; PE, phycoerythrin; PNGase F, peptide:N-glycosydase F; sE, DV-soluble E protein; SFV, Semliki forest virus; WT, wild type. is an arthropod-borne flavivirus that belongs to the Flaviviridae family (1Chambers T.J. Monath T.P. The Flavivirus: Pathogenesis and Immunity. Elsevier Science Publishing Co., New York2003Google Scholar). The four serotypes of DV (DV-1 to DV-4) are transmitted to humans by the mosquito vector Aedes aegypti (1Chambers T.J. Monath T.P. The Flavivirus: Pathogenesis and Immunity. Elsevier Science Publishing Co., New York2003Google Scholar, 2Weaver S.C. Barrett A.D. Nat. Rev. Microbiol. 2004; 2: 789-801Crossref PubMed Scopus (447) Google Scholar). DV infection results in a spectrum of illnesses, ranging from a flu-like disease (dengue fever) to dengue hemorrhagic fever that can progress to dengue shock syndrome and death (1Chambers T.J. Monath T.P. The Flavivirus: Pathogenesis and Immunity. Elsevier Science Publishing Co., New York2003Google Scholar).DV is a lipid-enveloped virus with a single-stranded, positive sense RNA genome, which replicates in the cytoplasm of infected cells (2Weaver S.C. Barrett A.D. Nat. Rev. Microbiol. 2004; 2: 789-801Crossref PubMed Scopus (447) Google Scholar, 3Mukhopadhyay S. Kuhn R.J. Rossmann M.G. Nat. Rev. Microbiol. 2005; 3: 13-22Crossref PubMed Scopus (869) Google Scholar). The 11-kb viral RNA encodes for a large polyprotein precursor, which is processed by both host and viral proteases to yield the non-structural proteins NS1 to NS5 and three structural proteins: C (core), prM (the intracellular precursor of the M protein), and E (envelope) glycoprotein (2Weaver S.C. Barrett A.D. Nat. Rev. Microbiol. 2004; 2: 789-801Crossref PubMed Scopus (447) Google Scholar, 3Mukhopadhyay S. Kuhn R.J. Rossmann M.G. Nat. Rev. Microbiol. 2005; 3: 13-22Crossref PubMed Scopus (869) Google Scholar). The E protein is assumed to bind cellular receptors that direct DV particles to the endocytic pathway. The acidic environment in the endosome is believed to trigger major conformational changes in the E protein, which induce fusion of the viral and host cell membranes, resulting in entry of the virion into the cytoplasm (3Mukhopadhyay S. Kuhn R.J. Rossmann M.G. Nat. Rev. Microbiol. 2005; 3: 13-22Crossref PubMed Scopus (869) Google Scholar).In the natural infection, DV is introduced into human skin by an infected mosquito during a blood meal (2Weaver S.C. Barrett A.D. Nat. Rev. Microbiol. 2004; 2: 789-801Crossref PubMed Scopus (447) Google Scholar). Immature dendritic cells (DCs) and Langerhans cells (LCs), which are normally resident in the skin, have been to infected by DV and are believed to the cells by the virus Nat. PubMed Scopus Google Scholar). We have the interactions between DV and human DCs to cellular for virus entry. We and S. S. PubMed Scopus Google Scholar, PubMed Scopus Google have DC-SIGN as an essential molecule for DV infection of immature DC-SIGN molecules DV is a C-type lectin that present on the surface of viral as human immunodeficiency virus and hepatitis C virus S. PubMed Scopus Google Scholar, S. PubMed Scopus Google Scholar, Nat. Rev. 3: PubMed Scopus Google Scholar). The DV E protein, which is the glycoprotein on the surface of DV is responsible for to the host cell surface and an in viral entry (3Mukhopadhyay S. Kuhn R.J. Rossmann M.G. Nat. Rev. Microbiol. 2005; 3: 13-22Crossref PubMed Scopus (869) Google Scholar, R.J. Rossmann M.G. S. PubMed Scopus Google Scholar). present on the E glycoprotein have been to for virus to host cells PubMed Scopus Google Scholar). In to viral that bind DC-SIGN and are DV E protein at and which are used by the four DV serotypes PubMed Scopus Google Scholar). The interactions of E protein with DC-SIGN, believed to a for DV entry into are to DC-SIGN, DV is believed to and to an acidic where fusion (3Mukhopadhyay S. Kuhn R.J. Rossmann M.G. Nat. Rev. Microbiol. 2005; 3: 13-22Crossref PubMed Scopus (869) Google Scholar). of cells with which the DV infection PubMed Scopus Google Scholar). DC-SIGN is an endocytic that been to Nat. Rev. 3: PubMed Scopus Google Scholar, R.J. S. 2004; PubMed Scopus Google Scholar, Immunity. PubMed Scopus Google Scholar, PubMed Scopus Google it is DC-SIGN is responsible for DV to it the of in the of a entry we have the of DC-SIGN in the of DV entry into DCs. results the E protein as the DV responsible for to DC-SIGN and a for N-glycans in interactions between DV E protein and We that DV endocytosis is dispensable for DV infection of target cells and propose that DC-SIGN as a DV factor that interaction of viral particles with an as yet unidentified cellular which to DV entry.

Host cell interactions of human adenovirus serotypes belonging to subgroups B (adenovirus type 3 [Ad3] and Ad7) and C (Ad2 and Ad5) were comparatively analyzed at three levels: (i) binding of virus particles with host cell receptors; (ii) cointernalization of macromolecules with adenovirions; and (iii) adenovirus-induced cytoskeletal alterations. The association constants with human cell receptors were found to be similar for Ad2 and Ad3 (8 x 10(9) to 9 x 10(9) M-1), and the number of receptor sites per cell ranged from 5,000 (Ad2) to 7,000 (Ad3). Affinity blottings, competition experiments, and immunofluorescence stainings suggested that the receptor sites for adenovirus were distinct for members of subgroups B and C. Adenovirions increased the permeability of cells to macromolecules. We showed that this global effect could be divided into two distinct events: (i) cointernalization of macromolecules and virions into endocytotic vesicles, a phenomenon that occurred in a serotype-independent way, and (ii) release of macromolecules into the cytoplasm upon adenovirus-induced lysis of endosomal membranes. The latter process was found to be type specific and to require unaltered and infectious virus particles of serotype 2 or 5. Perinuclear condensation of the vimentin filament network was observed at early stages of infection with Ad2 or Ad5 but not with Ad3, Ad7, and noninfectious particles of Ad2 or Ad5, obtained by heat inactivation of wild-type virions or with the H2 ts1 mutant. This phenomenon appeared to be a cytological marker for cytoplasmic transit of infectious virions within adenovirus-infected cells. It could be experimentally dissociated from vimentin proteolysis, which was found to be serotype dependent, occurring only with members of subgroup C, regardless of the infectivity of the input virus.

Hepatitis C virus (HCV), a major cause of chronic liver disease in humans, is the focus of intense research efforts worldwide. Yet structural data on the viral envelope glycoproteins E1 and E2 are scarce, in spite of their essential role in the viral life cycle. To obtain more information, we developed an efficient production system of recombinant E2 ectodomain (E2e), truncated immediately upstream its trans-membrane (TM) region, using Drosophila melanogaster cells. This system yields a majority of monomeric protein, which can be readily separated chromatographically from contaminating disulfide-linked aggregates. The isolated monomeric E2e reacts with a number of conformation-sensitive monoclonal antibodies, binds the soluble CD81 large external loop and efficiently inhibits infection of Huh7.5 cells by infectious HCV particles (HCVcc) in a dose-dependent manner, suggesting that it adopts a native conformation. These properties of E2e led us to experimentally determine the connectivity of its 9 disulfide bonds, which are strictly conserved across HCV genotypes. Furthermore, circular dichroism combined with infrared spectroscopy analyses revealed the secondary structure contents of E2e, indicating in particular about 28% beta-sheet, in agreement with the consensus secondary structure predictions. The disulfide connectivity pattern, together with data on the CD81 binding site and reported E2 deletion mutants, enabled the threading of the E2e polypeptide chain onto the structural template of class II fusion proteins of related flavi- and alphaviruses. The resulting model of the tertiary organization of E2 gives key information on the antigenicity determinants of the virus, maps the receptor binding site to the interface of domains I and III, and provides insight into the nature of a putative fusogenic conformational change.

Interferons (IFNs) encode a family of secreted proteins that provide the front-line defense against viral infections. Their diverse biological actions are thought to be mediated by the products of specific but usually overlapping sets of cellular genes induced in the target cells. We have recently isolated a new human IFN-induced gene that we have termed ISG20, which codes for a 3' to 5' exonuclease with specificity for single-stranded RNA and, to a lesser extent, for DNA. In this report, we demonstrate that ISG20 is involved in the antiviral functions of IFN. In the absence of IFN treatment, ISG20-overexpressing HeLa cells showed resistance to infections by vesicular stomatitis virus (VSV), influenza virus, and encephalomyocarditis virus (three RNA genomic viruses) but not to the DNA genomic adenovirus. ISG20 specifically interfered with VSV mRNA synthesis and protein production while leaving the expression of cellular control genes unaffected. No antiviral effect was observed in cells overexpressing a mutated ISG20 protein defective in exonuclease activity, demonstrating that the antiviral effects were due to the exonuclease activity of ISG20. In addition, the inactive mutant ISG20 protein, which is able to inhibit ISG20 exonuclease activity in vitro, significantly reduced the ability of IFN to block VSV development. Taken together, these data suggested that the antiviral activity of IFN against VSV is partly mediated by ISG20. We thus show that, besides RNase L, ISG20 has an antiviral activity, supporting the idea that it might represent a novel antiviral pathway in the mechanism of IFN action.

Rabies virus P protein is a cofactor of RNA polymerase. We investigated other potential roles of P (CVS strain) by searching for cellular partners using two-hybrid screening. We isolated a cDNA encoding the signal transducer and activator of transcription 1 (STAT1) that is a critical component of interferon type I (IFN-alpha/beta) and type II (IFN-gamma) signaling. We confirmed this interaction by glutathione S-transferase-pull-down assay. Deletion mutant analysis indicated that the carboxy-terminal part of P interacted with a region containing the DNA-binding domain and the coiled-coil domain of STAT1. The expression of P protein inhibits IFN-alpha- and IFN-gamma-induced transcriptional responses, thus impairing the IFN-induced antiviral state. Mechanistic studies indicate that P protein does not induce STAT1 degradation and does not interfere with STAT1 phosphorylation but prevents IFN-induced STAT1 nuclear accumulation. These results indicate that rabies P protein overcomes the antiviral response of the infected cells.

Replication-deficient adenovirus used in humans for gene therapy induces a strong immune response to the vector, resulting in transient recombinant protein expression and the blocking of gene transfer upon a second administration. Therefore, in this study we examined in detail the capsid-specific humoral immune response in sera of patients with lung cancer who had been given one dose of a replication-defective adenovirus. We analyzed the immune response to the three major components of the viral capsid, hexon (Hx), penton base (Pb), and fiber (Fi). A longitudinal study of the humoral response assayed on adenovirus particle-coated enzyme-linked immunosorbent assay plates showed that patients had preexisting immunity to adenovirus prior to the administration of adenovirus-beta-gal. The level of the response increased in three patients after adenovirus administration and remained at a maximum after three months. One patient had a strong immune response to adenovirus prior to treatment, and this response was unaffected by adenovirus administration. Sera collected from the patients were assayed for recognition of each individual viral capsid protein to determine more precisely the molecular basis of the humoral immune response. Clear differences existed in the humoral response to the three major components of the viral capsid in serum from humans. Sequential appearance of these antibodies was observed: anti-Fi antibodies appeared first, followed by anti-Pb antibodies and then by anti-Hx antibodies. Moreover, anti-Fi antibodies preferentially recognized the native trimeric form of Fi protein, suggesting that they recognized conformational epitopes. Our results showed that sera with no neutralizing activity contained only anti-Fi antibodies. In contrast, neutralizing activity was only obtained with sera containing anti-Fi and anti-Pb antibodies. More importantly, we showed that anti-native Fi and anti-Pb antibodies had a synergistic effect on neutralization. The application of these conclusions to human gene therapy with recombinant adenovirus should lead to the development of strategies to overcome the formation of such neutralization antibodies, which have been shown to limit the efficacy of gene transfer in humans.

BACKGROUND: The genetic diversity observed among bacteriophages remains a major obstacle for the identification of homologs and the comparison of their functional modules. In the structural module, although several classes of homologous proteins contributing to the head and tail structure can be detected, proteins of the head-to-tail connection (or neck) are generally more divergent. Yet, molecular analyses of a few tailed phages belonging to different morphological classes suggested that only a limited number of structural solutions are used in order to produce a functional virion. To challenge this hypothesis and analyze proteins diversity at the virion neck, we developed a specific computational strategy to cope with sequence divergence in phage proteins. We searched for homologs of a set of proteins encoded in the structural module using a phage learning database. RESULTS: We show that using a combination of iterative profile-profile comparison and gene context analyses, we can identify a set of head, neck and tail proteins in most tailed bacteriophages of our database. Classification of phages based on neck protein sequences delineates 4 Types corresponding to known morphological subfamilies. Further analysis of the most abundant Type 1 yields 10 Clusters characterized by consistent sets of head, neck and tail proteins. We developed Virfam, a webserver that automatically identifies proteins of the phage head-neck-tail module and assign phages to the most closely related cluster of phages. This server was tested against 624 new phages from the NCBI database. 93% of the tailed and unclassified phages could be assigned to our head-neck-tail based categories, thus highlighting the large representativeness of the identified virion architectures. Types and Clusters delineate consistent subgroups of Caudovirales, which correlate with several virion properties. CONCLUSIONS: Our method and webserver have the capacity to automatically classify most tailed phages, detect their structural module, assign a function to a set of their head, neck and tail genes, provide their morphologic subtype and localize these phages within a "head-neck-tail" based classification. It should enable analysis of large sets of phage genomes. In particular, it should contribute to the classification of the abundant unknown viruses found on assembled contigs of metagenomic samples.

Membrane fusion of enveloped viruses with cellular membranes is mediated by viral glycoproteins (GP). Interaction of GP with cellular receptors alone or coupled to exposure to the acidic environment of endosomes induces extensive conformational changes in the fusion protein which pull two membranes into close enough proximity to trigger bilayer fusion. The refolding process provides the energy for fusion and repositions both membrane anchors, the transmembrane and the fusion peptide regions, at the same end of an elongated hairpin structure in all fusion protein structures known to date. The fusion process follows several lipidic intermediate states, which are generated by the refolding process. Although the major principles of viral fusion are understood, the structures of fusion protein intermediates and their mode of lipid bilayer interaction, the structures and functions of the membrane anchors and the number of fusion proteins required for fusion, necessitate further investigations.

Adenovirus serotype 37 (Ad37) belongs to species D and can cause epidemic keratoconjunctivitis, whereas the closely related Ad19p does not. Primary cell attachment by adenoviruses is mediated through receptor binding of the knob domain of the fiber protein. The knobs of Ad37 and Ad19p differ at only two positions, Lys240Glu and Asn340Asp. We report the high-resolution crystal structures of the Ad37 and Ad19p knobs, both native and in complex with sialic acid, which has been proposed as a receptor for Ad37. Overall, the Ad37 and Ad19p knobs are very similar to previously reported knob structures, especially to that of Ad5, which binds the coxsackievirus-adenovirus receptor (CAR). Ad37 and Ad19p knobs are structurally identical with the exception of the changed side chains and are structurally most similar to CAR-binding knobs (e.g., that of Ad5) rather than non-CAR-binding knobs (e.g., that of Ad3). The two mutations in Ad19p result in a partial loss of the exceptionally high positive surface charge of the Ad37 knob but do not affect sialic acid binding. This site is located on the top of the trimer and binds both alpha(2,3) and alpha(2,6)-linked sialyl-lactose, although only the sialic acid residue makes direct contact. Amino acid alignment suggests that the sialic acid binding site is conserved in several species D serotypes. Our results show that the altered viral tropism and cell binding of Ad19p relative to those of Ad37 are not explained by a different binding ability toward sialyl-lactose.

The order Mononegavirales includes three virus families that replicate in the cytoplasm: the Paramyxoviridae, composed of two subfamilies, the Paramyxovirinae and Pneumovirinae, the Rhabdoviridae and the Filoviridae. These viruses, also called non-segmented negative-strand RNA viruses (NNV), contain five to ten tandemly linked genes, which are separated by conserved junctional sequences that act as mRNA start and poly(A)/stop sites. For the NNV, downstream mRNA synthesis depends on termination of the upstream mRNA, and all NNV RNA-dependent RNA polymerases reiteratively copy ("stutter" on) a short run of template uridylates during transcription to polyadenylate and terminate their mRNAs. The RNA-dependent RNA polymerase of a subset of the NNV, all members of the Paramyxovirinae, also stutter in a very controlled fashion to edit their phosphoprotein gene mRNA, and Ebola virus, a filovirus, carries out a related process on its glycoprotein mRNA. Remarkably, all viruses that edit their phosphoprotein mRNA are also governed by the "rule of six", i.e. their genomes must be of polyhexameric length (6n+0) to replicate efficiently. Why these two seemingly unrelated processes are so tightly linked in the Paramyxovirinae has been an enigma. This paper will review what is presently known about these two processes that are unique to viruses of this subfamily, and will discuss whether this enigmatic linkage could be due to the phenomenon of RNA virus error catastrophe.

Rhabdoviruses enter the cell via the endocytic pathway and subsequently fuse with a cellular membrane within the acidic environment of the endosome. Both receptor recognition and membrane fusion are mediated by a single transmembrane viral glycoprotein (G). Fusion is triggered via a low-pH induced structural rearrangement. G is an atypical fusion protein as there is a pH-dependent equilibrium between its pre- and post-fusion conformations. The elucidation of the atomic structures of these two conformations for the vesicular stomatitis virus (VSV) G has revealed that it is different from the previously characterized class I and class II fusion proteins. In this review, the pre- and post-fusion VSV G structures are presented in detail demonstrating that G combines the features of the class I and class II fusion proteins. In addition to these similarities, these G structures also reveal some particularities that expand our understanding of the working of fusion machineries. Combined with data from recent studies that revealed the cellular aspects of the initial stages of rhabdovirus infection, all these data give an integrated view of the entry pathway of rhabdoviruses into their host cell.