The identification and characterization of heparan sulfate as a cell surface attachment molecule for Ross River virus mutants with an expanded host range

The alphaviruses are blood-borne pathogen transmitted in the wild by a mosquito vector to a vertebrate host, which is usually a bird. Alphaviruses can infect humans, and these infections can cause serious illness. The symptoms of an alphavirus infection include fever, rash, arthralgia, and encephalitis. This broad host range and tissue tropism are attributed to the alphaviruses\u27 ability to utilize multiple host receptors. The purpose of the present study was: to define Ross River virus antireceptor amino acids: to identify a Ross River virus co-receptor: and to obtain structural information about the virus receptor interaction. In this study, it was determined that the enhanced entry of Ross River virus mutants. N218R and N218K, into cultures of chicken embryo fibroblasts was the result of an interaction between the mutants and cell surface heparan sulfate (HS) moieties. The mutants bound immobilized heparin (a highly sulfated form of HS), and were inhibited in growth in HS-deficient cell lines. These data suggested that a single amino acid change expanded the host range through an interaction with HS. Cryoelectron microscopy followed by image reconstruction determined the binding location of heparin, and by inference HS, on the surface of the particle. Heparin bound the distal portion of the spike. The footprints of heparin and Fab T10C9, which recognizes residue E2 216, overlapped each other suggesting heparin was interacting with residue 218. These data supported the idea that this region of the alphavirus spike is involved in cell surface recognition. An RRV mutant with a deletion of 7 amino acids in the E2 glycoprotein was shown here to bind HS. The location of HS binding on the mutant particle is unknown, but it is likely to be distinct from binding at residue 218. These studies suggested that there are multiple antireceptors for RRV

HTS-Driven Discovery of New Chemotypes with West Nile Virus Inhibitory Activity

West Nile virus (WNV) is a positive sense, single-stranded RNA virus that can cause illness in humans when transmitted via mosquito vectors. Unfortunately, no antivirals or vaccines are currently available, and therefore efficient and safe antivirals are urgently needed. We developed a high throughput screen to discover small molecule probes that inhibit virus infection of Vero E6 cells. A primary screen of a 13,001 compound library at a 10 µM final concentration was conducted using the 384-well format. Z′ values ranged from 0.54–0.83 with a median of 0.74. Average S/B was 17 and S/N for each plate ranged from 10.8 to 23.9. Twenty-six compounds showed a dose response in the HT screen and were further evaluated in a time of addition assay and in a titer reduction assay. Seven compounds showed potential as small molecule probes directed at WNV. The hit rate from the primary screen was 0.185% (24 compounds out of 13,001 compounds) and from the secondary screens was 0.053% (7 out of 13,001 compounds) respectively