scholarly journals Contribution of SARS-CoV-2 accessory proteins to viral pathogenicity in K18 hACE2 transgenic mice

2021 ◽  
Author(s):  
Jesus A. Silvas ◽  
Desarey Morales Vasquez ◽  
Jun-Gyu Park ◽  
Kevin Chiem ◽  
Anna Allué-Guardia ◽  
...  
Keyword(s):  
Severe Acute Respiratory Syndrome ◽  
Transgenic Mice ◽  
Reverse Genetics ◽  
Viral Pathogenesis ◽  
Open Reading Frame ◽  
World Health ◽  
Disease Outcome ◽  
Viral Pathogen ◽  
Reading Frame ◽  
Live Attenuated Vaccines

Severe Acute Respiratory Syndrome coronavirus 2 (SARS-CoV-2) is the viral pathogen responsible for the current coronavirus disease 2019 (COVID-19) pandemic. As of May 19 th 2021, John Hopkins University’s COVID-19 tracking platform has reported 3.3 million deaths associated with SARS-CoV-2 infection. Currently, the World Health Organization has granted emergency use listing (EUL) to six COVID-19 vaccines candidates. However much of the pathogenesis observed during SARS-CoV-2 infection remains elusive. To gain insight into the contribution of individual accessory open reading frame (ORF) proteins in SARS-CoV-2 pathogenesis, we used our recently described reverse genetics system approach to successfully engineer recombinant (r)SARS-CoV-2, where we individually removed viral 3a, 6, 7a, 7b, and 8 ORF proteins, and characterized these recombinant viruses in vitro and in vivo . Our results indicate differences in plaque morphology, with ORF deficient (ΔORF) viruses producing smaller plaques than those of the wild-type (rSARS-CoV-2/WT). However, growth kinetics of ΔORF viruses were like those of rSARS-CoV-2/WT. Interestingly, infection of K18 human angiotensin converting enzyme 2 (hACE2) transgenic mice with the ΔORF rSARS-CoV-2 identified ORF3a and ORF6 as the major contributors of viral pathogenesis, while ΔORF7a, ΔORF7b and ΔORF8 rSARS-CoV-2 induced comparable pathology to rSARS-CoV-2/WT. This study demonstrates the robustness of our reverse genetics system to generate rSARS-CoV-2 and the major role for ORF3a and ORF6 in viral pathogenesis, providing important information for the generation of attenuated forms of SARS-CoV-2 for their implementation as live-attenuated vaccines for the treatment of SARS-CoV-2 infection and associated COVID-19. IMPORTANCE Despite great efforts put forward worldwide to combat the current coronavirus disease 2019 (COVID-19) pandemic, Severe Acute Respiratory Syndrome coronavirus 2 (SARS-CoV-2) continues to be a human health and socioeconomic threat. Insights into the pathogenesis of SARS-CoV-2 and contribution of viral proteins to disease outcome remains elusive. Our study aims to determine the contribution of SARS-CoV-2 accessory open reading frame (ORF) proteins in viral pathogenesis and disease outcome, and develop a synergistic platform combining our robust reverse genetics system to generate recombinant (r)SARS-CoV-2 with a validated rodent model of infection and disease. We demonstrated that SARS-CoV-2 ORF3a and ORF6 contribute to lung pathology and ultimately disease outcome in K18 hACE2 transgenic mice, while ORF7a, ORF7b, and ORF8 have little impact on disease outcome. Moreover, our combinatory platform serves as the foundation to generate attenuated forms of the virus to develop live-attenuated vaccines for the treatment of SARS-CoV-2.

scholarly journals Contribution of SARS-CoV-2 accessory proteins to viral pathogenicity in K18 hACE2 transgenic mice

2021 ◽  
Author(s):  
Jesus Silvas ◽  
Desarey Morales-Vasquez ◽  
Jun-Gyu Park ◽  
Kevin Chiem ◽  
Jordi B. Torrelles ◽  
...  
Keyword(s):  
Severe Acute Respiratory Syndrome ◽  
Transgenic Mice ◽  
Reverse Genetics ◽  
Viral Pathogenesis ◽  
Open Reading Frame ◽  
Disease Outcome ◽  
Lung Pathology ◽  
Viral Pathogen ◽  
Reading Frame ◽  
Live Attenuated Vaccines

ABSTRACTSevere Acute Respiratory Syndrome coronavirus 2 (SARS-CoV-2) is the viral pathogen responsible for the current coronavirus disease 2019 (COVID-19) pandemic. To date, it is estimated that over 113 million individuals have been infected with SARS-CoV-2 and over 2.5 million human deaths have been recorded worldwide. Currently, three vaccines have been approved by the Food and Drug Administration for emergency use only. However much of the pathogenesis observed during SARS-CoV-2 infection remains elusive. To gain insight into the contribution of individual accessory open reading frame (ORF) proteins in SARS-CoV-2 pathogenesis, we used our recently described reverse genetics system approach to successfully engineer recombinant (r)SARS-CoV-2, where we individually removed viral 3a, 6, 7a, 7b, and 8 ORF proteins, and characterized these recombinant viruses in vitro and in vivo. Our results indicate differences in plaque morphology, with ORF deficient (ΔORF) viruses producing smaller plaques than those of the wild-type (rSARS-CoV-2/WT). However, growth kinetics of ΔORF viruses were like those of rSARS-CoV-2/WT. Interestingly, infection of K18 human angiotensin converting enzyme 2 (hACE2) transgenic mice with the ΔORF rSARS-CoV-2 identified ORF3a and ORF6 as the major contributors of viral pathogenesis, while ΔORF7a, ΔORF7b and ΔORF8 rSARS-CoV-2 induced comparable pathology to rSARS-CoV-2/WT. This study demonstrates the robustness of our reverse genetics system to generate rSARS-CoV-2 and the major role for ORF3a and ORF6 in viral pathogenesis, providing important information for the generation of attenuated forms of SARS-CoV-2 for their implementation as live-attenuated vaccines for the treatment of SARS-CoV-2 infection and associated COVID-19.IMPORTANCEDespite great efforts put forward worldwide to combat the current coronavirus disease 2019 (COVID-19) pandemic, Severe Acute Respiratory Syndrome coronavirus 2 (SARS-CoV-2) continues to be a human health and socioeconomic threat. Insights into the pathogenesis of SARS-CoV-2 and contribution of viral proteins to disease outcome remains elusive. Our study aims to determine the contribution of SARS-CoV-2 accessory open reading frame (ORF) proteins in viral pathogenesis and disease outcome, and develop a synergistic platform combining our robust reverse genetics system to generate recombinant (r)SARS-CoV-2 with a validated rodent model of infection and disease. We demonstrated that SARS-CoV-2 ORF3a and ORF6 contribute to lung pathology and ultimately disease outcome in K18 hACE2 transgenic mice, while ORF7a, ORF7b, and ORF8 have little impact on disease outcome. Moreover, our combinatory platform serves as the foundation to generate attenuated forms of the virus to develop live-attenuated vaccines for the treatment of SARS-CoV-2.

scholarly journals Mapping of Transcription Termination within the S Segment of SFTS Phlebovirus Facilitated Generation of NSs Deletant Viruses

2017 ◽  
Vol 91 (16) ◽  
Author(s):  
Benjamin Brennan ◽  
Veronica V. Rezelj ◽  
Richard M. Elliott
Keyword(s):  
Virus Infection ◽  
Reverse Genetics ◽  
Fluorescent Protein ◽  
Nonstructural Protein ◽  
Transcription Termination ◽  
Severe Disease ◽  
Open Reading Frame ◽  
Coding Region ◽  
Reading Frame ◽  
Green Fluorescent

ABSTRACT SFTS phlebovirus (SFTSV) is an emerging tick-borne bunyavirus that was first reported in China in 2009. Here we report the generation of a recombinant SFTSV (rHB29NSsKO) that cannot express the viral nonstructural protein (NSs) upon infection of cells in culture. We show that rHB29NSsKO replication kinetics are greater in interferon (IFN)-incompetent cells and that the virus is unable to suppress IFN induced in response to viral replication. The data confirm for the first time in the context of virus infection that NSs acts as a virally encoded IFN antagonist and that NSs is dispensable for virus replication. Using 3′ rapid amplification of cDNA ends (RACE), we mapped the 3′ end of the N and NSs mRNAs, showing that the mRNAs terminate within the coding region of the opposite open reading frame. We show that the 3′ end of the N mRNA terminates upstream of a 5′-GCCAGCC-3′ motif present in the viral genomic RNA. With this knowledge, and using virus-like particles, we could demonstrate that the last 36 nucleotides of the NSs open reading frame (ORF) were needed to ensure the efficient termination of the N mRNA and were required for recombinant virus rescue. We demonstrate that it is possible to recover viruses lacking NSs (expressing just a 12-amino-acid NSs peptide or encoding enhanced green fluorescent protein [eGFP]) or an NSs-eGFP fusion protein in the NSs locus. This opens the possibility for further studies of NSs and potentially the design of attenuated viruses for vaccination studies. IMPORTANCE SFTS phlebovirus (SFTSV) and related tick-borne viruses have emerged globally since 2009. SFTSV has been shown to cause severe disease in humans. For bunyaviruses, it has been well documented that the nonstructural protein (NSs) enables the virus to counteract the human innate antiviral defenses and that NSs is one of the major determinants of virulence in infection. Therefore, the use of reverse genetics systems to engineer viruses lacking NSs is an attractive strategy to rationally attenuate bunyaviruses. Here we report the generation of several recombinant SFTS viruses that cannot express the NSs protein or have the NSs open reading frame replaced with a reporter gene. These viruses cannot antagonize the mammalian interferon (IFN) response mounted to virus infection. The generation of NSs-lacking viruses was achieved by mapping the transcriptional termination of two S-segment-derived subgenomic mRNAs, which revealed that transcription termination occurs upstream of a 5′-GCCAGCC-3′ motif present in the virus genomic S RNA.

scholarly journals Insights into SARS-CoV-2, the Coronavirus Underlying COVID-19: Recent Genomic Data and the Development of Reverse Genetics Systems

2020 ◽  
Vol 101 (10) ◽  
pp. 1021-1024
Author(s):  
Severino Jefferson Ribeiro da Silva ◽  
Renata Pessôa Germano Mendes ◽  
Caroline Targino Alves da Silva ◽  
Alessio Lorusso ◽  
Alain Kohl ◽  
...  
Keyword(s):  
Severe Acute Respiratory Syndrome ◽  
Reverse Genetics ◽  
World Health ◽  
Close Relative ◽  
Sequence Information ◽  
Recombinant Viruses ◽  
Protein Functions ◽  
Genomic Studies ◽  
Health Organization

The emergence and rapid worldwide spread of a novel pandemic of acute respiratory disease – eventually named coronavirus disease 2019 (COVID-19) by the World Health Organization (WHO) – across the human population has raised great concerns. It prompted a mobilization around the globe to study the underlying pathogen, a close relative of severe acute respiratory syndrome coronavirus (SARS-CoV) called severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Numerous genome sequences of SARS-CoV-2 are now available and in-depth analyses are advancing. These will allow detailed characterization of sequence and protein functions, including comparative studies. Care should be taken when inferring function from sequence information alone, and reverse genetics systems can be used to unequivocally identify key features. For example, the molecular markers of virulence, host range and transmissibility of SARS-CoV-2 can be compared to those of related viruses in order to shed light on the biology of this emerging pathogen. Here, we summarize some recent insights from genomic studies and strategies for reverse genetics systems to generate recombinant viruses, which will be useful to investigate viral genome properties and evolution.

scholarly journals Targeted Inactivation of a Tobacco Intron–containing Open Reading Frame Reveals a Novel Chloroplast-encoded Photosystem I–related Gene

1997 ◽  
Vol 139 (1) ◽  
pp. 95-102 ◽  
Author(s):  
Stephanie Ruf ◽  
Hans Kössel ◽  
Ralph Bock
Keyword(s):  
Photosystem I ◽  
Reverse Genetics ◽  
Higher Plants ◽  
Single Copy ◽  
Open Reading Frame ◽  
Higher Plant ◽  
Reading Frame ◽  
Chloroplast Genomes ◽  
Plant Chloroplast ◽  
Targeted Inactivation

The chloroplast genome of all higher plants encodes, in its large single-copy region, a conserved open reading frame of unknown function (ycf3), which is split by two group II introns and undergoes RNA editing in monocotyledonous plants. To elucidate the function of ycf3 we have deleted the reading frame from the tobacco plastid genome by biolistic transformation. We show here that homoplasmic Δycf3 plants display a photosynthetically incompetent phenotype. Molecular analyses indicate that this phenotype is not due to a defect in any of the general functions of the plastid genetic apparatus. Instead, the mutant plants specifically lack detectable amounts of all photosystem I (PSI) subunits analyzed. In contrast, at least under low light conditions, photosystem II subunits are still present and assemble into a physiologically active complex. Faithful transcription of photosystem I genes as well as correct mRNA processing and efficient transcript loading with ribosomes in the Δycf3 plants suggest a posttranslational cause of the PSI-defective phenotype. We therefore propose that ycf3 encodes an essential protein for the assembly and/or stability of functional PSI units. This study provides a first example for the suitability of reverse genetics approaches to complete our picture of the coding capacity of higher plant chloroplast genomes.

scholarly journals Rescue of Rabies Virus from Cloned cDNA and Identification of the Pathogenicity-Related Gene: Glycoprotein Gene Is Associated with Virulence for Adult Mice

2001 ◽  
Vol 75 (19) ◽  
pp. 9121-9128 ◽  
Author(s):  
Naoto Ito ◽  
Mutsuyo Takayama ◽  
Kentaro Yamada ◽  
Makoto Sugiyama ◽  
Nobuyuki Minamoto
Keyword(s):  
Rabies Virus ◽  
Reverse Genetics ◽  
Biological Properties ◽  
Open Reading Frame ◽  
Chimeric Virus ◽  
Viral Gene ◽  
Reading Frame ◽  
Intracerebral Inoculation ◽  
Glycoprotein Gene ◽  
Adult Mice

ABSTRACT In order to identify the viral gene related to the pathogenicity of rabies virus, we tried to establish a reverse genetics system of the attenuated RC-HL strain, which causes nonlethal infection in adult mice after intracerebral inoculation. A full-length genome plasmid encoding the complete antigenomic cDNA of the RC-HL strain and helper plasmids containing cDNAs of the complete open reading frame of the N, P, and L genes, respectively, were constructed. After transfection of these plasmids into BHK-21 cells infected with the T7 RNA polymerase-expressing vaccinia virus, infectious rabies virus with almost the same biological properties as those of the wild-type RC-HL strain was rescued. Using this reverse genetics system of the RC-HL strain, we generated a chimeric virus with the open reading frame of the glycoprotein gene from the parent Nishigahara strain, which kills adult mice after intracerebral inoculation, in the background of the RC-HL genome. Since the chimeric virus killed adult mice following intracerebral inoculation, it became evident that the open reading frame of the glycoprotein gene is related to the pathogenicity of the Nishigahara strain for adult mice.

scholarly journals Processing of Open Reading Frame 1a Replicase Proteins nsp7 to nsp10 in Murine Hepatitis Virus Strain A59 Replication

2007 ◽  
Vol 81 (19) ◽  
pp. 10280-10291 ◽  
Author(s):  
Damon J. Deming ◽  
Rachel L. Graham ◽  
Mark R. Denison ◽  
Ralph S. Baric
Keyword(s):  
Reverse Genetics ◽  
Hepatitis Virus ◽  
Proteolytic Processing ◽  
Mutant Virus ◽  
Open Reading Frame ◽  
Cleavage Sites ◽  
Nonstructural Proteins ◽  
Murine Hepatitis Virus ◽  
Reading Frame ◽  
Genes Encoding

ABSTRACT Coronaviruses express open reading frame 1a (ORF1a) and ORF1b polyproteins from which 16 nonstructural proteins (nsp) are derived. The highly conserved region at the carboxy terminus of ORF1a is processed by the nsp5 proteinase (Mpro) into mature products, including nsp7, nsp8, nsp9, and nsp10, proteins with predicted or identified activities involved in RNA synthesis. Although continuous translation and proteolytic processing of ORF1ab by Mpro is required for replication, it is unknown whether specific cleavage events within the polyprotein are dispensable. We determined the requirement for the nsp7 to nsp10 proteins and their processing during murine hepatitis virus (MHV) replication. Through use of an MHV reverse genetics system, in-frame deletions of the coding sequences for nsp7 to nsp10, or ablation of their flanking Mpro cleavage sites, were made and the effects upon replication were determined. Viable viruses were characterized by analysis of Mpro processing, RNA transcription, and growth fitness. Deletion of any of the regions encoding nsp7 to nsp10 was lethal. Disruption of the cleavage sites was lethal with the exception of that of the nsp9-nsp10 site, which resulted in a mutant virus with attenuated replication. Passage of the attenuated nsp9-nsp10 cleavage mutant increased fitness to near-wild-type kinetics without reversion to a virus capable of processing nsp9-nsp10. We also confirmed the presence of a second cleavage site between nsp7 and nsp8. In order to determine whether a distinct function could be attributed to preprocessed forms of the polyprotein, including nsp7 to nsp10, the genes encoding nsp7 and nsp8 were rearranged. The mutant virus was not viable, suggesting that the uncleaved protein may be essential for replication or proteolytic processing.

scholarly journals Coding-Complete Genome Sequences of Alpha and Delta SARS-CoV-2 Variants from Kamphaeng Phet Province, Thailand, from May to July 2021

2021 ◽  
Vol 10 (48) ◽  
Author(s):  
Kanyarat Phutthasophit ◽  
Darunee Buddhari ◽  
Piyawan Chinnawirotpisan ◽  
Khajohn Joonlasak ◽  
Wudtichai Manasatienkij ◽  
...  
Keyword(s):  
Severe Acute Respiratory Syndrome ◽  
Complete Genome ◽  
Open Reading Frame ◽  
Genome Sequences ◽  
Nonsense Mutations ◽  
Reading Frame ◽  
Alpha Variant

We report coding-complete genome sequences of 44 severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) strains of the alpha and delta variants identified from patients in Kamphaeng Phet, Thailand. Two nonsense mutations in open reading frame 3a (ORF3a) (G254*) and ORF8 (K68*) were found in the alpha variant sequences. Two lineages of the delta variant, B.1.617.2 and AY.30, were found.

scholarly journals In-Frame 12-Nucleotide Deletion within Open Reading Frame 3a in a SARS-CoV-2 Strain Isolated from a Patient Hospitalized with COVID-19

2021 ◽  
Vol 10 (8) ◽  
Author(s):  
John A. Lednicky ◽  
Kartikeya Cherabuddi ◽  
Massimiliano S. Tagliamonte ◽  
Maha A. Elbadry ◽  
Kuttichantran Subramaniam ◽  
...  
Keyword(s):  
Severe Acute Respiratory Syndrome ◽  
Open Reading Frame ◽  
Reading Frame ◽  
Nucleotide Deletion

ABSTRACT Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) strain UF-8, with an in-frame 12-nucleotide deletion within open reading frame 3a (ORF3a), was isolated from a 78-year-old COVID-19 patient in March 2020.

scholarly journals Reverse Genetics Identifies the Product of Open Reading Frame 4 as an Essential Particle Assembly Factor of Nyamanini Virus

2013 ◽  
Vol 87 (14) ◽  
pp. 8257-8260 ◽  
Author(s):  
M. Herrel ◽  
L. Haag ◽  
J. Nilsson ◽  
P. Staeheli ◽  
U. Schneider
Keyword(s):  
Reverse Genetics ◽  
Open Reading Frame ◽  
Particle Assembly ◽  
Reading Frame ◽  
Assembly Factor

scholarly journals Efficient Transmission of Two Different Sheep Scrapie Isolates in Transgenic Mice Expressing the Ovine PrP Gene

2001 ◽  
Vol 75 (11) ◽  
pp. 5328-5334 ◽  
Author(s):  
Carole Crozet ◽  
Frederic Flamant ◽  
Anna Bencsik ◽  
Denise Aubert ◽  
Jacques Samarut ◽  
...  
Keyword(s):  
Transgenic Mice ◽  
Prion Protein ◽  
Neuron Specific Enolase ◽  
Open Reading Frame ◽  
Protein Accumulation ◽  
Spongiform Encephalopathies ◽  
Reading Frame ◽  
Neurological Signs ◽  
Prp Gene ◽  
Sheep Scrapie

ABSTRACT We produced transgenic mice expressing the sheep prion protein to obtain a sensitive model for sheep spongiform encephalopathies (scrapie). The complete open reading frame, with alanine, arginine, and glutamine at susceptibility codons 136, 154, and 171, respectively, was inserted downstream from the neuron-specific enolase promoter. A mouse line, Tg(OvPrP4), devoid of the murine PrP gene, was obtained by crossing with PrP knockout mice. Tg(OvPrP4) mice were shown to selectively express sheep PrP in their brains, as demonstrated in mRNA and protein analysis. We showed that these mice were susceptible to infection by sheep scrapie following intracerebral inoculation with two natural sheep scrapie isolates, as demonstrated not only by the occurrence of neurological signs but also by the presence of the spongiform changes and abnormal prion protein accumulation in their brains. Mean times to death of 238 and 290 days were observed with these isolates, but the clinical course of the disease was strikingly different in the two cases. One isolate led to a very early onset of neurological signs which could last for prolonged periods before death. Independently of the incubation periods, some of the mice inoculated with this isolate showed low or undetectable levels of PrPsc, as detected by both Western blotting and immunohistochemistry. The development of experimental scrapie in these mice following inoculation of the scrapie infectious agent further confirms that neuronal expression of the PrP open reading frame alone is sufficient to mediate susceptibility to spongiform encephalopathies. More importantly, these mice provide a new and promising tool for studying the infectious agents in sheep spongiform encephalopathies.

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