Replication of Synthetic Defective Interfering RNAs Derived from Coronavirus Mouse Hepatitis Virus-A59

AbstractWe have analyzed the replication of deletion mutants of defective interfering (DI) RNAs derived from the coronavirus mouse hepatitis virus (MHV)-A59 in the presence of MHV-A59. Using two parental DI RNAs, MIDI and MIDIΔH, a twin set of deletion mutants was generated with progressively shorter stretches of 5′ sequence colinear with the genomic RNA. All deletion mutants contained in-frame ORFs. We show that in transfected cells and after one passage the DI RNAs were detectable and that their accumulation was positively correlated with the length of 5′ sequence they contained. However, accumulation of two twin mutants, Δ2, in which sequences from nucleotide position 467 were fused to those from position 801, was undetectable. In passage 4 cells, but not in transfected or in passage 1 cells, recombination with genomic RNA led to the appearance of the parental DI RNAs. The accumulation of these parental RNAs was inversely correlated with the length of 5′ sequence on the deletion mutants and was highest in the Δ2 samples. In sharp contrast to the data reported for MHV-JHM-derived DI RNAs, we show that MHV-A59-derived mutant RNAs do not require an internal sequence domain for replication. The data suggest that coronavirus replication involves an RNA superstructure at the 5′ end of the genome or one comprising both ends of the genomic RNA. We also conclude from the recombination data that in-frame mutants with impaired replication signals are more fit than out-frame mutants with intact replication signals

Hepatitis C virus NS4B carboxy terminal domain is a membrane binding domain

<p>Abstract</p> <p>Background</p> <p>Hepatitis C virus (HCV) induces membrane rearrangements during replication. All HCV proteins are associated to membranes, pointing out the importance of membranes for HCV. Non structural protein 4B (NS4B) has been reported to induce cellular membrane alterations like the membranous web. Four transmembrane segments in the middle of the protein anchor NS4B to membranes. An amphipatic helix at the amino-terminus attaches to membranes as well. The carboxy-terminal domain (CTD) of NS4B is highly conserved in Hepaciviruses, though its function remains unknown.</p> <p>Results</p> <p>A cytosolic localization is predicted for the NS4B-CTD. However, using membrane floatation assays and immunofluorescence, we now show targeting of the NS4B-CTD to membranes. Furthermore, a profile-profile search, with an HCV NS4B-CTD multiple sequence alignment, indicates sequence similarity to the membrane binding domain of prokaryotic D-lactate dehydrogenase (d-LDH). The crystal structure of E. coli d-LDH suggests that the region similar to NS4B-CTD is located in the membrane binding domain (MBD) of d-LDH, implying analogy in membrane association. Targeting of d-LDH to membranes occurs via electrostatic interactions of positive residues on the outside of the protein with negative head groups of lipids. To verify that anchorage of d-LDH MBD and NS4B-CTD is analogous, NS4B-CTD mutants were designed to disrupt these electrostatic interactions. Membrane association was confirmed by swopping the membrane contacting helix of d-LDH with the corresponding domain of the 4B-CTD. Furthermore, the functionality of these residues was tested in the HCV replicon system.</p> <p>Conclusion</p> <p>Together these data show that NS4B-CTD is associated to membranes, similar to the prokaryotic d-LDH MBD, and is important for replication.</p

Regulation of Relative Abundance of Arterivirus Subgenomic mRNAs

The subgenomic (sg) mRNAs of arteriviruses (order Nidovirales) form a 5′- and 3′-coterminal nested set with the viral genome. Their 5′ common leader sequence is derived from the genomic 5′-proximal region. Fusion of sg RNA leader and “body” segments involves a discontinuous transcription step. Presumably during minus-strand synthesis, the nascent RNA strand is transferred from one site in the genomic template to another, a process guided by conserved transcription-regulating sequences (TRSs) at these template sites. Subgenomic RNA species are produced in different but constant molar ratios, with the smallest RNAs usually being most abundant. Factors thought to influence sg RNA synthesis are size differences between sg RNA species, differences in sequence context between body TRSs, and the mutual influence (or competition) between strand transfer reactions occurring at different body TRSs. Using an Equine arteritis virus infectious cDNA clone, we investigated how body TRS activity affected sg RNA synthesis from neighboring body TRSs. Flanking sequences were standardized by head-to-tail insertion of several copies of an RNA7 body TRS cassette. A perfect gradient of sg RNA abundance, progressively favoring smaller RNA species, was observed. Disruption of body TRS function by mutagenesis did not have a significant effect on the activity of other TRSs. However, deletion of body TRS-containing regions enhanced synthesis of sg RNAs from upstream TRSs but not of those produced from downstream TRSs. The results of this study provide considerable support for the proposed discontinuous extension of minus-strand RNA synthesis as a crucial step in sg RNA synthesis

Nidovirus transcription: how to make sense...?

Many positive-stranded RNA viruses use subgenomic mRNAs to express part of their genetic information. To produce structural and accessory proteins, members of the order Nidovirales (corona-, toro-, arteri- and roniviruses) generate a 3' co-terminal nested set of at least three and often seven to nine mRNAs. Coronavirus and arterivirus subgenomic transcripts are not only 3' co-terminal but also contain a common 5' leader sequence, which is derived from the genomic 5' end. Their synthesis involves a process of discontinuous RNA synthesis that resembles similarity-assisted RNA recombination. Most models proposed over the past 25 years assume co-transcriptional fusion of subgenomic RNA leader and body sequences, but there has been controversy over the question of whether this occurs during plus- or minus-strand synthesis. In the latter model, which has now gained considerable support, subgenomic mRNA synthesis takes place from a complementary set of subgenome-size minus-strand RNAs, produced by discontinuous minus-strand synthesis. Sense-antisense base-pairing interactions between short conserved sequences play a key regulatory role in this process. In view of the presumed common ancestry of nidoviruses, the recent finding that ronivirus and torovirus mRNAs do not contain a common 5' leader sequence is surprising. Apparently, major mechanistic differences must exist between nidoviruses, which raises questions about the functions of the common leader sequence and nidovirus transcriptase proteins and the evolution of nidovirus transcription. In this review, nidovirus transcription mechanisms are compared, the experimental systems used are critically assessed and, in particular, the impact of recently developed reverse genetic systems is discusse

GxxxG Motif of Severe Acute Respiratory Syndrome Coronavirus Spike Glycoprotein Transmembrane Domain Is Not Involved in Trimerization and Is Not Important for Entry▿

Recently, a paper was published in which it was proposed that the GxxxG motif of the severe acute respiratory syndrome (SARS) coronavirus spike (S) protein transmembrane domain plays a vital role in oligomerization of the protein (E. Arbely, Z. Granot, I. Kass, J. Orly, and I. T. Arkin, Biochemistry 45:11349-11356, 2006). Here, we show that the GxxxG motif is not involved in SARS S oligomerization by trimerization analysis of S GxxxG mutant proteins. In addition, the capability of S to mediate entry of SARS S-pseudotyped particles overall was affected moderately in the mutant proteins, also arguing for a nonvital role for the GxxxG motif in SARS coronavirus entry