scholarly journals Systemic Polyomavirus Genome Increase and Dissemination of Capsid-Defective Genomes in Mammary Gland Tumor-Bearing Mice

2000 ◽  
Vol 74 (15) ◽  
pp. 6975-6983 ◽  
Author(s):  
Julie J. Wirth ◽  
Li Chen ◽  
Michele M. Fluck
Keyword(s):  
Mammary Gland ◽  
Viral Genome ◽  
Wild Type Strain ◽  
Neonatal Infection ◽  
Wild Type ◽  
Coding Region ◽  
Live Virus ◽  
Viral Genomes ◽  
Mammary Gland Tumors ◽  
Low Levels

ABSTRACT BALB/c mice that developed tumors 7 to 8 months following neonatal infection by polyomavirus (PYV) wild-type strain A2 were characterized with respect to the abundance and integrity of the viral genome in the tumors and in 12 nontumorous organs. These patterns were compared to those found in tumor-free mice infected in parallel. Six mice were analyzed in detail including four sibling females with mammary gland tumors. In four of five mammary gland tumors, the viral genome had undergone a unique deletion and/or rearrangement. Three tumor-resident genomes with an apparently intact large T coding region were present in abundant levels in an unintegrated state. Two of these had undergone deletions and rearrangements involving the capsid genes and therefore lacked the capacity to produce live virus. In the comparative organ survey, the tumors harboring replication-competent genomes contained by far the highest levels of genomes of any tissue. However, the levels of PYV genomes in other organs were elevated by up to 1 to 2 orders of magnitude compared to those detected in the same organs of tumor-free mice. The genomes found in the nontumorous organs had the same rearrangements as the genomes residing in the tumors. The original wild-type genome was detected at low levels in a few organs, particularly in the kidneys. The data indicate that a systemic increase in the level of viral genomes occurred in conjunction with the induction of tumors by PYV. The results suggest two novel hypotheses: (i) that genomes may spread from the tumors to the usual PYV target tissues and (ii) that this dissemination may take place in the absence of capsids, providing an important path for a virus to escape from the immune response. This situation may offer a useful model for the spread of HPV accompanying HPV-induced oncogenesis.

scholarly journals Complementation of the yeast deletion mutant DeltaNCE103 by members of the beta class of carbonic anhydrases is dependent on carbonic anhydrase activity rather than on antioxidant activity

2004 ◽  
Vol 379 (3) ◽  
pp. 609-615 ◽  
Author(s):  
Daniel CLARK ◽  
Roger S. ROWLETT ◽  
John R. COLEMAN ◽  
Daniel F. KLESSIG
Keyword(s):  
Antioxidant Activity ◽  
Type Strain ◽  
Deletion Mutant ◽  
Wild Type Strain ◽  
Carbonic Anhydrase Activity ◽  
Site Directed Mutagenesis ◽  
Carbonic Anhydrases ◽  
Wild Type ◽  
Tobacco Chloroplast ◽  
Low Levels

In recent years, members of the β class of CAs (carbonic anhydrases) have been shown to complement ΔNCE103, a yeast strain unable to grow under aerobic conditions. The activity required for complementation of ΔNCE103 by tobacco chloroplast CA was studied by site-directed mutagenesis. E196A (Glu196→Ala), a mutated tobacco CA with low levels of CA activity, complemented ΔNCE103. To determine whether restoration of ΔNCE103 was due to residual levels of CA activity or whether it was related to previously proposed antioxidant activity of CAs [Götz, Gnann and Zimmermann (1999) Yeast 15, 855–864], additional complementation analysis was performed using human CAII, an α CA structurally unrelated to the β class of CAs to which the tobacco protein belongs. Human CAII complemented ΔNCE103, strongly arguing that CA activity is responsible for the complementation of ΔNCE103. Consistent with this conclusion, recombinant NCE103 synthesized in Escherichia coli shows CA activity, and ΔNCE103 expressing the tobacco chloroplast CA exhibits the same sensitivity to H2O2 as the wild-type strain.

scholarly journals Characterisation of SARS-CoV-2 Lentiviral Pseudotypes and Correlation between Pseudotype-Based Neutralisation Assays and Live Virus-Based Micro Neutralisation Assays

Viruses ◽  
2020 ◽  
Vol 12 (9) ◽  
pp. 1011 ◽  
Author(s):  
Inesa Hyseni ◽  
Eleonora Molesti ◽  
Linda Benincasa ◽  
Pietro Piu ◽  
Elisa Casa ◽  
...  
Keyword(s):  
Wild Type Strain ◽  
Spike Protein ◽  
Wild Type Virus ◽  
Wild Type ◽  
Serum Samples ◽  
Live Virus ◽  
Rapid Spread ◽  
Recent Outbreak ◽  
Human Sera ◽  
Novel Coronavirus

The recent outbreak of a novel Coronavirus (SARS-CoV-2) and its rapid spread across the continents has generated an urgent need for assays to detect the neutralising activity of human sera or human monoclonal antibodies against SARS-CoV-2 spike protein and to evaluate the serological immunity in humans. Since the accessibility of live virus microneutralisation (MN) assays with SARS-CoV-2 is limited and requires enhanced bio-containment, the approach based on “pseudotyping” can be considered a useful complement to other serological assays. After fully characterising lentiviral pseudotypes bearing the SARS-CoV-2 spike protein, we employed them in pseudotype-based neutralisation assays in order to profile the neutralising activity of human serum samples from an Italian sero-epidemiological study. The results obtained with pseudotype-based neutralisation assays mirrored those obtained when the same panel of sera was tested against the wild type virus, showing an evident convergence of the pseudotype-based neutralisation and MN results. The overall results lead to the conclusion that the pseudotype-based neutralisation assay is a valid alternative to using the wild-type strain, and although this system needs to be optimised and standardised, it can not only complement the classical serological methods, but also allows serological assessments to be made when other methods cannot be employed, especially in a human pandemic context.

scholarly journals Persistence of an Oncogenic Papillomavirus Genome RequirescisElements from the Viral Transcriptional Enhancer

mBio ◽  
2017 ◽  
Vol 8 (6) ◽  
Author(s):  
Koenraad Van Doorslaer ◽  
Dan Chen ◽  
Sandra Chapman ◽  
Jameela Khan ◽  
Alison A. McBride
Keyword(s):  
Persistent Infection ◽  
Viral Genome ◽  
Replication Origin ◽  
Human Papillomaviruses ◽  
Wild Type ◽  
Transcriptional Enhancer ◽  
Complementation Assay ◽  
Viral Genomes ◽  
Stable Maintenance

ABSTRACTHuman papillomavirus (HPV) genomes are replicated and maintained as extrachromosomal plasmids during persistent infection. The viral E2 proteins are thought to promote stable maintenance replication by tethering the viral DNA to host chromatin. However, this has been very difficult to prove genetically, as the E2 protein is involved in transcriptional regulation and initiation of replication, as well as its assumed role in genome maintenance. This makes mutational analysis of viraltransfactors andciselements in the background of the viral genome problematic and difficult to interpret. To circumvent this problem, we have developed a complementation assay in which the complete wild-type HPV18 genome is transfected into primary human keratinocytes along with subgenomic or mutated replicons that contain the minimal replication origin. The wild-type genome provides the E1 and E2 proteins intrans, allowing us to determine additionalciselements that are required for long-term replication and partitioning of the replicon. We found that, in addition to the core replication origin (and the three E2 binding sites located therein), additional sequences from the transcriptional enhancer portion of the URR (upstream regulatory region) are required incisfor long-term genome replication.IMPORTANCEHuman papillomaviruses infect cutaneous and mucosal epithelial cells of the host, and this results in very-long-lived, persistent infection. The viral genomes are small, circular, double-stranded DNA molecules that replicate extrachromosomally in concert with cellular DNA. This replication strategy requires that the virus has a robust mechanism to partition and retain the viral genomes in dividing cells. This has been difficult to study, because viral transcription, replication, and partitioning are regulated by the same viral proteins and involve overlapping elements in the viral genome. We developed a complementation assay that allows us to separate these functions and define the elements required for long-term replication and stable maintenance replication of the HPV genome. This has important implications, as disruption of viral maintenance replication can eliminate viral genomes from infected cells, thus curing persistent HPV infection.

scholarly journals Restriction of Foamy Viruses by APOBEC Cytidine Deaminases

2006 ◽  
Vol 80 (2) ◽  
pp. 605-614 ◽  
Author(s):  
Frédéric Delebecque ◽  
Rodolphe Suspène ◽  
Sara Calattini ◽  
Nicoletta Casartelli ◽  
Ali Saïb ◽  
...  
Keyword(s):  
Antiviral Activity ◽  
Apolipoprotein B ◽  
Viral Genome ◽  
Potent Inhibitor ◽  
Wild Type ◽  
Cytidine Deaminases ◽  
Foamy Viruses ◽  
Low Levels ◽  
Apobec Family ◽  
Human Apobec3g

ABSTRACT Foamy viruses (FVs) are nonpathogenic retroviruses infecting many species of mammals, notably primates, cattle, and cats. We have examined whether members of the apolipoprotein B-editing catalytic polypeptide-like subunit (APOBEC) family of antiviral cytidine deaminases restrict replication of simian FV. We show that human APOBEC3G is a potent inhibitor of FV infectivity in cell culture experiments. This antiviral activity is associated with cytidine editing of the viral genome. Both molecular FV clones and primary uncloned viruses were susceptible to APOBEC3G, and viral infectivity was also inhibited by murine and simian APOBEC3G homologues, as well as by human APOBEC3F. Wild-type and bet-deleted viruses were similarly sensitive to this antiviral activity, suggesting that Bet does not significantly counteract APOBEC proteins. Moreover, we did not detect FV sequences that may have been targeted by APOBEC in naturally infected macaques, but we observed a few G-to-A substitutions in humans that have been accidentally contaminated by simian FV. In infected hosts, the persistence strategy employed by FV might be based on low levels of replication, as well as avoidance of cells expressing large amounts of active cytidine deaminases.

Binding of a 30-kDa protein to the pyruvate kinase gene of Neurospora crassa

1988 ◽  
Vol 66 (9) ◽  
pp. 958-966
Author(s):  
Medhavinee R. Devchand ◽  
M. Kapoor
Keyword(s):  
Neurospora Crassa ◽  
Pyruvate Kinase ◽  
Wild Type Strain ◽  
Wild Type ◽  
Coding Region ◽  
Dna Complexes ◽  
Kinase Gene ◽  
Total N ◽  
Flanking Region ◽  
Kinetics Of

Extracts of a wild-type strain of Neurospora crassa, electrophoresed on SDS–polyacrylamide gels and electroblotted onto nitrocellulose sheets, were hybridized to an end-labelled pyruvate kinase (PK) gene fragment containing the 5′ noncoding sequence and a large part of the coding region. A 30-kDa protein was found to bind strongly to the PK gene DNA, while binding weakly to plasmid pUC12 DNA and to total N. crassa DNA. Probing of blots with individual restriction fragments derived from the PK gene showed that the protein binding occurred primarily to the 5′ noncoding region. Nonspecific DNA from pUC12, PK gene DNA from the recombinant plasmid pNP460 (pUC12 containing a 1.8-kilobase EcoRI insert of the PK gene DNA), along with a 0.7-kilobase EcoRI-AccI restriction fragment containing the 5′ flanking region, were used in filter-binding experiments to analyze the kinetics of binding. Formation of protein–DNA complexes was demonstrated by monitoring the electrophoretic mobility of this fragment on nondenaturing gels.

Growth properties of afolAnull mutant ofEscherichia coliK12

1999 ◽  
Vol 45 (3) ◽  
pp. 191-200 ◽  
Author(s):  
Muriel B Herrington ◽  
Neema T Chirwa
Keyword(s):  
Escherichia Coli ◽  
Dihydrofolate Reductase ◽  
Type Strain ◽  
Wild Type Strain ◽  
De Novo ◽  
Kanamycin Resistance ◽  
Wild Type ◽  
Coding Region ◽  
Growth Properties ◽  
One Carbon Metabolism

In Escherichia coli, dihydrofolate reductase is required for both the de novo synthesis of tetrahydrofolate and the recycling of dihydrofolate produced during the synthesis of thymidylate. The coding region of the dihydrofolate reductase gene, folA, was replaced with a kanamycin resistance determinant. Unlike earlier deletions, this mutation did not disrupt flanking genes. When the mutation was transferred into a wild-type strain and a thymidine- (thy) requiring strain, the resulting strains were viable but slow growing on rich medium. Both synthesized less folate than their parents, as judged by the incorporation of radioactive para-aminobenzoic acid. The derivative of the wild-type strain did not grow on any defined minimal media tested. In contrast, the derivative of the thy-requiring strain grew slowly on minimal medium with thy but exhibited auxotrophies on some combinations of supplements. These results suggest that when folates are limited, they can be distributed appropriately to folate-dependent biosynthetic reactions only under some conditions. Key words: dihydrofolate reductase, Escherichia coli, biosynthesis, folates, one-carbon metabolism.

scholarly journals Use of Transposon Tn5367 Mutagenesis and a Nitroimidazopyran-Based Selection System To Demonstrate a Requirement for fbiA and fbiB in Coenzyme F420 Biosynthesis by Mycobacterium bovis BCG

2001 ◽  
Vol 183 (24) ◽  
pp. 7058-7066 ◽  
Author(s):  
Kwang-Pil Choi ◽  
Thomas B. Bair ◽  
Young-Min Bae ◽  
Lacy Daniels
Keyword(s):  
Mycobacterium Bovis ◽  
Wild Type Strain ◽  
Selection System ◽  
Coenzyme F420 ◽  
Wild Type ◽  
Reverse Transcription Pcr ◽  
Low Levels ◽  
Resistant Mutants ◽  
Specific Reactions ◽  
Insertion Mutants

ABSTRACT Three transposon Tn5367 mutagenesis vectors (phAE94, pPR28, and pPR29) were used to create a collection of insertion mutants of Mycobacterium bovis strain BCG. A strategy to select for transposon-generated mutants that cannot make coenzyme F420 was developed using the nitroimidazopyran-based antituberculosis drug PA-824. One-third of 134 PA-824-resistant mutants were defective in F420 accumulation. Two mutants that could not make F420-5,6 but which made the biosynthesis intermediate FO were examined more closely. These mutants contained transposons inserted in two adjacent homologues of Mycobacterium tuberculosis genes, which we have named fbiA andfbiB for F420 biosynthesis. Homologues offbiA were found in all seven microorganisms that have been fully sequenced and annotated and that are known to make F420. fbiB homologues were found in all but one such organism. Complementation of the fbiA mutant with fbiAB and complementation of thefbiB mutant with fbiB both restored the F420-5,6 phenotype. Complementation of thefbiA mutant with fbiA orfbiB alone did not restore the F420-5,6 phenotype, but the fbiA mutant complemented withfbiA produced F420-2,3,4 at levels similar to F420-5,6 made by the wild-type strain, but produced much less F420-5. These data demonstrate that both genes are essential for normal F420-5,6 production and suggest that the fbiA mutation has a partial polar effect onfbiB. Reverse transcription-PCR data demonstrated thatfbiA and fbiB constitute an operon. However, very low levels of fbiB mRNA are produced by the fbiA mutant, suggesting that a low-level alternative start site is located upstream of fbiB. The specific reactions catalyzed by FbiA and FbiB are unknown, but both function between FO and F420-5,6, since FO is made by both mutants.

scholarly journals The evolution of collective infectious units in viruses

2019 ◽  
Author(s):  
Asher Leeks ◽  
Rafael Sanjuán ◽  
Stuart A. West
Keyword(s):  
Viral Genome ◽  
Social Evolution ◽  
Viral Pathogenesis ◽  
Wild Type Virus ◽  
Antiviral Resistance ◽  
Costs And Benefits ◽  
Wild Type ◽  
Viral Genomes ◽  
Viral Sequences ◽  
Infectious Unit

Viruses frequently spread among cells or hosts in groups, with multiple viral genomes inside the same infectious unit. These collective infectious units can consist of multiple viral genomes inside the same virion, or multiple virions inside a larger structure such as a vesicle. Collective infectious units deliver multiple viral genomes to the same cell simultaneously, which can have important implications for viral pathogenesis, antiviral resistance, and social evolution. However, little is known about why some viruses transmit in collective infectious units, whereas others do not. We used a simple evolutionary approach to model the potential costs and benefits of transmitting in a collective infectious unit. We found that collective infectious units could be favoured if cells infected by multiple viral genomes were significantly more productive than cells infected by just one viral genome, and especially if there were also efficiency benefits to packaging multiple viral genomes inside the same infectious unit. We also found that if some viral sequences are defective, then collective infectious units could evolve to become very large, but that if these defective sequences interfered with wild-type virus replication, then collective infectious units were disfavoured.

scholarly journals Antibody response to SARS-CoV-2 mRNA vaccine in lung cancer patients: Reactivity to vaccine antigen and variants of concern.

2022 ◽  
Author(s):  
Rajesh Valanparambil ◽  
Jennifer Carlisle ◽  
Susanne Linderman ◽  
Akil Akthar ◽  
Ralph Linwood Millett ◽  
...  
Keyword(s):  
Antibody Response ◽  
Type Strain ◽  
Wild Type Strain ◽  
Specific Binding ◽  
Neutralizing Antibody ◽  
Wild Type ◽  
Live Virus ◽  
Antibody Titers ◽  
Binding Antibody ◽  
Nsclc Patients

Purpose: We investigated SARS-CoV-2 mRNA vaccine-induced binding and live-virus neutralizing antibody response in NSCLC patients to the SARS-CoV-2 wild type strain and the emerging Delta and Omicron variants. Methods: 82 NSCLC patients and 53 healthy adult volunteers who received SARS-CoV-2 mRNA vaccines were included in the study. Blood was collected longitudinally, and SARS-CoV-2-specific binding and live-virus neutralization response to 614D (WT), B.1.617.2 (Delta), B.1.351 (Beta) and B.1.1.529 (Omicron) variants were evaluated by Meso Scale Discovery (MSD) assay and Focus Reduction Neutralization Assay (FRNT) respectively. We determined the longevity and persistence of vaccine-induced antibody response in NSCLC patients. The effect of vaccine-type, age, gender, race and cancer therapy on the antibody response was evaluated. Results: Binding antibody titer to the mRNA vaccines were lower in the NSCLC patients compared to the healthy volunteers (P=<0.0001). More importantly, NSCLC patients had reduced live-virus neutralizing activity compared to the healthy vaccinees (P=<0.0001). Spike and RBD-specific binding IgG titers peaked after a week following the second vaccine dose and declined after six months (P=<0.001). While patients >70 years had lower IgG titers (P=<0.01), patients receiving either PD-1 monotherapy, chemotherapy or a combination of both did not have a significant impact on the antibody response. Binding antibody titers to the Delta and Beta variants were lower compared to the WT strain (P=<0.0001). Importantly, we observed significantly lower FRNT50 titers to Delta (6-fold), and Omicron (79-fold) variants (P=<0.0001) in NSCLC patients. Conclusions: Binding and live-virus neutralizing antibody titers to SARS-CoV-2 mRNA vaccines in NSCLC patients were lower than the healthy vaccinees, with significantly lower live-virus neutralization of B.1.617.2 (Delta), and more importantly, the B.1.1.529 (Omicron) variant compared to the wild-type strain. These data highlight the concern for cancer patients given the rapid spread of SARS-CoV-2 Omicron variant.

Excretion of ammonium by Azospirillum brasilense mutants resistant to ethylenediamine

1991 ◽  
Vol 37 (7) ◽  
pp. 549-553 ◽  
Author(s):  
H. B. Machado ◽  
S. Funayama ◽  
L. U. Rigo ◽  
F. O. Pedrosa
Keyword(s):  
Glutamine Synthetase ◽  
Azospirillum Brasilense ◽  
Type Strain ◽  
Wild Type Strain ◽  
Synthetase Activity ◽  
Ammonium Excretion ◽  
Glutamine Synthetase Activity ◽  
Wild Type ◽  
High Concentration ◽  
Low Levels

Several spontaneous ethylenediamine-resistant mutants of Azospirillum brasilense strain FP2 (Sp7, NalR SmR) were isolated. Four mutants, HM053, HM14, HM26, and HM210, were found to fix nitrogen constitutively in the presence of high concentration of NH4+ and to excrete NH4+ derived from nitrogen fixation. They also showed lower rates of NH4+ uptake than the wild-type strain, FP2. All of the mutants were prototrophic for glutamine or glutamate. Their glutamate synthase and glutamate dehydrogenase activities were similar to those of the wild-type strain. However, they presented different patterns of glutamine synthetase activity. Mutant HM14 showed low levels of normally regulated glutamine synthetase activity, while the other mutants showed low levels (HM053) or wild-type levels (HM26 and HM210) of constitutively adenylylated glutamine synthetase activity. The mutants are probably defective in the adenylylation system. Key words: Azospirillum brasilense, ammonium excretion, ethylenediamine resistance, glutamine synthetase.

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