SARS-Coronavirus Replication/Transcription Complexes Are Membrane-Protected and Need a Host Factor for Activity In Vitro

Abstract

SARS-coronavirus (SARS-CoV) replication and transcription are mediated by a replication/transcription complex (RTC) of which virus-encoded, non-structural proteins (nsps) are the primary constituents. The 16 SARS-CoV nsps are produced by autoprocessing of two large precursor polyproteins. The RTC is believed to be associated with characteristic virus-induced double-membrane structures in the cytoplasm of SARS-CoV-infected cells. To investigate the link between these structures and viral RNA synthesis, and to dissect RTC organization and function, we isolated active RTCs from infected cells and used them to develop the first robust assay for their in vitro activity. The synthesis of genomic RNA and all eight subgenomic mRNAs was faithfully reproduced by the RTC in this in vitro system. Mainly positive-strand RNAs were synthesized and protein synthesis was not required for RTC activity in vitro. All RTC activity, enzymatic and putative membrane-spanning nsps, and viral RNA cosedimented with heavy membrane structures. Furthermore, the pelleted RTC required the addition of a cytoplasmic host factor for reconstitution of its in vitro activity. Newly synthesized subgenomic RNA appeared to be released, while genomic RNA remained predominantly associated with the RTC-containing fraction. RTC activity was destroyed by detergent treatment, suggesting an important role for membranes. The RTC appeared to be protected by membranes, as newly synthesized viral RNA and several replicase/transcriptase subunits were protease- and nuclease-resistant and became susceptible to degradation only upon addition of a non-ionic detergent. Our data establish a vital functional dependence of SARS-CoV RNA synthesis on virus-induced membrane structures.

Changes in nonstructural protein 3 are associated with attenuation in avian coronavirus infectious bronchitis virus

“…The Mpro contained within nsp 5 cleaves nsps 5 through 16. The biological characteristics of many nsps have been previously reported [9,10,[12][13][14][15][16][17]. In addition to nsps 3 and 5, which contain proteases PL2 and Mpro, respectively, nsps 2, 4, and 6 contain hydrophobic residues predicted to play a role in anchoring the RTC to the Golgi.…”

“…The polyprotein 1ab is translated through a-1 frame-shift translation mechanism that occurs approximately 20-40% of the time [9]. The IBV 1a and 1ab polyproteins are post-translationally cleaved into 15 nonstructural proteins (nsps), nsps 2 through 16 by a papain-like protease (PLP) and the main protease (Mpro), also referred to as the 3C-like protease [10]. IBV does not have an nsp 1 equivalent found in some other coronaviruses.…”

Suramin inhibits chikungunya virus replication through multiple mechanisms

“…RNA isolation from infected cells, denaturing agarose gel electrophoresis, detection of 32 P-RNA or viral RNA by hybridization with (strand-) specific probes have been described previously (Albulescu et al, 2014;van Hemert et al, 2008). CHIKV genome copy numbers were determined by internally-controlled TaqMan multiplex RT-qPCR as described (Scholte et al, 2015).…”

Cell-based antiviral screening against coronaviruses: Developing virus-specific and broad-spectrum inhibitors

“…A previous study by van Hemert et al established a protocol to isolate and monitor the activity SARS-CoV replication complexes in vitro (van Hemert et al, 2008). The study performed by te Velthuis et al used this protocol and showed that zinc-ionophores inhibited the synthesis of RNA by SARS-CoV RdRp.…”