scholarly journals Essential Role of the NV Protein of Novirhabdovirus for Pathogenicity in Rainbow Trout

2004 ◽  
Vol 78 (8) ◽  
pp. 4098-4107 ◽  
Author(s):  
Maria-Isabel Thoulouze ◽  
Edwige Bouguyon ◽  
Catherine Carpentier ◽  
Michel Brémont
Keyword(s):  
Cell Culture ◽  
Essential Role ◽  
Reverse Genetics ◽  
Fluorescent Protein ◽  
Normal Growth ◽  
Viral Hemorrhagic Septicemia Virus ◽  
Fish Cell ◽  
Reading Frame ◽  
Juvenile Trout

ABSTRACT Novirhabdovirus, infectious hematopoietic necrosis virus (IHNV), and viral hemorrhagic septicemia virus (VHSV) are fish rhabdoviruses that, in comparison to the other rhabdoviruses, contain an additional gene coding for a small nonvirion (NV) protein of unassigned function. A recombinant IHNV with the NV gene deleted but expressing the green fluorescent protein (rIHNV-ΔNV) has previously been shown to be efficiently recovered by reverse genetics (S. Biacchesi et al., J. Virol. 74:11247-11253, 2000). However, preliminary experiments suggested that the growth in cell culture of rIHNV-ΔNV was affected by the NV deletion. In the present study, we show that the growth in cell culture of rIHNV-ΔNV is indeed severely impaired but that a normal growth of rIHNV-ΔNV can be restored when NV is provided in trans by using fish cell clones constitutively expressing the NV protein. These results indicate that NV is a protein that has a crucial biological role for optimal replication of IHNV in cell culture. Although IHNV and VHSV NV proteins do not share any significant identity, we show here that both NV proteins play a similar role since a recombinant IHNV virus, rIHNV-NVVHSV, in which the IHNV NV open reading frame has been replaced by that of VHSV, was shown to replicate as well as the wild-type (wt) IHNV into fish cells. Finally, data provided by experimental fish infections with the various recombinant viruses strongly suggest an essential role of the NV protein for the pathogenicity of IHNV. Furthermore, we show that juvenile trout immunized with NV-knockout IHNV were protected against challenge with wt IHNV. That opens a new perspective for the development of IHNV attenuated live vaccines.

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 Potassium Uptake Modulates Staphylococcus aureus Metabolism

mSphere ◽  
2016 ◽  
Vol 1 (3) ◽  
Author(s):  
Casey M. Gries ◽  
Marat R. Sadykov ◽  
Logan L. Bulock ◽  
Sujata S. Chaudhari ◽  
Vinai C. Thomas ◽  
...  
Keyword(s):  
Carbon Flux ◽  
Fluorescent Protein ◽  
Critical Role ◽  
Normal Growth ◽  
Cytoplasmic Ph ◽  
Carbon Utilization ◽  
Content Type ◽  
Alkaline Conditions ◽  
Green Fluorescent

ABSTRACT Previous studies describing mechanisms for K+ uptake in S. aureus revealed that the Ktr-mediated K+ transport system was required for normal growth under alkaline conditions but not under neutral or acidic conditions. This work focuses on the effect of K+ uptake on S. aureus metabolism, including intracellular pH and carbon flux, and is the first to utilize a pH-dependent green fluorescent protein (GFP) to measure S. aureus cytoplasmic pH. These studies highlight the role of K+ uptake in supporting proton efflux under alkaline conditions and uncover a critical role for K+ uptake in establishing efficient carbon utilization. As a leading cause of community-associated and nosocomial infections, Staphylococcus aureus requires sophisticated mechanisms that function to maintain cellular homeostasis in response to its exposure to changing environmental conditions. The adaptation to stress and maintenance of homeostasis depend largely on membrane activity, including supporting electrochemical gradients and synthesis of ATP. This is largely achieved through potassium (K+) transport, which plays an essential role in maintaining chemiosmotic homeostasis, affects antimicrobial resistance, and contributes to fitness in vivo. Here, we report that S. aureus Ktr-mediated K+ uptake is necessary for maintaining cytoplasmic pH and the establishment of a proton motive force. Metabolite analyses revealed that K+ deficiency affects both metabolic and energy states of S. aureus by impairing oxidative phosphorylation and directing carbon flux toward substrate-level phosphorylation. Taken together, these results underline the importance of K+ uptake in maintaining essential components of S. aureus metabolism. IMPORTANCE Previous studies describing mechanisms for K+ uptake in S. aureus revealed that the Ktr-mediated K+ transport system was required for normal growth under alkaline conditions but not under neutral or acidic conditions. This work focuses on the effect of K+ uptake on S. aureus metabolism, including intracellular pH and carbon flux, and is the first to utilize a pH-dependent green fluorescent protein (GFP) to measure S. aureus cytoplasmic pH. These studies highlight the role of K+ uptake in supporting proton efflux under alkaline conditions and uncover a critical role for K+ uptake in establishing efficient carbon utilization.

scholarly journals The requirement of glycoprotein C (gC) for interindividual spread is a conserved function of gC for avian herpesviruses

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Widaliz Vega-Rodriguez ◽  
Huai Xu ◽  
Nagendraprabhu Ponnuraj ◽  
Haji Akbar ◽  
Taejoong Kim ◽  
...  
Keyword(s):  
Cell Culture ◽  
Virus Replication ◽  
Vaccine Strain ◽  
Fluorescent Protein ◽  
Natural Infection ◽  
Fluorescent Microscopy ◽  
Open Reading Frame ◽  
Reading Frame ◽  
Glycoprotein C ◽  
Essential Function

AbstractWe have formerly shown that glycoprotein C (gC) of Gallid alphaherpesvirus 2, better known as Marek’s disease (MD) alphaherpesvirus (MDV), is required for interindividual spread in chickens. Since gC is conserved within the Alphaherpesvirinae subfamily, we hypothesized gC was important for interindividual spread of other alphaherpesviruses. To test this hypothesis, we first generated a fluorescent protein tagged clone of Gallid alphaherpesvirus 3 MD vaccine strain 301B/1 to track virus replication in cell culture and chickens using fluorescent microscopy. Following validation of this system, we removed the open reading frame of 301B/1 gC from the genome and determined whether it was required for interindividual spread using experimental and natural infection studies. Interindividual spread of MD vaccine 301B/1 was abrogated by removal of 301B/1 gC. Rescuent virus in which 301B/1 gC was inserted back into the genome efficiently spread among chickens. To further study the conserved function of gC, we replaced 301B/1 gC with MDV gC and this virus also efficiently spread in chickens. These data suggest the essential function of alphaherpesvirus gC proteins is conserved and can be exploited during the generation of future vaccines against MD that affects the poultry industry worldwide.

scholarly journals Restriction of SARS-CoV-2 replication by targeting programmed −1 ribosomal frameshifting

2021 ◽  
Vol 118 (26) ◽  
pp. e2023051118
Author(s):  
Yu Sun ◽  
Laura Abriola ◽  
Rachel O. Niederer ◽  
Savannah F. Pedersen ◽  
Mia M. Alfajaro ◽  
...  
Keyword(s):  
Essential Role ◽  
Viral Gene Expression ◽  
Open Reading Frame ◽  
Viral Gene ◽  
Ribosomal Frameshift ◽  
Ribosomal Frameshifting ◽  
Reading Frame ◽  
Rna Pseudoknot ◽  
Proof Of Principle

Translation of open reading frame 1b (ORF1b) in severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) requires a programmed −1 ribosomal frameshift (−1 PRF) promoted by an RNA pseudoknot. The extent to which SARS-CoV-2 replication may be sensitive to changes in −1 PRF efficiency is currently unknown. Through an unbiased, reporter-based high-throughput compound screen, we identified merafloxacin, a fluoroquinolone antibacterial, as a −1 PRF inhibitor for SARS-CoV-2. Frameshift inhibition by merafloxacin is robust to mutations within the pseudoknot region and is similarly effective on −1 PRF of other betacoronaviruses. Consistent with the essential role of −1 PRF in viral gene expression, merafloxacin impedes SARS-CoV-2 replication in Vero E6 cells, thereby providing proof-of-principle for targeting −1 PRF as a plausible and effective antiviral strategy for SARS-CoV-2 and other coronaviruses.

scholarly journals Horizontal transfer of whole mitochondria restores tumorigenic potential in mitochondrial DNA-deficient cancer cells

eLife ◽  
2017 ◽  
Vol 6 ◽  
Author(s):  
Lan-Feng Dong ◽  
Jaromira Kovarova ◽  
Martina Bajzikova ◽  
Ayenachew Bezawork-Geleta ◽  
David Svec ◽  
...  
Keyword(s):  
Complex I ◽  
Essential Role ◽  
Fluorescent Protein ◽  
Mitochondrial Complex I ◽  
Red Fluorescent Protein ◽  
Mitochondrial Complex ◽  
Host Animal ◽  
Mt Dna ◽  
Tumorigenic Potential

Recently, we showed that generation of tumours in syngeneic mice by cells devoid of mitochondrial (mt) DNA (ρ0 cells) is linked to the acquisition of the host mtDNA. However, the mechanism of mtDNA movement between cells remains unresolved. To determine whether the transfer of mtDNA involves whole mitochondria, we injected B16ρ0 mouse melanoma cells into syngeneic C57BL/6Nsu9-DsRed2 mice that express red fluorescent protein in their mitochondria. We document that mtDNA is acquired by transfer of whole mitochondria from the host animal, leading to normalisation of mitochondrial respiration. Additionally, knockdown of key mitochondrial complex I (NDUFV1) and complex II (SDHC) subunits by shRNA in B16ρ0 cells abolished or significantly retarded their ability to form tumours. Collectively, these results show that intact mitochondria with their mtDNA payload are transferred in the developing tumour, and provide functional evidence for an essential role of oxidative phosphorylation in cancer.

scholarly journals Deletion of the SH gene from avian metapneumovirus has a greater impact on virus production and immunogenicity in turkeys than deletion of the G gene or M2-2 open reading frame

2008 ◽  
Vol 89 (2) ◽  
pp. 525-533 ◽  
Author(s):  
Roger Ling ◽  
Sabrina Sinkovic ◽  
Didier Toquin ◽  
Olivier Guionie ◽  
Nicolas Eterradossi ◽  
...  
Keyword(s):  
Cell Culture ◽  
Antibody Response ◽  
Antibody Production ◽  
Reverse Genetics ◽  
Open Reading Frame ◽  
Virus Growth ◽  
Reading Frame ◽  
Viral Yield

Subgroup A avian metapneumoviruses lacking either the SH or G gene or the M2-2 open reading frame were generated by using a reverse-genetics approach. The growth properties of these viruses were studied in vitro and in vivo in their natural host. Deletion of the SH gene alone resulted in the generation of a syncytial-plaque phenotype and this was reversed by the introduction of the SH gene from a subgroup B, but not a subgroup C, virus. Infected turkeys were assessed for antibody production and the presence of viral genomic RNA in tracheal swabs. The virus with a deleted SH gene also showed the greatest impairment of replication both in cell culture and in infected turkeys. This contrasts with the situation with other pneumoviruses in culture and in model animals, where deletion of the SH gene results in little effect upon viral yield and a good antibody response. Replication of the G- and M2-2-deleted viruses was impaired more severely in turkeys than in cell culture, with only some animals showing evidence of virus growth and antibody production. There was no correlation between virus replication and antibody response, suggesting that replication sites other than the trachea may be important for induction of antibody responses.

scholarly journals Murine norovirus virulence factor 1 (VF1) protein contributes to viral fitness during persistent infection

2019 ◽  
Author(s):  
Constantina Borg ◽  
Aminu S. Jahun ◽  
Lucy Thorne ◽  
Frédéric Sorgeloos ◽  
Dalan Bailey ◽  
...  
Keyword(s):  
Virulence Factor ◽  
Persistent Infection ◽  
Reverse Genetics ◽  
Viral Fitness ◽  
Murine Norovirus ◽  
Reading Frame ◽  
Viral Loads ◽  
Immunocompetent Mice

AbstractBackgroundMurine norovirus (MNV) is widely used as a model for studying norovirus biology. While MNV isolates vary in their pathogenesis, infection of immunocompetent mice mostly results in persistent infection. The ability of a virus to establish a persistent infection is dependent on its ability to subvert or avoid the host immune response. Previously, we described the identification and characterization of virulence factor 1 (VF1) in MNV, and established that it acts as an innate immune antagonist. Here, we explore the role of VF1 during persistent MNV infection in an immunocompetent host.MethodsUsing reverse genetics, we generated VF1-knockout MNV-3 that contained a single or a triple termination codon inserted in the VF1 open reading frame.ResultsVF1-knockout MNV-3 replicated to comparable levels to the wildtype virus in tissue culture. Comparative studies between MNV-3 and an acute MNV-1 strain show that MNV-3 VF1 exerts the same functions as MNV-1 VF1, but with reduced potency. Mice infected with VF1-knockout MNV-3 showed significantly reduced replication kinetics during the acute phase of the infection, but viral loads rapidly reached the levels seen in mice infected with wildtype virus after phenotypic restoration of VF1 expression. Infection with an MNV-3 mutant that had three termination codons inserted into VF1, in which reversion was suppressed, resulted in consistently lower replication throughout a three-month persistent infection in mice, suggesting a role for VF1 in viral fitness in vivo.ConclusionOur results indicate that VF1 expressed by a persistent strain of MNV also functions to antagonise the innate response to infection. We found that VF1 is not essential for viral persistence, but instead contributes to viral fitness in mice. These data fit with the hypothesis that noroviruses utilise multiple mechanisms to avoid and/or control the host response to infection and that VF1 is just one component of this.

On the nature of the circadian clock in mammals

1993 ◽  
Vol 264 (5) ◽  
pp. R821-R832 ◽  
Author(s):  
J. D. Miller
Keyword(s):  
Cell Culture ◽  
Functional Analysis ◽  
Circadian Clock ◽  
Suprachiasmatic Nucleus ◽  
Response Curve ◽  
Essential Role ◽  
Phase Response Curve ◽  
Coupling Model ◽  
Clock Mechanism

The evidence for the essential role of the suprachiasmatic nucleus (SCN) for the generation and maintenance of circadian rhythms in mammals is briefly reviewed. The pharmacology of the phase-response curve is considered and a new circadian measure, the phase-dose-response surface (PDRS), is introduced. The role of neurotransmission, ion fluxes, and non-neuronal cellular elements in the generation and maintenance of circadian rhythmicity is considered. Cell culture of the SCN is proposed as a tool for the functional analysis of clock mechanism. The critical contribution of coupling and synchronization of clock elements is reviewed in the context of the explicit predictions generated by a strong coupling model of the circadian clock. Finally, the nature of the circadian output signal is analyzed from a phylogenetic viewpoint.

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