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

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

Sequence requirements for RNA strand transfer during nidovirus discontinuous subgenomic RNA synthesis

Nidovirus subgenomic mRNAs contain a leader sequence derived from the 5′ end of the genome fused to different sequences (‘bodies’) derived from the 3′ end. Their generation involves a unique mechanism of discontinuous subgenomic RNA synthesis that resembles copy-choice RNA recombination. During this process, the nascent RNA strand is transferred from one site in the template to another, during either plus or minus strand synthesis, to yield subgenomic RNA molecules. Central to this process are transcription-regulating sequences (TRSs), which are present at both template sites and ensure the fidelity of strand transfer. Here we present results of a comprehensive co-variation mutagenesis study of equine arteritis virus TRSs, demonstrating that discontinuous RNA synthesis depends not only on base pairing between sense leader TRS and antisense body TRS, but also on the primary sequence of the body TRS. While the leader TRS merely plays a targeting role for strand transfer, the body TRS fulfils multiple functions. The sequences of mRNA leader–body junctions of TRS mutants strongly suggested that the discontinuous step occurs during minus strand synthesis

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