Structural insights into hepatitis C virus receptor binding and entry.

Hepatitis C virus (HCV) infection is a causal agent of chronic liver disease, cirrhosis and hepatocellular carcinoma in humans, and afflicts more than 70 million people worldwide. The HCV envelope glycoproteins E1 and E2 are responsible for the binding of the virus to the host cell, but the exact entry process remains undetermined1. The majority of broadly neutralizing antibodies block interaction between HCV E2 and the large extracellular loop (LEL) of the cellular receptor CD81 (CD81-LEL)2. Here we show that low pH enhances the binding of CD81-LEL to E2, and we determine the crystal structure of E2 in complex with an antigen-binding fragment (2A12) and CD81-LEL (E2-2A12-CD81-LEL); E2 in complex with 2A12 (E2-2A12); and CD81-LEL alone. After binding CD81, residues 418-422 in E2 are displaced, which allows for the extension of an internal loop consisting of residues 520-539. Docking of the E2-CD81-LEL complex onto a membrane-embedded, full-length CD81 places the residues Tyr529 and Trp531 of E2 proximal to the membrane. Liposome flotation assays show that low pH and CD81-LEL increase the interaction of E2 with membranes, whereas structure-based mutants of Tyr529, Trp531 and Ile422 in the amino terminus of E2 abolish membrane binding. These data support a model in which acidification and receptor binding result in a conformational change in E2 in preparation for membrane fusion.

Characterization and biodistribution of under-employed gene therapy vector AAV7.

IMPORTANCE: The use of adeno-associated viruses (AAVs) as gene delivery vectors has vast potential for the treatment of many severe human diseases. Over one hundred naturally existing AAV capsid variants have been described and classified into phylogenetic clades based on their sequences. AAV8, AAV9, AAVrh.10, and other intensively studied capsids have been propelled into pre-clinical and clinical use, and more recently, marketed products; however, less-studied capsids may also have desirable properties (e.g., potency differences, tissue tropism, reduced immunogenicity, etc.) that have yet to be thoroughly described. These data will help build a broader structure-function knowledge base in the field, present capsid engineering opportunities, and enable the use of novel capsids with unique properties.

Structure of the core ectodomain of the hepatitis C virus envelope glycoprotein 2.

Hepatitis C virus (HCV) is a significant public health concern with approximately 160 million people infected worldwide. HCV infection often results in chronic hepatitis, liver cirrhosis and hepatocellular carcinoma. No vaccine is available and current therapies are effective against some, but not all, genotypes. HCV is an enveloped virus with two surface glycoproteins (E1 and E2). E2 binds to the host cell through interactions with scavenger receptor class B type I (SR-BI) and CD81, and serves as a target for neutralizing antibodies. Little is known about the molecular mechanism that mediates cell entry and membrane fusion, although E2 is predicted to be a class II viral fusion protein. Here we describe the structure of the E2 core domain in complex with an antigen-binding fragment (Fab) at 2.4 Å resolution. The E2 core has a compact, globular domain structure, consisting mostly of ß-strands and random coil with two small α-helices. The strands are arranged in two, perpendicular sheets (A and B), which are held together by an extensive hydrophobic core and disulphide bonds. Sheet A has an IgG-like fold that is commonly found in viral and cellular proteins, whereas sheet B represents a novel fold. Solution-based studies demonstrate that the full-length E2 ectodomain has a similar globular architecture and does not undergo significant conformational or oligomeric rearrangements on exposure to low pH. Thus, the IgG-like fold is the only feature that E2 shares with class II membrane fusion proteins. These results provide unprecedented insights into HCV entry and will assist in developing an HCV vaccine and new inhibitors.

Structural and functional insights into alphavirus polyprotein processing and pathogenesis.

Alphaviruses, a group of positive-sense RNA viruses, are globally distributed arboviruses capable of causing rash, arthritis, encephalitis, and death in humans. The viral replication machinery consists of four nonstructural proteins (nsP1-4) produced as a single polyprotein. Processing of the polyprotein occurs in a highly regulated manner, with cleavage at the P2/3 junction influencing RNA template use during genome replication. Here, we report the structure of P23 in a precleavage form. The proteins form an extensive interface and nsP3 creates a ring structure that encircles nsP2. The P2/3 cleavage site is located at the base of a narrow cleft and is not readily accessible, suggesting a highly regulated cleavage. The nsP2 protease active site is over 40 Å away from the P2/3 cleavage site, supporting a trans cleavage mechanism. nsP3 contains a previously uncharacterized protein fold with a zinc-coordination site. Known mutations in nsP2 that result in formation of noncytopathic viruses or a temperature sensitive phenotype cluster at the nsP2/nsP3 interface. Structure-based mutations in nsP3 opposite the location of the nsP2 noncytopathic mutations prevent efficient cleavage of P23, affect RNA infectivity, and alter viral RNA production levels, highlighting the importance of the nsP2/nsP3 interaction in pathogenesis. A potential RNA-binding surface, spanning both nsP2 and nsP3, is proposed based on the location of ion-binding sites and adaptive mutations. These results offer unexpected insights into viral protein processing and pathogenesis that may be applicable to other polyprotein-encoding viruses such as HIV, hepatitis C virus (HCV), and Dengue virus.

Structure of the AAVhu.37 capsid by cryoelectron microscopy.

Adeno-associated viruses (AAVs) are used as in vivo gene-delivery vectors in gene-therapy products and have been heavily investigated for numerous indications. Over 100 naturally occurring AAV serotypes and variants have been isolated from primate samples. Many reports have described unique properties of these variants (for instance, differences in potency, target cell or evasion of the immune response), despite high amino-acid sequence conservation. AAVhu.37 is of interest for clinical applications owing to its proficient transduction of the liver and central nervous system. The sequence identity of the AAVhu.37 VP1 to the well characterized AAVrh.10 serotype, for which no structure is available, is greater than 98%. Here, the structure of the AAVhu.37 capsid at 2.56 Šresolution obtained via single-particle cryo-electron microscopy is presented.

Overcoming Challenges of Hepatitis C Virus Envelope Glycoprotein Production in Mammalian Cells.

Posttranslational modifications (PTMs) are often required for proper folding and physiological function of proteins, including the envelope glycoproteins 1 and 2 (E1 and E2) of hepatitis C virus (HCV). Commonly used expression systems such as bacteria, yeast, and baculovirus produce soluble HCV E1 and E2 at very low yields, as the cellular environment and molecular machinery necessary for PTMs may be suboptimal or missing. Here, we describe an expression system for HCV E2 ectodomain (eE2) with 11 N-linked glycans and eight disulfide bonds, which combines lentivirus transduction of mammalian cells and a continuous growth, adherent cell bioreactor. It is environmentally friendly, as well as cost- and time-efficient compared to other methods of recombinant protein expression in mammalian systems with final yields of eE2 approaching 60 mg/L of cell culture supernatant. eE2 produced by this system is amenable to a variety of biophysical studies, including structural determination by X-ray crystallography. Considering the ease of use and flexibility, this method can be applied to express an array of difficult target proteins in a variety of mammalian cell lines.

Hepatitis C Virus Envelope Glycoproteins: A Balancing Act of Order and Disorder.

Chronic hepatitis C virus infection often leads to liver cirrhosis and primary liver cancer. In 2015, an estimated 71 million people were living with chronic HCV. Although infection rates have decreased in many parts of the world over the last several decades, incidence of HCV infection doubled between 2010 and 2014 in the United States mainly due to increases in intravenous drug use. The approval of direct acting antiviral treatments is a necessary component in the elimination of HCV, but inherent barriers to treatment (e.g., cost, lack of access to healthcare, adherence to treatment, resistance, etc.) prevent dramatic improvements in infection rates. An effective HCV vaccine would significantly slow the spread of the disease. Difficulties in the development of an HCV culture model system and expression of properly folded- and natively modified-HCV envelope glycoproteins E1 and E2 have hindered vaccine development efforts. The recent structural and biophysical studies of these proteins have demonstrated that the binding sites for the cellular receptor CD-81 and neutralizing antibodies are highly flexible in nature, which complicate vaccine design. Furthermore, the interactions between E1 and E2 throughout HCV infection is poorly understood, and structural flexibility may play a role in shielding antigenic epitopes during infection. Here we discuss the structural complexities of HCV E1 and E2.

Viral precursor polyproteins: keys of regulation from replication to maturation.

Many viruses use a replication strategy involving the translation of a large polyprotein, which is cleaved by viral and/or cellular proteases. Several of these viruses severely impact human health around the globe, including HIV, HCV, Dengue virus, and West Nile virus. This method of genome organization has many benefits to the virus such as condensation of genetic material, as well as temporal and spatial regulation of protein activity depending on polyprotein cleavage state. The study of polyprotein precursors is necessary to fully understand viral infection, and identify possible new drug targets; however, few atomic structures are currently available. Presented here are structures of four recent polyprotein precursors from viruses with a positive sense RNA genome.

Effects of blood meal source on the reproduction of Culex pipiens quinquefasciatus (Diptera: Culicidae).

Culex pipiens quinquefasciatus were fed blood meals from a live chicken (LC), chicken blood in Alsever's (AC) solution, defibrinated bovine blood (DB), or bovine blood in citrate (CB) and incubated at 28° C. The effects of different blood meal sources were evaluated with respect to rates of blood feeding and reproduction (i.e., fecundity and fertility) over two gonotrophic cycles. Mosquitoes that fed on the first blood meal were subjected to a second blood meal as follows (first blood meal / second blood meal): LC/LC, LC/DB, DB/DB, CB/CB, AC/AC. Fecundity and fertility of Cx. p. quinquefasciatus were significantly (P < 0.05) greater in mosquitoes fed LC blood; however, fecundity and fertility in different treatment groups varied by gonotrophic cycle. These results contribute to our understanding of the impact of blood meal source on feeding and reproduction in Cx. p. quinquefasciatus. The potential impacts of blood meal source on virus transmission experiments are discussed.