Generation of Measles Virus with a Segmented RNA Genome

ABSTRACT Viruses classified in the order Mononegavirales have a single nonsegmented RNA molecule as the genome and employ similar strategies for genome replication and gene expression. Infectious particles of Measles virus (MeV), a member of the family Paramyxoviridae in the order Mononegavirales, with two or three RNA genome segments (2 seg- or 3 seg-MeV) were generated using a highly efficient reverse genetics system. All RNA segments of the viruses were designed to have authentic 3′ and 5′ self-complementary termini, similar to those of negative-stranded RNA viruses that intrinsically have multiple RNA genome segments. The 2 seg- and 3 seg-MeV were viable and replicated well in cultured cells. 3 seg-MeV could accommodate up to six additional transcriptional units, five of which were shown to be capable of expressing foreign proteins efficiently. These data indicate that the MeV genome can be segmented, providing an experimental insight into the divergence of the negative-stranded RNA viruses with nonsegmented or segmented RNA genomes. They also illustrate a new strategy to develop mononegavirus-derived vectors harboring multiple additional transcriptional units.

Download Full-text

Proofreading-Deficient Coronaviruses Adapt for Increased Fitness over Long-Term Passage without Reversion of Exoribonuclease-Inactivating Mutations

mBio ◽

10.1128/mbio.01503-17 ◽

2017 ◽

Vol 8 (6) ◽

Cited By ~ 16

Author(s):

Kevin W. Graepel ◽

Xiaotao Lu ◽

James Brett Case ◽

Nicole R. Sexton ◽

Everett Clinton Smith ◽

...

Keyword(s):

Rna Viruses ◽

Hepatitis Virus ◽

Nucleoside Analogues ◽

High Fidelity ◽

Genome Replication ◽

Murine Hepatitis Virus ◽

Replicase Proteins ◽

Rna Genome ◽

Inactivating Mutations

ABSTRACT The coronavirus (CoV) RNA genome is the largest among the single-stranded positive-sense RNA viruses. CoVs encode a proofreading 3′-to-5′ exoribonuclease within nonstructural protein 14 (nsp14-ExoN) that is responsible for CoV high-fidelity replication. Alanine substitution of ExoN catalytic residues [ExoN(-)] in severe acute respiratory syndrome-associated coronavirus (SARS-CoV) and murine hepatitis virus (MHV) disrupts ExoN activity, yielding viable mutant viruses with defective replication, up to 20-fold-decreased fidelity, and increased susceptibility to nucleoside analogues. To test the stability of the ExoN(-) genotype and phenotype, we passaged MHV-ExoN(-) 250 times in cultured cells (P250), in parallel with wild-type MHV (WT-MHV). Compared to MHV-ExoN(-) P3, MHV-ExoN(-) P250 demonstrated enhanced replication and increased competitive fitness without reversion at the ExoN(-) active site. Furthermore, MHV-ExoN(-) P250 was less susceptible than MHV-ExoN(-) P3 to multiple nucleoside analogues, suggesting that MHV-ExoN(-) was under selection for increased replication fidelity. We subsequently identified novel amino acid changes within the RNA-dependent RNA polymerase and nsp14 of MHV-ExoN(-) P250 that partially accounted for the reduced susceptibility to nucleoside analogues. Our results suggest that increased replication fidelity is selected in ExoN(-) CoVs and that there may be a significant barrier to ExoN(-) reversion. These results also support the hypothesis that high-fidelity replication is linked to CoV fitness and indicate that multiple replicase proteins could compensate for ExoN functions during replication. IMPORTANCE Uniquely among RNA viruses, CoVs encode a proofreading exoribonuclease (ExoN) in nsp14 that mediates high-fidelity RNA genome replication. Proofreading-deficient CoVs with disrupted ExoN activity [ExoN(-)] either are nonviable or have significant defects in replication, RNA synthesis, fidelity, fitness, and virulence. In this study, we showed that ExoN(-) murine hepatitis virus can adapt during long-term passage for increased replication and fitness without reverting the ExoN-inactivating mutations. Passage-adapted ExoN(-) mutants also demonstrate increasing resistance to nucleoside analogues that is explained only partially by secondary mutations in nsp12 and nsp14. These data suggest that enhanced resistance to nucleoside analogues is mediated by the interplay of multiple replicase proteins and support the proposed link between CoV fidelity and fitness. IMPORTANCE Uniquely among RNA viruses, CoVs encode a proofreading exoribonuclease (ExoN) in nsp14 that mediates high-fidelity RNA genome replication. Proofreading-deficient CoVs with disrupted ExoN activity [ExoN(-)] either are nonviable or have significant defects in replication, RNA synthesis, fidelity, fitness, and virulence. In this study, we showed that ExoN(-) murine hepatitis virus can adapt during long-term passage for increased replication and fitness without reverting the ExoN-inactivating mutations. Passage-adapted ExoN(-) mutants also demonstrate increasing resistance to nucleoside analogues that is explained only partially by secondary mutations in nsp12 and nsp14. These data suggest that enhanced resistance to nucleoside analogues is mediated by the interplay of multiple replicase proteins and support the proposed link between CoV fidelity and fitness.

Download Full-text

Rescue of Tomato spotted wilt tospovirus entirely from cDNA clones, establishment of the first reverse genetics system for a segmented (-)RNA plant virus

10.1101/680900 ◽

2019 ◽

Author(s):

Mingfeng Feng ◽

Ruixiang Cheng ◽

Minglong Chen ◽

Rong Guo ◽

Luyao Li ◽

...

Keyword(s):

Life Cycle ◽

Reverse Genetics ◽

Molecular Genetic ◽

Cdna Clones ◽

Genome Replication ◽

Negative Strand ◽

Replication System ◽

Ribonucleoprotein Complexes ◽

Tomato Spotted Wilt ◽

Rna Genome

AbstractThe group of negative strand RNA viruses (NSVs) includes not only dangerous pathogens of medical importance but also serious plant pathogens of agronomical importance. Tomato spotted wilt tospovirus (TSWV) is one of those plant NSVs that cause severe diseases on agronomic crops and pose major threats to global food security. Its negative-strand segmented RNA genome has, however, always posed a major obstacle to molecular genetic manipulation. In this study, we report the complete recovery of infectious TSWV entirely from cDNA clones, the first reverse genetics (RG) system for a segmented plant NSV. First, a replication and transcription competent mini-genome replication system was established based on 35S-driven constructs of the S(-)-genomic (g) or S(+)-antigenomic (ag) RNA template, flanked by a 5’ Hammerhead and 3’ Ribozyme sequence of Hepatitis Delta virus, a nucleocapsid (N) protein gene and codon-optimized viral RNA dependent RNA polymerase (RdRp) gene. Next, a movement competent mini-genome replication system was developed based on M(-)-gRNA, which was able to complement cell-to-cell and systemic movement of reconstituted ribonucleoprotein complexes (RNPs) of S RNA replicon. After further optimization, infectious TSWV and derivatives carrying eGFP reporters were successfully rescuedin plantavia simultaneous expression of full-length cDNA constructs coding for S(+)-agRNA, M(-)-gRNA and L(+)-agRNA. Viral rescue occurred in the additional presence of various viral suppressors of RNAi, but TSWV NSs interfered with the rescue of genomic RNA. The establishment of a RG system for TSWV now allows detailed molecular genetic analysis of all aspects of tospovirus life cycle and their pathogenicity.SignificanceFor many different animal-infecting segmented negative-strand viruses (NSVs), a reverse genetics system has been established that allows the generation of mutant viruses to study disease pathology and the role ofcis- andtrans-acting elements in the virus life cycle. In contrast to the relative ease to establish RG systems for animal-infecting NSVs, establishment of such system for the plant-infecting NSVs with a segmented RNA genome so far has not been successful. Here we report the first reverse genetics system for a segmented plant NSV, the Tomato spotted wilt tospovirus, a virus with a tripartite RNA genome. The establishment of this RG system now provides us with a new and powerful platform to study their disease pathology during a natural infection.

Download Full-text

Reduced Nucleoprotein availability impairs negative-sense RNA virus replication and promotes host recognition

Journal of Virology ◽

10.1128/jvi.02274-20 ◽

2021 ◽

Author(s):

Benjamin E. Nilsson-Payant ◽

Daniel Blanco-Melo ◽

Skyler Uhl ◽

Beatriz Escudero-Pérez ◽

Silke Olschewski ◽

...

Keyword(s):

Host Response ◽

Measles Virus ◽

Influenza A ◽

Ebola Virus ◽

Rna Viruses ◽

Viral Rna ◽

Genome Replication ◽

Replication Machinery ◽

Molecular Patterns ◽

Negative Sense

Negative-sense RNA viruses (NSVs) rely on prepackaged viral RNA-dependent RNA polymerases (RdRp) to replicate and transcribe their viral genomes. Their replication machinery consists of an RdRp bound to viral RNA which is wound around a nucleoprotein (NP) scaffold, forming a viral ribonucleoprotein complex. NSV NP is known to regulate transcription and replication of genomic RNA, however its role in maintaining and protecting the viral genetic material is unknown. Here, we exploited host microRNA expression to target NP of influenza A virus and Sendai virus to ascertain how this would impact genomic levels and the host response to infection. We find that in addition to inducing a drastic decrease in genome replication, the antiviral host response in the absence of NP is dramatically enhanced. Additionally, our data shows that insufficient levels of NP prevent the replication machinery of these NSVs to process full-length genomes, resulting in aberrant replication products which form pathogen-associated molecular patterns in the process. These dynamics facilitate immune recognition by cellular pattern recognition receptors leading to a strong host antiviral response. Moreover, we observe that the consequences of limiting NP levels are universal amongst NSVs including Ebola virus, Lassa virus and Measles virus. Overall, these results provide new insights into viral genome replication of negative-sense RNA viruses and highlight novel avenues towards developing effective antiviral strategies, adjuvants, and/or live-attenuated vaccines. IMPORTANCE Negative-sense RNA viruses comprise some of the most important known human pathogens, including influenza A virus, measles virus and Ebola virus. These viruses possess RNA genomes that are unreadable to the host as they require specific viral RNA dependent RNA polymerases in conjunction with other viral proteins such as nucleoprotein to be replicated and transcribed. As this process generates a significant amount of pathogen-associated molecular patterns, this phylum of viruses can result in a robust induction of the intrinsic host cellular response. To circumvent these defenses, these viruses form tightly regulated ribonucleoprotein replication complexes in order to protect their genomes from detection and to prevent excessive aberrant replication. Here we demonstrate the balance that negative-sense RNA viruses must achieve to both replicate efficiently and to avoid induction of the host defenses.

Download Full-text

Regulation of RNA Virus Processes by Viral Genome Structure

Proceedings ◽

10.3390/proceedings2020050068 ◽

2020 ◽

Vol 50 (1) ◽

pp. 68

Author(s):

K. Andrew White

Keyword(s):

Viral Genome ◽

Rna Viruses ◽

Rna Virus ◽

Genome Structure ◽

Viral Proteins ◽

Viral Rna ◽

Rna Sequences ◽

Virus Reproduction ◽

The Family ◽

Rna Genome

The genomes of RNA viruses contain a variety of RNA sequences and structures that regulate different steps in virus reproduction. Events that are controlled by RNA elements include (i) the translation of viral proteins, (ii) the replication of viral RNA genomes, and (iii) the transcription of viral subgenomic mRNAs. Studies of members of the family Tombusviridae, which possess plus-strand RNA genomes, have revealed novel ways in which the RNA genome structure is utilized to control different viral processes. Recent advances in our understanding of RNA-based viral regulation in select tombusvirids will be presented.

Download Full-text

ICTV Virus Taxonomy Profile: Paramyxoviridae

Journal of General Virology ◽

10.1099/jgv.0.001328 ◽

2019 ◽

Vol 100 (12) ◽

pp. 1593-1594 ◽

Cited By ~ 30

Author(s):

Bert Rima ◽

Anne Balkema-Buschmann ◽

William G. Dundon ◽

Paul Duprex ◽

Andrew Easton ◽

...

Keyword(s):

Measles Virus ◽

Rna Viruses ◽

Nipah Virus ◽

International Committee ◽

Mumps Virus ◽

Hendra Virus ◽

Virus Taxonomy ◽

Link Type ◽

Parainfluenza Viruses ◽

The Family

The family Paramyxoviridae consists of large enveloped RNA viruses infecting mammals, birds, reptiles and fish. Many paramyxoviruses are host-specific and several, such as measles virus, mumps virus, Nipah virus, Hendra virus and several parainfluenza viruses, are pathogenic for humans. The transmission of paramyxoviruses is horizontal, mainly through airborne routes; no vectors are known. This is a summary of the current International Committee on Taxonomy of Viruses (ICTV) Report on the family Paramyxoviridae. which is available at ictv.global/report/paramyxoviridae.

Download Full-text

Snapshots of a Non-Canonical RdRP in Action

Viruses ◽

10.3390/v13071260 ◽

2021 ◽

Vol 13 (7) ◽

pp. 1260

Author(s):

Diego S. Ferrero ◽

Michela Falqui ◽

Nuria Verdaguer

Keyword(s):

Metal Ion ◽

Rna Viruses ◽

Ion Binding ◽

Genome Replication ◽

Metal Ion Binding ◽

Insect Virus ◽

X Ray ◽

Template Strand ◽

Nucleotide Incorporation ◽

Elongation Process

RNA viruses typically encode their own RNA-dependent RNA polymerase (RdRP) to ensure genome replication and transcription. The closed “right hand” architecture of RdRPs encircles seven conserved structural motifs (A to G) that regulate the polymerization activity. The four palm motifs, arranged in the sequential order A to D, are common to all known template dependent polynucleotide polymerases, with motifs A and C containing the catalytic aspartic acid residues. Exceptions to this design have been reported in members of the Permutotetraviridae and Birnaviridae families of positive single stranded (+ss) and double-stranded (ds) RNA viruses, respectively. In these enzymes, motif C is located upstream of motif A, displaying a permuted C–A–B–D connectivity. Here we study the details of the replication elongation process in the non-canonical RdRP of the Thosea asigna virus (TaV), an insect virus from the Permutatetraviridae family. We report the X-ray structures of three replicative complexes of the TaV polymerase obtained with an RNA template-primer in the absence and in the presence of incoming rNTPs. The structures captured different replication events and allowed to define the critical interactions involved in: (i) the positioning of the acceptor base of the template strand, (ii) the positioning of the 3’-OH group of the primer nucleotide during RNA replication and (iii) the recognition and positioning of the incoming nucleotide. Structural comparisons unveiled a closure of the active site on the RNA template-primer binding, before rNTP entry. This conformational rearrangement that also includes the repositioning of the motif A aspartate for the catalytic reaction to take place is maintained on rNTP and metal ion binding and after nucleotide incorporation, before translocation.

Download Full-text

Recent Progress in Torovirus Molecular Biology

Viruses ◽

10.3390/v13030435 ◽

2021 ◽

Vol 13 (3) ◽

pp. 435

Author(s):

Makoto Ujike ◽

Fumihiro Taguchi

Keyword(s):

Molecular Biology ◽

Recent Progress ◽

Reverse Genetics ◽

Cultured Cells ◽

Host Adaptation ◽

Economic Losses ◽

New Family ◽

Enteric Diseases ◽

Long Time ◽

Medical Importance

Torovirus (ToV) has recently been classified into the new family Tobaniviridae, although it belonged to the Coronavirus (CoV) family historically. ToVs are associated with enteric diseases in animals and humans. In contrast to CoVs, which are recognised as pathogens of veterinary and medical importance, little attention has been paid to ToVs because their infections are usually asymptomatic or not severe; for a long time, only one equine ToV could be propagated in cultured cells. However, bovine ToVs, which predominantly cause diarrhoea in calves, have been detected worldwide, leading to economic losses. Porcine ToVs have also spread globally; although they have not caused serious economic losses, coinfections with other pathogens can exacerbate their symptoms. In addition, frequent inter- or intra-recombination among ToVs can increase pathogenesis or unpredicted host adaptation. These findings have highlighted the importance of ToVs as pathogens and the need for basic ToV research. Here, we review recent progress in the study of ToV molecular biology including reverse genetics, focusing on the similarities and differences between ToVs and CoVs.

Download Full-text

Structure and assembly of double-headed Sendai virus nucleocapsids

Communications Biology ◽

10.1038/s42003-021-02027-y ◽

2021 ◽

Vol 4 (1) ◽

Author(s):

Na Zhang ◽

Hong Shan ◽

Mingdong Liu ◽

Tianhao Li ◽

Rui Luo ◽

...

Keyword(s):

Electron Microscopy ◽

Measles Virus ◽

Sendai Virus ◽

Nipah Virus ◽

Mumps Virus ◽

Genome Replication ◽

Direct Visualization ◽

Closed State ◽

Cryo Electron Microscopy ◽

Negative Sense

AbstractParamyxoviruses, including the mumps virus, measles virus, Nipah virus and Sendai virus (SeV), have non-segmented single-stranded negative-sense RNA genomes which are encapsidated by nucleoproteins into helical nucleocapsids. Here, we reported a double-headed SeV nucleocapsid assembled in a tail-to-tail manner, and resolved its helical stems and clam-shaped joint at the respective resolutions of 2.9 and 3.9 Å, via cryo-electron microscopy. Our structures offer important insights into the mechanism of the helical polymerization, in particular via an unnoticed exchange of a N-terminal hole formed by three loops of nucleoproteins, and unveil the clam-shaped joint in a hyper-closed state for nucleocapsid dimerization. Direct visualization of the loop from the disordered C-terminal tail provides structural evidence that C-terminal tail is correlated to the curvature of nucleocapsid and links nucleocapsid condensation and genome replication and transcription with different assembly forms.

Download Full-text

An Efficient Weighted Residual Time Integration Family

International Journal of Structural Stability and Dynamics ◽

10.1142/s0219455421501066 ◽

2021 ◽

pp. 2150106

Author(s):

Mohammad Rezaiee-Pajand ◽

S. A. H. Esfehani ◽

H. Ehsanmanesh

Keyword(s):

Degrees Of Freedom ◽

Time Integration ◽

Nonlinear Damping ◽

Implicit Methods ◽

New Family ◽

Error Analyses ◽

Damping Behavior ◽

The Family ◽

New Strategy ◽

Time Integration Methods

A new family of time integration methods is formulated. The recommended technique is useful and robust for the loads with large variations and the systems with nonlinear damping behavior. It is also applicable for the structures with lots of degrees of freedom, and can handle general nonlinear dynamic systems. By comparing the presented scheme with the fourth-order Runge–Kutta and the Newmark algorithms, it is concluded that the new strategy is more stable. The authors’ formulations have good results on amplitude decay and dispersion error analyses. Moreover, the family orders of accuracy are [Formula: see text] and [Formula: see text] for even and odd values of [Formula: see text], respectively. Findings demonstrate the superiority of the new family compared to explicit and implicit methods and dissipative and non-dissipative algorithms.

Download Full-text