scholarly journals Effects of Defective Interfering Viruses on Virus Replication and Pathogenesis In Vitro and In Vivo

1991 ◽  
pp. 181-211 ◽  
Author(s):  
Laurent Roux ◽  
Anne E. Simon ◽  
John J. Holland
Keyword(s):  
Virus Replication

scholarly journals Towards plant resistance to viruses using protein-only RNase P

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Anthony Gobert ◽  
Yifat Quan ◽  
Mathilde Arrivé ◽  
Florent Waltz ◽  
Nathalie Da Silva ◽  
...  
Keyword(s):  
Mosaic Virus ◽  
Virus Replication ◽  
Yield Loss ◽  
Rnase P ◽  
Plant Viruses ◽  
Yellow Mosaic Virus ◽  
Resistance To Viruses ◽  
Target Plant

AbstractPlant viruses cause massive crop yield loss worldwide. Most plant viruses are RNA viruses, many of which contain a functional tRNA-like structure. RNase P has the enzymatic activity to catalyze the 5′ maturation of precursor tRNAs. It is also able to cleave tRNA-like structures. However, RNase P enzymes only accumulate in the nucleus, mitochondria, and chloroplasts rather than cytosol where virus replication takes place. Here, we report a biotechnology strategy based on the re-localization of plant protein-only RNase P to the cytosol (CytoRP) to target plant viruses tRNA-like structures and thus hamper virus replication. We demonstrate the cytosol localization of protein-only RNase P in Arabidopsis protoplasts. In addition, we provide in vitro evidences for CytoRP to cleave turnip yellow mosaic virus and oilseed rape mosaic virus. However, we observe varied in vivo results. The possible reasons have been discussed. Overall, the results provided here show the potential of using CytoRP for combating some plant viral diseases.

scholarly journals Dominant-Negative FADD Rescues the In Vivo Fitness of a Cytomegalovirus Lacking an Antiapoptotic Viral Gene

2007 ◽  
Vol 82 (5) ◽  
pp. 2056-2064 ◽  
Author(s):  
Luka Čičin-Šain ◽  
Zsolt Ruzsics ◽  
Juergen Podlech ◽  
Ivan Bubić ◽  
Carine Menard ◽  
...  
Keyword(s):  
Virus Replication ◽  
Control Cell ◽  
Death Receptor ◽  
Formal Proof ◽  
Dominant Negative ◽  
Viral Fitness ◽  
Viral Gene ◽  
Apoptosis Inhibition

ABSTRACT Genes that inhibit apoptosis have been described for many DNA viruses. Herpesviruses often contain even more than one gene to control cell death. Apoptosis inhibition by viral genes is postulated to contribute to viral fitness, although a formal proof is pending. To address this question, we studied the mouse cytomegalovirus (MCMV) protein M36, which binds to caspase-8 and blocks death receptor-induced apoptosis. The growth of MCMV recombinants lacking M36 (ΔM36) was attenuated in vitro and in vivo. In vitro, caspase inhibition by zVAD-fmk blocked apoptosis in ΔM36-infected macrophages and rescued the growth of the mutant. In vivo, ΔM36 infection foci in liver tissue contained significantly more apoptotic hepatocytes and Kupffer cells than did revertant virus foci, and apoptosis occurred during the early phase of virus replication prior to virion assembly. To further delineate the mode of M36 function, we replaced the M36 gene with a dominant-negative FADD (FADDDN) in an MCMV recombinant. FADDDN was expressed in cells infected with the recombinant and blocked the death-receptor pathway, replacing the antiapoptotic function of M36. Most importantly, FADDDN rescued ΔM36 virus replication, both in vitro and in vivo. These findings have identified the biological role of M36 and define apoptosis inhibition as a key determinant of viral fitness.

scholarly journals The recently identified flavivirus Bamaga virus is transmitted horizontally by Culex mosquitoes and interferes with West Nile virus replication in vitro and transmission in vivo

2018 ◽  
Vol 12 (10) ◽  
pp. e0006886 ◽  
Author(s):  
Agathe M. G. Colmant ◽  
Sonja Hall-Mendelin ◽  
Scott A. Ritchie ◽  
Helle Bielefeldt-Ohmann ◽  
Jessica J. Harrison ◽  
...  
Keyword(s):  
West Nile Virus ◽  
Virus Replication ◽  
West Nile ◽  
Culex Mosquitoes

scholarly journals Vector competence is strongly affected by a small deletion or point mutations in bluetongue virus

2019 ◽  
Vol 12 (1) ◽  
Author(s):  
René G. P. van Gennip ◽  
Barbara S. Drolet ◽  
Paula Rozo Lopez ◽  
Ashley J. C. Roost ◽  
Jan Boonstra ◽  
...  
Keyword(s):  
Virus Replication ◽  
Bluetongue Virus ◽  
Vector Competence ◽  
Outer Shell ◽  
Virus Propagation ◽  
Virus Growth ◽  
Shell Protein ◽  
Vector Borne

Abstract Background Transmission of vector-borne virus by insects is a complex mechanism consisting of many different processes; viremia in the host, uptake, infection and dissemination in the vector, and delivery of virus during blood-feeding leading to infection of the susceptible host. Bluetongue virus (BTV) is the prototype vector-borne orbivirus (family Reoviridae). BTV serotypes 1–24 (typical BTVs) are transmitted by competent biting Culicoides midges and replicate in mammalian (BSR) and midge (KC) cells. Previously, we showed that genome segment 10 (S10) encoding NS3/NS3a protein is required for virus propagation in midges. BTV serotypes 25–27 (atypical BTVs) do not replicate in KC cells. Several distinct BTV26 genome segments cause this so-called ‘differential virus replication’ in vitro. Methods Virus strains were generated using reverse genetics and their growth was examined in vitro. The midge feeding model has been developed to study infection, replication and disseminations of virus in vivo. A laboratory colony of C. sonorensis, a known competent BTV vector, was fed or injected with BTV variants and propagation in the midge was examined using PCR testing. Crossing of the midgut infection barrier was examined by separate testing of midge heads and bodies. Results A 100 nl blood meal containing ±105.3 TCID50/ml of BTV11 which corresponds to ±20 TCID50 infected 50% of fully engorged midges, and is named one Midge Alimentary Infective Dose (MAID50). BTV11 with a small in-frame deletion in S10 infected blood-fed midge midguts but virus release from the midgut into the haemolymph was blocked. BTV11 with S1[VP1] of BTV26 could be adapted to virus growth in KC cells, and contained mutations subdivided into ‘corrections’ of the chimeric genome constellation and mutations associated with adaptation to KC cells. In particular one amino acid mutation in outer shell protein VP2 overcomes differential virus replication in vitro and in vivo. Conclusion Small changes in NS3/NS3a or in the outer shell protein VP2 strongly affect virus propagation in midges and thus vector competence. Therefore, spread of disease by competent Culicoides midges can strongly differ for very closely related viruses.

scholarly journals Role of Initiating Nucleoside Triphosphate Concentrations in the Regulation of Influenza Virus Replication and Transcription

2008 ◽  
Vol 82 (14) ◽  
pp. 6902-6910 ◽  
Author(s):  
Frank T. Vreede ◽  
Hugh Gifford ◽  
George G. Brownlee
Keyword(s):  
Influenza Virus ◽  
Virus Replication ◽  
Influenza A ◽  
De Novo ◽  
Nucleoside Triphosphate ◽  
Ribonucleoprotein Complexes ◽  
Influenza Virus Replication ◽  
High Concentrations

ABSTRACT The mechanisms regulating the synthesis of mRNA, cRNA, and viral genomic RNA (vRNA) by the influenza A virus RNA-dependent RNA polymerase are not fully understood. Previous studies in our laboratory have shown that virion-derived viral ribonucleoprotein complexes synthesize both mRNA and cRNA in vitro and early in the infection cycle in vivo. Our continued studies showed that de novo synthesis of cRNA in vitro is more sensitive to the concentrations of ATP, CTP, and GTP than capped-primer-dependent synthesis of mRNA. Using rescued recombinant influenza A/WSN/33 viruses, we now demonstrate that the 3′-terminal sequence of the vRNA promoter dictates the requirement for a high nucleoside triphosphate (NTP) concentration during de novo-initiated replication to cRNA, whereas this is not the case for the extension of capped primers during transcription to mRNA. In contrast to some other viral polymerases, for which only the initiating NTP is required at high concentrations, influenza virus polymerase requires high concentrations of the first three NTPs. In addition, we show that base pair mutations in the vRNA promoter can lead to nontemplated dead-end mutations during replication to cRNA in vivo. Based on our observations, we propose a new model for the de novo initiation of influenza virus replication.

scholarly journals N-linked glycosylation plays an important role in budding of NA protein and virulence of influenza viruses

2020 ◽  
Author(s):  
Danqi Bao ◽  
Ruixue Xue ◽  
Min Zhang ◽  
Chenyang Lu ◽  
Tianxin Ma ◽  
...  
Keyword(s):  
Influenza Virus ◽  
Virus Replication ◽  
Immune Escape ◽  
H1n1 Influenza ◽  
Influenza Viruses ◽  
H1n1 Influenza Virus ◽  
Protein Loss ◽  
Knock Out

Neuraminidase (NA) has multiple functions in the life cycle of influenza virus, especially in the late stage of virus replication. Both of Hemagglutinin (HA) and NA are highly glycosylated proteins. N-linked glycosylation (NLG) of HA has been reported to contribute to immune escape and virulence of influenza viruses. However, the function of NLG of NA remains largely unclear. In this study, we found that NLG is critical for budding ability of NA. Tunicamycin treatment or NLG knock-out significantly inhibited the budding of NA. Further studies showed that the NLG knock-out caused attenuation of virus in vitro and in vivo. Notably the NLG at 219 position plays an important role in budding, replication, and virulence of H1N1 influenza virus. To explore the underlying mechanism, unfolded protein response (UPR) was determined in NLG knock-out NA overexpressed cells, which showed that the mutant NA was mainly located in ER, and the UPR markers BIP and p-eIF2α were upregulated, and XBP1 was downregulated. All the results indicated that NLG knock-out NA was stacked in ER and triggered UPR, which might shut down the budding process of NA. Overall, the study shed light on the function of NLG of NA in virus replication and budding. IMPORTANCE NA is a highly glycosylated protein. Nevertheless, how the NLG affects the function of NA protein remains largely unclear. In this study, we found that NLG plays important roles in budding and Neuraminidase activity of NA protein. Loss of NLG attenuated viral budding and replication. Especially the 219 NLG site mutation significantly attenuated the replication and virulence of H1N1 influenza virus in vitro and in vivo, which suggested that NLG of NA protein is a novel virulence marker for influenza viruses.

scholarly journals Identification of Common Deletions in the Spike Protein of Severe Acute Respiratory Syndrome Coronavirus 2

2020 ◽  
Vol 94 (17) ◽  
Author(s):  
Zhe Liu ◽  
Huanying Zheng ◽  
Huifang Lin ◽  
Mingyue Li ◽  
Runyu Yuan ◽  
...  
Keyword(s):  
Severe Acute Respiratory Syndrome ◽  
Virus Replication ◽  
Receptor Binding ◽  
Cleavage Site ◽  
Spike Protein ◽  
Clinical Samples ◽  
Receptor Binding Domain ◽  
Flanking Sequence

ABSTRACT Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a novel coronavirus first identified in December 2019. Notable features that make SARS-CoV-2 distinct from most other previously identified betacoronaviruses include a receptor binding domain and a unique insertion of 12 nucleotides or 4 amino acids (PRRA) at the S1/S2 boundary. In this study, we identified two deletion variants of SARS-CoV-2 that either directly affect the polybasic cleavage site itself (NSPRRAR) or a flanking sequence (QTQTN). These deletions were verified by multiple sequencing methods. In vitro results showed that the deletion of NSPRRAR likely does not affect virus replication in Vero and Vero-E6 cells; however, the deletion of QTQTN may restrict late-phase viral replication. The deletion of QTQTN was detected in 3 of 68 clinical samples and 12 of 24 in vitro-isolated viruses, while the deletion of NSPRRAR was identified in 3 in vitro-isolated viruses. Our data indicate that (i) there may be distinct selection pressures on SARS-CoV-2 replication or infection in vitro and in vivo; (ii) an efficient mechanism for deleting this region from the viral genome may exist, given that the deletion variant is commonly detected after two rounds of cell passage; and (iii) the PRRA insertion, which is unique to SARS-CoV-2, is not fixed during virus replication in vitro. These findings provide information to aid further investigation of SARS-CoV-2 infection mechanisms and a better understanding of the NSPRRAR deletion variant observed here. IMPORTANCE The spike protein determines the infectivity and host range of coronaviruses. Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has two unique features in its spike protein, the receptor binding domain and an insertion of 12 nucleotides at the S1/S2 boundary resulting in a furin-like cleavage site. Here, we identified two deletion variants of SARS-CoV-2 that either directly affect the furin-like cleavage site itself (NSPRRAR) or a flanking sequence (QTQTN), and we investigated these deletions in cell isolates and clinical samples. The absence of the polybasic cleavage site in SARS-CoV-2 did not affect virus replication in Vero or Vero-E6 cells. Our data indicate the PRRAR sequence and the flanking QTQTN sequence are not fixed in vitro; thus, there appears to be distinct selection pressures on SARS-CoV-2 sequences in vitro and in vivo. Further investigation of the mechanism of generating these deletion variants and their infectivity in different animal models would improve our understanding of the origin and evolution of this virus.

scholarly journals The rat cytomegalovirus homologue of parvoviral rep genes, r127, encodes a nuclear protein with single- and double-stranded DNA-binding activity that is dispensable for virus replication

2004 ◽  
Vol 85 (7) ◽  
pp. 2001-2013 ◽  
Author(s):  
Koen W. R. van Cleef ◽  
Wendy M. A. Scaf ◽  
Karen Maes ◽  
Suzanne J. F. Kaptein ◽  
Erik Beuken ◽  
...  
Keyword(s):  
Dna Binding ◽  
Virus Replication ◽  
Deletion Mutant ◽  
Nuclear Protein ◽  
Human Herpesvirus ◽  
Binding Activity ◽  
Dna Binding Activity ◽  
Double Stranded Dna

An intriguing feature of the rat cytomegalovirus (RCMV) genome is open reading frame (ORF) r127, which shows similarity to the rep genes of parvoviruses as well as the U94 genes of human herpesvirus type 6A (HHV-6A) and 6B (HHV-6B). Counterparts of these genes have not been found in other herpesviruses. Here, it is shown that the r127 gene is transcribed during the early and late phases of virus replication in vitro as an unspliced 1·1 kb transcript containing the complete r127 ORF. Transcripts of r127 were also detected in various organs of RCMV-infected rats at 1 week post-infection (p.i.), but only in the salivary gland at 4 months p.i. Using rabbit polyclonal antibodies raised against the r127-encoded protein (pr127), pr127 was found to be expressed as early as 12 h p.i. within the nuclei of RCMV-infected cells in vitro. Expression of pr127 was also observed within the nuclei of cells in various organs of RCMV-infected rats at 3 weeks p.i. Moreover, pr127 was demonstrated to bind single- as well as double-stranded DNA. Finally, an RCMV r127 deletion mutant (RCMVΔr127) was generated, in which the r127 ORF was disrupted. This deletion mutant, however, was shown to replicate with a similar efficiency as wild-type RCMV (wt RCMV), both in vitro and in vivo. Taken together, it is concluded that the RCMV r127 gene encodes a nuclear protein with single- and double-stranded DNA-binding activity that is dispensable for virus replication, not only in vitro, but also during the acute phase of infection in vivo.

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