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

Equine Arteritis Virus Subgenomic RNA Transcription: UV Inactivation and Translation Inhibition Studies

AbstractThe expression of the genetic information of equine arteritis virus (EAV), an arterivirus, involves the synthesis of six subgenomic (sg) mRNAs. These are 5′ and 3′ coterminal since they are composed of a leader and a body sequence, which are identical to the 5′ and 3′ ends of the genome, respectively. Previously, it has been suggested thatcis-splicing of a genome-length precursor RNA is involved in their synthesis. This was reevaluated in a comparative analysis of the sg RNA synthesis of EAV, the coronavirus mouse hepatitis virus (MHV), and the alphavirus Sindbis virus. UV transcription mapping showed that the majority of the EAV sg RNAs made at later stages of infection is not derived from a genome-length precursor. However, complete independence of sg RNA synthesis from that of genomic RNA was never observed during the course of infection. The possibility that this resulted from UV irradiation-induced effects on the synthesis of the viral replicase was investigated by inhibiting translation using cycloheximide. For EAV, ongoing protein synthesis was found to be more important for the synthesis of sg RNA than for that of genomic RNA. In general, MHV transcription was extremely sensitive to translation inhibition, whereas EAV genomic RNA synthesis became independent ofde novoprotein synthesis late in infection

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

Evolution of naturally occurring 5' non-translated region variants of hepatitis C virus genotype 1b in selectable replicons

Quasispecies shifts are essential for the development of persistent hepatitis C virus (HCV) infection. Naturally occurring sequence variations in the 5' non-translated region (NTR) of the virus could lead to changes in protein expression levels, reflecting selective forces on the virus. The extreme 5' end of the virus' genome, containing signals essential for replication, is followed by an internal ribosomal entry site (IRES) essential for protein translation as well as replication. The 5' NTR is highly conserved and has a complex RNA secondary structure consisting of several stem-loops. This report analyses the quasispecies distribution of the 5' NTR of an HCV genotype 1b clinical isolate and found a number of sequences differing from the consensus sequence. The consensus sequence, as well as a major variant located in stem-loop IIIa of the IRES, was investigated using self-replicating HCV RNA molecules in human hepatoma cells. The stem-loop IIIa mutation, which is predicted to disrupt the stem structure, showed slightly lower translation efficiency but was severely impaired in the colony formation of selectable HCV replicons. Interestingly, during selection of colonies supporting autonomous replication, mutations emerged that restored the base pairing in the stem-loop. Recloning of these altered IRESs confirmed that these second site revertants were more efficient in colony formation. In conclusion, naturally occurring variants in the HCV 5' NTR can lead to changes in their replication ability. Furthermore, IRES quasispecies evolution was observed in vitro under the selective pressure of the replicon system

A recombinant Yellow Fever 17D vaccine expressing Lassa virus glycoproteins

AbstractThe Yellow Fever Vaccine 17D (YFV17D) has been used as a vector for the Lassa virus glycoprotein precursor (LASV-GPC) resulting in construction of YFV17D/LASV-GPC recombinant virus. The virus was replication-competent and processed the LASV-GPC in cell cultures. The recombinant replicated poorly in guinea pigs but still elicited specific antibodies against LASV and YFV17D antigens. A single subcutaneous injection of the recombinant vaccine protected strain 13 guinea pigs against fatal Lassa Fever. This study demonstrates the potential to develop an YFV17D-based bivalent vaccine against two viruses that are endemic in the same area of Africa

Mutagenesis of the transmembrane domain of the SARS coronavirus spike glycoprotein: refinement of the requirements for SARS coronavirus cell entry

Abstract Background The spike protein (S) of SARS Coronavirus (SARS-CoV) mediates entry of the virus into target cells, including receptor binding and membrane fusion. Close to or in the viral membrane, the S protein contains three distinct motifs: a juxtamembrane aromatic part, a central highly hydrophobic stretch and a cysteine rich motif. Here, we investigate the role of aromatic and hydrophobic parts of S in the entry of SARS CoV and in cell-cell fusion. This was investigated using the previously described SARS pseudotyped particles system (SARSpp) and by fluorescence-based cell-cell fusion assays. Results Mutagenesis showed that the aromatic domain was crucial for SARSpp entry into cells, with a likely role in pore enlargement. Introduction of lysine residues in the hydrophobic stretch of S also resulted in a block of entry, suggesting the borders of the actual transmembrane domain. Surprisingly, replacement of a glycine residue, situated close to the aromatic domain, with a lysine residue was tolerated, whereas the introduction of a lysine adjacent to the glycine, was not. In a model, we propose that during fusion, the lateral flexibility of the transmembrane domain plays a critical role, as do the tryptophans and the cysteines. Conclusions The aromatic domain plays a crucial role in the entry of SARS CoV into target cells. The positioning of the aromatic domain and the hydrophobic domain relative to each other is another essential characteristic of this membrane fusion process.</p

Heterodimerization of the Two Major Envelope Proteins Is Essential for Arterivirus Infectivity

The two major envelope proteins of arteriviruses, the membrane protein (M) and the major glycoprotein (GP(5)), associate into a disulfide-linked heterodimer that is incorporated into the virion and has been assumed to be a prerequisite for virus assembly. Using an equine arteritis virus (EAV) infectious cDNA clone, we have analyzed the requirement for GP(5)-M heterodimerization and have identified the Cys residues involved in the formation of the GP(5)-M disulfide bond. The single Cys residue (Cys-8) in the M ectodomain was crucial for heterodimerization and virus infectivity. Mutagenesis of any of the five Cys residues in the GP(5) ectodomain or removal of the single GP(5) N-glycosylation site also rendered the full-length clone noninfectious. However, an analysis of revertants yielded an exceptional pseudorevertant in which residues 52 to 79 of the GP(5) ectodomain had been deleted and the original Cys-80→Ser mutation had been maintained. Consequently, this revertant lacked the GP(5) N-glycosyation site (Asn-56) and retained only a single cysteine residue (Cys-34). By using this GP(5) deletion, we confirmed that Cys-34 of GP(5) and Cys-8 of M are essential for GP(5)-M heterodimerization, a key event in the assembly of the EAV envelope