Severe acute respiratory syndrome (SARS) is a newly emerging infectious disease, and an effective vaccine is not available. In this study, we compared the immunogenicity and protection efficacy of recombinant proteins corresponding to different domains of the SARS-coronavirus spike protein. Trimeric recombinant proteins were created by fusing the foldon domain derived from T4 bacteriophage to the carboxy-termini of individual domains of the spike protein. While the full-length ectodomain (S) of the spike protein, the full-length ectodomain fused to foldon (S-foldon), the S1 domain (S1), S1-foldon, and the S2 domain(S2) antigens all elicited comparable antibody titers as measured by ELISA, S-foldon induced a significantly higher titer of neutralizing antibody and S2 protein did not elicit virus neutralizing antibodies. When tested in a mouse virus replication model, all the mice vaccinated with the S1, S1-foldon, S, or S-foldon were completely protected.
Hepatitis A virus (HAV), an atypical member of the Picornaviridae that causes acute hepatitis in humans (for a review, see reference 16), has a positive-sense RNA genome of approximately 7,500 bases encapsidated in a shell formed by 60 copies of at least three viral proteins (VP1, VP2, and VP3). HAV codes for a very small VP4, the fourth picornavirus structural protein, which has not been detected in mature virions. Most wild-type strains of HAV do not grow in cell culture; however, attenuated variants that grow efficiently in primate cell culture have been isolated on serial passaging of the virus (4, 5, 8, 10-12, 15, 30). HAV has also been adapted to grow in guinea pig, pig, and dolphin cell cultures (9), indicating that the cellular factors required for HAV replication are not restricted to primates.Like other picornaviruses, the first step in the life cycle of HAV is its interaction with a cellular receptor that allows it to enter the cell. Using protective monoclonal antibody (MAb) 190/4 as a probe, Kaplan et al. (18) identified the HAV cellular receptor-1 (havcr-1) in African green monkey kidney cells as a receptor for HAV. Nucleotide sequence analysis revealed that havcr-1 is a class I integral membrane glycoprotein of unknown natural function. The extracellular domain of havcr-1 contains an N-terminal cysteine-rich region (D1), which has homology to members of the immunoglobulin superfamily, followed by a threonine-, serine-, and proline-rich (TSP-rich) region, which is characteristic of O-glycosylated mucin-like glycoproteins (27). D1, which is required for binding of HAV and MAb 190/4 (35), is most probably extended well above the cell surface by the TSP-rich region.Immunoadhesins are antibody-like molecules resulting from the fusion of the hinge and Fc portion of an immunoglobulin and the ligand-binding region of a receptor or adhesion molecule (for a review, see reference 3). These chimeric immunoglobulins are frequently used as research tools because they are easy to construct, express, and purify through protein A or G columns. In addition, the structure and function of the fused receptors are usually maintained in the immunoadhesins as a result of the flexibility and separation provided by the hinge region. Further, due to their homomultimeric characteristics, immunoadhesins have higher ligand avidity than do the monomeric receptors from which they were derived. To study the interaction of HAV with havcr-1, we constructed immunoadhesins fusing the hinge and Fc region of human IgG1 to D1 (D1-Fc) or the ectodomain of the poliovirus receptor (PVRFc) and expressed them in CHO cells. These immunoadhesins were secreted to the cell culture medium and purified using protein A columns. Here we report that D1-Fc binds specifically and neutralizes HAV whereas PVR-Fc has no effect on the HAV titers. The data presented in this work indicate that D1 is sufficient for HAV receptor function and provide further evidence that havcr-1 is a functional receptor for HAV.
Background: Hepatitis C virus (HCV) NS5B is an essential component of the viral replication machinery and an important target for antiviral intervention. Aurintricarboxylic acid (ATA), a broad-spectrum antiviral agent, was evaluated and characterized for its anti-NS5B activity in vitro and in HCV replicon cells. Methods: Recombinant NS5B, HCV replicase and Huh-7 cells harbouring the subgenomic HCV replicon of genotype 1b were employed for biochemical and mechanistic investigations. Results: Analysis of ATA activity in vitro yielded equipotent inhibition of recombinant NS5B and HCV replicase in the submicromolar range (50% inhibition concentration [IC 50 ] approximately 150 nM). Biochemical and mechanistic studies revealed a bimodal mechanism of ATA inhibition with characteristics of pyrophosphate mimics and non-nucleoside inhibitors. Molecular modelling and competition displacement studies were consistent with these parameters, suggesting that ATA might bind to the benzothiadiazine allosteric pocket 3 of NS5B or at its catalytic centre. Kinetic studies revealed a mixed mode of ATA inhibition with respect to both RNA and UTP substrates. Under single-cycle assay conditions, ATA inhibited HCV NS5B initiation and elongation from pre-bound RNA, but with ≥fivefold decreased potency compared with continuous polymerization conditions. The IC 50 value of ATA for the native replicase complex was 145 nM. In HCV replicon cells, ATA treatment ablated HCV RNA replication (50% effective concentration =75 nM) with concomitant decrease in NS5B expression and no apparent cytotoxic effects. Conclusions: This study identified ATA as a potent anti-NS5B inhibitor and suggests that its unique mode of action might be exploited for structural refinement and development of novel anti-NS5B agents.Hepatitis C virus (HCV) is a significant human bloodborne pathogen affecting an estimated 3% of the world's population, of whom 80% progress to chronic infection [1,2]. Sequelae include fibrosis, cirrhosis and hepatocellular carcinoma, making HCV the leading cause of liver transplantation in the USA [2,3]. The currently approved anti-HCV therapy of pegylated interferon-α (PEG-IFN-α) in combination with ribavirin exhibit sustained virological response (SVR) rates of 40-50% for genotype 1 and ≤80% for genotypes 2 and 3, and are associated with severe side effects resulting in limited patient compliance [4]. Additional challenges are posed by the high cost of therapy and the emergence of HCV quasispecies during treatment [5]; therefore, it is paramount to develop new and improved anti-HCV therapeutic agents. A prime area of focus is the development of small molecule inhibitors targeting the essential viral enzymes.HCV is an enveloped, single-stranded RNA virus with approximately 9.6 kb genome of positive polarity that encodes three structural and seven non-structural (NS) proteins [6,7]. Among these, the NS5B RNAdependent RNA polymerase (RdRp), a crucial and unique component of the viral replication machinery with no functional equivalent in the ho...
Hepatitis A virus (HAV) infects African green monkey kidney cells via HAV cellular receptor 1 (havcr-1).The ectodomain of havcr-1 contains an N-terminal cysteine-rich immunoglobulin-like region (D1), followed by a mucin-like region that extends D1 well above the cell surface. D1 is required for binding of HAV, and a soluble construct containing D1 fused to the hinge and Fc portions of human immunoglobulin G1 (IgG1), D1-Fc, bound and neutralized HAV inefficiently. However, D1-Fc did not alter the virions. To determine whether additional regions of havcr-1 are required to trigger uncoating of HAV, we constructed D1muc-Fc containing D1 and two-thirds of the mucin-like region fused to the Fc and hinge portions of human IgG1. D1muc-Fc neutralized 10 times more HAV than did D1-Fc. Sedimentation analysis in sucrose gradients showed that treatment of HAV with 20 to 200 nM D1muc-Fc disrupted the majority of the virions, whereas treatment with 2 nM D1muc-Fc had no effect on the sedimentation of the particles. Treatment of HAV with 100 nM D1muc-Fc resulted in low-level accumulation of 100-to 125S particles. Negative-stain electron microscopy analysis revealed that the 100-to 125S particles had the characteristics of disrupted virions, such as internal staining and diffuse edges. Quantitative PCR analysis showed that the 100-to 125S particles contained viral RNA. These results indicate that D1 and the mucin-like region of havcr-1 are required to induce conformational changes leading to HAV uncoating. Hepatitis A virus (HAV) is an atypical member of the familyPicornaviridae that causes acute hepatitis in humans (for a review, see reference 20). HAV has a positive-strand genomic RNA of approximately 7.5 kb that is covalently linked to a small virus-encoded VPg protein at its 5Ј end (38) and contains a poly(A) tail at its 3Ј end. The mature HAV capsid is formed by 60 copies of at least three viral proteins, VP1, VP2, and VP3. A small unmyristoylated protein, VP4, of 23 amino acids plays a signal role in capsid assembly (29) but has not been detected in mature virions. Nonstructural protein 2A remains associated with the structural proteins and serves as a signal for the assembly of pentamers, which are precursors involved in the morphogenesis of the capsid (29).Wild-type HAV usually does not grow in cell culture. The virus was adapted to in vitro growth by serial passage in cell cultures of primate origin, which resulted in the establishment of persistent infections and attenuation (7, 8, 10, 12-14, 17, 30). HAV has also been adapted to growth in guinea pig, pig, and dolphin cell cultures (11), indicating that the cellular factors required for HAV replication are not restricted to primates.Picornaviruses have different cell entry mechanisms. For instance, cellular receptors bind differently to a depression around the fivefold axis of poliovirus and the major group of rhinovirus (2, 18, 39) and induce conformational changes in the virions that result in the accumulation of 135S A particles and other uncoating intermediates (for...
Chagas disease, caused by the intracellular protozoan Trypanosoma cruzi, affects 8–10 million people worldwide and represents a major public health challenge. There is no effective treatment or vaccine to control the disease that is characterized by a mild acute phase followed by a chronic life-long infection. Approximately 30% of chronically infected individuals develop cardiac and/or digestive pathologies. T. cruzi can invade a wide variety of nucleated cells, but only persists at specific tissues in the host. However, the mechanisms that determine tissue tropism and the progression of the infection have not been fully described. Identification of infection niches in animal models has been difficult due to the limited quantity of parasite-infected cells and their focal distribution in tissues during the chronic phase. To better understand the course of chronic infections and parasite dissemination, we developed a bioluminescence imaging system based on the use of transgenic T. cruzi Colombiana strain parasites expressing nanoluciferase. Swiss Webster mice were infected with luminescent trypomastigotes and monitored for 126 days. Whole animal in vivo imaging showed parasites predominantly distributed in the abdominal cavity and surrounding areas throughout the infection. Bioluminescence signal reached a peak between 14 to 21 days post infection (dpi) and decreased progressively over time. Total animal luminescence could still be measured 126 dpi while parasites remained undetectable in blood by microscopy in most animals. Ex vivo imaging of specific tissues and organs dissected post-mortem at 126 dpi revealed a widespread parasite distribution in the skeletal muscle, heart, intestines and mesenteric fat. Parasites were also detected in lungs and liver. This noninvasive imaging model represents a novel tool to study host-parasite interactions and to identify parasite reservoirs of chronic Chagas Disease.
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