Differential modulation of prM cleavage, extracellular particle distribution, and virus infectivity by conserved residues at nonfurin consensus positions of the dengue virus pr-M junction.

In the generation of flavivirus particles, an internal cleavage of the envelope glycoprotein prM by furin is required for the acquisition of infectivity. Unlike cleavage of the prM of other flaviviruses, cleavage of dengue virus prM is incomplete in many cell lines; the partial cleavage reflects the influence of residues at furin nonconsensus positions of the pr-M junction, as flaviviruses share basic residues at positions P1, P2, and P4, recognized by furin. In this study, viruses harboring the alanine-scanning and other multiple-point mutations of the pr-M junction were generated, employing a dengue virus background that exhibited 60 to 70% prM cleavage and a preponderance of virion-sized extracellular particles.

Mutation in murine coronavirus replication protein nsp4 alters assembly of double membrane vesicles.

Coronaviruses are positive-strand RNA viruses that replicate in the cytoplasm of infected cells by generating a membrane-associated replicase complex. The replicase complex assembles on double membrane vesicles (DMVs). Here, we studied the role of a putative replicase anchor, nonstructural protein 4 (nsp4), in the assembly of murine coronavirus DMVs.

Characterization of dengue virus NS1 stably expressed in 293T cell lines.

Dengue virus NS1 is a viral nonstructural protein detected in sera of infected individuals and in infected cells. Multiple NS1 structural forms have been reported but the functional characteristics of these forms remain unknown. In this study, a set of 293T cell lines stably expressing recombinant dengue NS1 without additional C-terminal sequence (rNS1s), with a heterologous transmembrane segment (rNS1tm), or with the 26-residue N-terminal portion of NS2A (rNS1v1) was established to aid in the characterization of different NS1 forms.

Membrane topology of murine coronavirus replicase nonstructural protein 3.

Mouse hepatitis virus (MHV) is a member of the family Coronaviridae. These positive strand RNA viruses encode a replicase polyprotein that is processed into 16 nonstructural proteins (nsps). The nsps assemble with membranes to generate double membrane vesicles, which are the sites of viral RNA synthesis.

Identification of severe acute respiratory syndrome coronavirus replicase products and characterization of papain-like protease activity.

Gene 1 of the coronavirus associated with severe acute respiratory syndrome (SARS) encodes replicase polyproteins that are predicted to be processed into 16 nonstructural proteins (nsps 1 to 16) by two viral proteases, a papain-like protease (PLpro) and a 3C-like protease (3CLpro). Here, we identify SARS coronavirus amino-terminal replicase products nsp1, nsp2, and nsp3 and describe trans-cleavage assays that characterize the protease activity required to generate these products. We generated polyclonal antisera to glutathione S-transferase-replicase fusion proteins and used the antisera to detect replicase intermediates and products in pulse-chase experiments.

Identification of the murine coronavirus MP1 cleavage site recognized by papain-like proteinase 2.

The replicase polyprotein of murine coronavirus is extensively processed by three proteinases, two papain-like proteinases (PLPs), termed PLP1 and PLP2, and a picornavirus 3C-like proteinase (3CLpro). Previously, we established a trans-cleavage assay and showed that PLP2 cleaves the replicase polyprotein between p210 and membrane protein 1 (MP1) (A. Kanjanahaluethai and S.

RNA replication of mouse hepatitis virus takes place at double-membrane vesicles.

The replication complexes (RCs) of positive-stranded RNA viruses are intimately associated with cellular membranes. To investigate membrane alterations and to characterize the RC of mouse hepatitis virus (MHV), we performed biochemical and ultrastructural studies using MHV-infected cells. Biochemical fractionation showed that all 10 of the MHV gene 1 polyprotein products examined pelleted with the membrane fraction, consistent with membrane association of the RC.