scholarly journals The avian retroviral integration protein cleaves the terminal sequences of linear viral DNA at the in vivo sites of integration.

1989 ◽  
Vol 63 (12) ◽  
pp. 5319-5327 ◽  
Author(s):  
M Katzman ◽  
R A Katz ◽  
A M Skalka ◽  
J Leis
Keyword(s):  
Viral Dna ◽  
Retroviral Integration

scholarly journals The free energy landscape of retroviral integration

2019 ◽  
Vol 10 (1) ◽  
Author(s):  
Willem Vanderlinden ◽  
Tine Brouns ◽  
Philipp U. Walker ◽  
Pauline J. Kolbeck ◽  
Lukas F. Milles ◽  
...  
Keyword(s):  
Energy Landscape ◽  
Forward Rate ◽  
Strand Transfer ◽  
Viral Dna ◽  
Retroviral Integration ◽  
Target Capture ◽  
Point Of No Return ◽  
Efficient Integration ◽  
Site Binding

Abstract Retroviral integration, the process of covalently inserting viral DNA into the host genome, is a point of no return in the replication cycle. Yet, strand transfer is intrinsically iso-energetic and it is not clear how efficient integration can be achieved. Here we investigate the dynamics of strand transfer and demonstrate that consecutive nucleoprotein intermediates interacting with a supercoiled target are increasingly stable, resulting in a net forward rate. Multivalent target interactions at discrete auxiliary interfaces render target capture irreversible, while allowing dynamic site selection. Active site binding is transient but rapidly results in strand transfer, which in turn rearranges and stabilizes the intasome in an allosteric manner. We find the resulting strand transfer complex to be mechanically stable and extremely long-lived, suggesting that a resolving agent is required in vivo.

290 Cytokine and immune subset signatures in patients with various solid and hematological malignancies treated with oncolytic vaccinia virus delivered by autologous stromal vascular fraction cells

2020 ◽  
Vol 8 (Suppl 3) ◽  
pp. A317-A317
Author(s):  
Dobrin Draganov ◽  
Antonio Santidrian ◽  
Ivelina Minev ◽  
Duong Nguyen ◽  
Dmitriy Zamarin ◽  
...  
Keyword(s):  
T Cell ◽  
Vaccinia Virus ◽  
Stromal Vascular Fraction ◽  
Oncolytic Viruses ◽  
Multiparameter Flow Cytometry ◽  
Viral Dna ◽  
Cell Responses ◽  
The Impact ◽  
Immune Subset

BackgroundThe development of oncolytic viruses for the treatment of cancer has been significantly hampered by their rapid clearance in circulation due to complement and antibody-mediated neutralization. In our recent first-in-human Phase I clinical trial, we evaluated the safety and feasibility of our approach to enhance virus delivery and improve tumor targeting by utilizing an autologous stromal vascular fraction (SVF) based cell delivery system. Patient sample analysis demonstrated that patients could be stratified based on the level of vaccinia virus amplification in vivo, as evidenced by analysis of persistent viral DNA in the blood.MethodsIn the current study, we evaluated the immunomodulatory potential of vaccinia virus delivered by autologous stromal vascular fraction (SVF)-derived cells and attempted to identify immunological correlates of successful vaccinia virus amplification in vivo. To this end, we performed an extensive time-course analysis of cytokines in patients‘ plasma as well as various peripheral blood immune subpopulations using Luminex multi-analyte profiling and multiparameter flow cytometry, respectively. We also analyzed the impact of this therapeutic approach on the innate and adaptive immune subpopulations, including NK cells, myeloid cells, as well as effector, regulatory and memory T cells.ResultsTherapy with SFV-delivered oncolytic vaccinia virus induced a coordinated activation of cytokine, T cell and NK cell responses in patients as early as 1 day after treatment, which peaked around 1-week and lasted for up to 1-month post treatment. The ability of the oncolytic virus to effectively amplify in cancer patients correlated with significant changes of multiple innate (NK) and adaptive (T cell) immunological parameters. Interestingly, patient stratification into groups with transient versus persistent viral DNA was linked to opposing and mutually exclusive patterns of robust activation of NK versus T cell responses, respectively. Our study also identified intriguing cytokine and immune subset frequency signatures present at baseline and associated with successful amplification and persistence of oncolytic vaccinia virus in vivo.ConclusionsOverall, this study establishes the timeline of treatment-related immunological changes and identifies biomarkers present at baseline and potential immunological correlates associated with the persistence of virus amplification in vivo. Therefore, our findings provide new insights into the role of interpatient immunological variability and will contribute to the proper evaluation of the therapeutic potency of oncolytic virotherapy in future clinical trials.

scholarly journals The Phage φ29 Membrane Protein p16.7, Involved in DNA Replication, Is Required for Efficient Ejection of the Viral Genome

2007 ◽  
Vol 189 (15) ◽  
pp. 5542-5549 ◽  
Author(s):  
Martín Alcorlo ◽  
Víctor González-Huici ◽  
José M. Hermoso ◽  
Wilfried J. J. Meijer ◽  
Margarita Salas
Keyword(s):  
Dna Replication ◽  
Membrane Protein ◽  
Second Step ◽  
Host Proteins ◽  
Viral Dna ◽  
Dna Ejection ◽  
Phage Dna ◽  
Ejection Mechanism ◽  
The Right

ABSTRACT It is becoming clear that in vivo phage DNA ejection is not a mere passive process. In most cases, both phage and host proteins seem to be involved in pulling at least part of the viral DNA inside the cell. The DNA ejection mechanism of Bacillus subtilis bacteriophage φ29 is a two-step process where the linear DNA penetrates the cell with a right-left polarity. In the first step ∼65% of the DNA is pushed into the cell. In the second step, the remaining DNA is actively pulled into the cytoplasm. This step requires protein p17, which is encoded by the right-side early operon that is ejected during the first push step. The membrane protein p16.7, also encoded by the right-side early operon, is known to play an important role in membrane-associated phage DNA replication. In this work we show that, in addition, p16.7 is required for efficient execution of the second pull step of DNA ejection.

scholarly journals Characterization of the Uracil-DNA Glycosylase Activity of Epstein-Barr Virus BKRF3 and Its Role in Lytic Viral DNA Replication

2006 ◽  
Vol 81 (3) ◽  
pp. 1195-1208 ◽  
Author(s):  
Chih-Chung Lu ◽  
Ho-Ting Huang ◽  
Jiin-Tarng Wang ◽  
Geir Slupphaug ◽  
Tsai-Kun Li ◽  
...  
Keyword(s):  
Dna Replication ◽  
Epstein Barr Virus ◽  
Efficient Production ◽  
Transcription Activator ◽  
Viral Dna ◽  
Barr Virus ◽  
Epstein Barr ◽  
Lytic Dna Replication

ABSTRACT Uracil-DNA glycosylases (UDGs) of the uracil-N-glycosylase (UNG) family are the primary DNA repair enzymes responsible for removal of inappropriate uracil from DNA. Recent studies further suggest that the nuclear human UNG2 and the UDGs of large DNA viruses may coordinate with their DNA polymerase accessory factors to enhance DNA replication. Based on its amino acid sequence, the putative UDG of Epstein-Barr virus (EBV), BKRF3, belongs to the UNG family of proteins, and it was demonstrated previously to enhance oriLyt-dependent DNA replication in a cotransfection replication assay. However, the expression and enzyme activity of EBV BKRF3 have not yet been characterized. In this study, His-BKRF3 was expressed in bacteria and purified for biochemical analysis. Similar to the case for the Escherichia coli and human UNG enzymes, His-BKRF3 excised uracil from single-stranded DNA more efficiently than from double-stranded DNA and was inhibited by the purified bacteriophage PBS1 inhibitor Ugi. In addition, BKRF3 was able to complement an E. coli ung mutant in rifampin and nalidixic acid resistance mutator assays. The expression kinetics and subcellular localization of BKRF3 products were detected in EBV-positive lymphoid and epithelial cells by using BKRF3-specific mouse antibodies. Expression of BKRF3 is regulated mainly by the immediate-early transcription activator Rta. The efficiency of EBV lytic DNA replication was slightly affected by BKRF3 small interfering RNA (siRNA), whereas cellular UNG2 siRNA or inhibition of cellular and viral UNG activities by expressing Ugi repressed EBV lytic DNA replication. Taking these results together, we demonstrate the UNG activity of BKRF3 in vitro and in vivo and suggest that UNGs may participate in DNA replication or repair and thereby promote efficient production of viral DNA.

scholarly journals Concurrent administration of IFNα14 and cART in TKO-BLT mice enhances suppression of HIV-1 viremia but does not eliminate the latent reservoir

2019 ◽  
Vol 9 (1) ◽  
Author(s):  
Kathrin Sutter ◽  
Kerry J. Lavender ◽  
Ronald J. Messer ◽  
Marek Widera ◽  
Katie Williams ◽  
...  
Keyword(s):  
Infection Type ◽  
Treatment Interruption ◽  
Type I Interferons ◽  
Latent Reservoir ◽  
Type I ◽  
Concurrent Administration ◽  
Viral Dna ◽  
Therapy Interruption ◽  
Hiv 1

AbstractCombination antiretroviral therapy (cART) prevents HIV-1 replication but does not eliminate the latent reservoir and cure the infection. Type I interferons (IFN) mediate antiviral effects through different mechanisms than cART. We previously showed that IFNα14 is the most potent IFNα subtype against HIV-1 and that it can significantly reduce the HIV-1 proviral reservoir. This study sought to determine whether combining cART with IFNα14 therapy would produce greater reductions in HIV-1 viral and proviral loads than ART alone. Immunodeficient Rag2−/−γc−/−CD47−/− C57BL/6 mice were humanized by the BLT method, infected with HIV-1JR-CSF and the in vivo efficacy of cART was compared with combined cART/IFNα14 therapy. Infection was allowed to establish for 6 weeks prior to 4 weeks of treatment with oral cART either with or without IFNα14. Plasma viral RNA and splenic CD4+ T cell viral DNA levels were measured immediately after treatment and after 2 weeks of therapy interruption. Augmentation of cART with IFNα14 resulted in significantly enhanced suppression of HIV-1 plasma viremia. However, no significant reduction in total viral DNA was detectable. Furthermore, virus rebounded after treatment interruption to similar levels in both groups. Thus, augmentation of cART with IFNα14 resulted in a more pronounced reduction of HIV viremia levels over cART alone, but the effect was not potent enough to be detected at the viral DNA level or to prevent virus rebound following therapy interruption in immune system-humanized mice.

scholarly journals Direct Evidence of Lower Viral Replication Rates In Vivo in Human Immunodeficiency Virus Type 2 (HIV-2) Infection than in HIV-1 Infection

2007 ◽  
Vol 81 (10) ◽  
pp. 5325-5330 ◽  
Author(s):  
Adam MacNeil ◽  
Abdoulaye Dieng Sarr ◽  
Jean-Louis Sankalé ◽  
Seema Thakore Meloni ◽  
Souleymane Mboup ◽  
...  
Keyword(s):  
Human Immunodeficiency Virus ◽  
Human Immunodeficiency Virus Type ◽  
Virus Type ◽  
Viral Mrna ◽  
Viral Dna ◽  
Viral Loads ◽  
Immunodeficiency Virus ◽  
Hiv 1

ABSTRACT Studies have shown that human immunodeficiency virus type 2 (HIV-2) is less pathogenic than HIV-1, with a lower rate of disease progression. Similarly, plasma viral loads are lower in HIV-2 infection, suggesting that HIV-2 replication is restricted in vivo in comparison to that of HIV-1. However, to date, in vivo studies characterizing replication intermediates in the viral life cycle of HIV-2 have been limited. In order to test the hypothesis that HIV-2 has a lower replication rate in vivo than HIV-1 does, we quantified total viral DNA, integrated proviral DNA, cell-associated viral mRNA, and plasma viral loads in peripheral blood samples from groups of therapy-naïve HIV-1-infected (n = 21) and HIV-2-infected (n = 18) individuals from Dakar, Senegal, with CD4+ T-cell counts of >200/μl. Consistent with our previous findings, total viral DNA loads were similar between HIV-1 and HIV-2 and plasma viral loads were higher among HIV-1-infected individuals. Proportions of DNA in the integrated form were also similar between these viruses. In contrast, levels of viral mRNA were lower in HIV-2 infection. Our study indicates that HIV-2 is able to establish a stable, integrated proviral infection in vivo, but that accumulation of viral mRNA is attenuated in HIV-2 infection relative to that in HIV-1 infection. The differences in viral mRNA are consistent with the differences in plasma viral loads between HIV-1 and HIV-2 and suggest that lower plasma viral loads, and possibly the attenuated pathogenesis of HIV-2, can be explained by lower rates of viral replication in vivo.

scholarly journals In vitro initiation of transcription by RNA polymerase II on in vivo-assembled chromatin templates.

1992 ◽  
Vol 12 (4) ◽  
pp. 1639-1651 ◽  
Author(s):  
S C Batson ◽  
R Sundseth ◽  
C V Heath ◽  
M Samuels ◽  
U Hansen
Keyword(s):  
Transcription Factors ◽  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Viral Dna ◽  
Dna Templates ◽  
Initiation Of Transcription ◽  
Transcription In Vitro ◽  
Alpha Amanitin

We have studied the initiation of transcription in vitro by RNA polymerase II on simian virus 40 (SV40) minichromosomal templates isolated from infected cells. The efficiency and pattern of transcription from the chromatin templates were compared with those from viral DNA templates by using two in vitro transcription systems, either HeLa whole-cell extract or basal transcription factors, RNA polymerase II, and one of two SV40 promoter-binding transcription factors, LSF and Sp1. Dramatic increases in numbers of transcripts upon addition of transcription extract and different patterns of usage of the multiple SV40 initiation sites upon addition of Sp1 versus LSF strongly suggested that transcripts were being initiated from the minichromosomal templates in vitro. That the majority of transcripts from the minichromosomes were due to initiation de novo was demonstrated by the efficient transcription observed in the presence of alpha-amanitin, which inhibited minichromosome-associated RNA polymerase II, and an alpha-amanitin-resistant RNA polymerase II, which initiated transcription in vitro. The pattern of transcription from the SV40 late and early promoters on the minichromosomal templates was similar to the in vivo pattern of transcription during the late stages of viral infection and was distinct from the pattern of transcription generated from viral DNA in vitro. In particular, the late promoter of the minichromosomal templates was transcribed with high efficiency, similar to viral DNA templates, while the early-early promoter of the minichromosomal templates was inhibited 10- to 15-fold. Finally, the number of minichromosomes competent to initiate transcription in vitro exceeded the amount actively being transcribed in vivo.

scholarly journals Histone H2AX Is Phosphorylated at Sites of Retroviral DNA Integration but Is Dispensable for Postintegration Repair

2004 ◽  
Vol 279 (44) ◽  
pp. 45810-45814 ◽  
Author(s):  
René Daniel ◽  
Joseph Ramcharan ◽  
Emmy Rogakou ◽  
Konstantin D. Taganov ◽  
James G. Greger ◽  
...  
Keyword(s):  
Chromatin Immunoprecipitation ◽  
Direct Evidence ◽  
Active Role ◽  
Viral Dna ◽  
Dna Integration ◽  
Retroviral Integration ◽  
Histone H2ax ◽  
Damage Response ◽  
Integration Sites ◽  
Increased Sensitivity

The histone variant H2AX is rapidly phosphorylated (denoted γH2AX) in large chromatin domains (foci) flanking double strand DNA (dsDNA) breaks that are produced by ionizing radiation or genotoxic agents and during V(D)J recombination. H2AX-deficient cells and mice demonstrate increased sensitivity to dsDNA break damage, indicating an active role for γH2AX in DNA repair; however, γH2AX formation is not required for V(D)J recombination. The latter finding has suggested a greater dependence on γH2AX for anchoring free broken endsversusends that are held together during programmed breakage-joining reactions. Retroviral DNA integration produces a unique intermediate in which a dsDNA break in host DNA is held together by the intervening viral DNA, and such a reaction provides a useful model to distinguish γH2AX functions. We found that integration promotes transient formation of γH2AX at retroviral integration sites as detected by both immunocytological and chromatin immunoprecipitation methods. These results provide the first direct evidence for the association of newly integrated viral DNA with a protein species that is an established marker for the onset of a DNA damage response. We also show that H2AX is not required for repair of the retroviral integration intermediate as determined by stable transduction. These observations provide independent support for an anchoring model for the function of γH2AX in chromatin repair.

scholarly journals Characterization of Human Herpesvirus 8 ORF59 Protein (PF-8) and Mapping of the Processivity and Viral DNA Polymerase-Interacting Domains

2000 ◽  
Vol 74 (23) ◽  
pp. 10920-10929 ◽  
Author(s):  
Szeman Ruby Chan ◽  
Bala Chandran
Keyword(s):  
Dna Polymerase ◽  
Human Herpesvirus ◽  
Human Herpesvirus 8 ◽  
Dependent Manner ◽  
Cell Nuclei ◽  
Viral Dna ◽  
Herpesvirus 8 ◽  
Barr Virus

ABSTRACT Human herpesvirus 8 (HHV-8) or Kaposi's sarcoma-associated herpesvirus (KSHV) ORF59 protein (PF-8) is a processivity factor for HHV-8 DNA polymerase (Pol-8) and is homologous to processivity factors expressed by other herpesviruses, such as herpes simplex virus type 1 UL42 and Epstein-Barr virus BMRF1. The interaction of UL42 and BMRF1 with their corresponding DNA polymerases is essential for viral DNA replication and the subsequent production of infectious virus. Using HHV-8-specific monoclonal antibody 11D1, we have previously identified the cDNA encoding PF-8 and showed that it is an early-late gene product localized to HHV-8-infected cell nuclei (S. R. Chan, C. Bloomer, and B. Chandran, Virology 240:118–126, 1998). Here, we have further characterized PF-8. This viral protein was phosphorylated both in vitro and in vivo. PF-8 bound double-stranded DNA (dsDNA) and single-stranded DNA independent of DNA sequence; however, the affinity for dsDNA was approximately fivefold higher. In coimmunoprecipitation reactions, PF-8 also interacted with Pol-8. In in vitro processivity assays with excess poly(dA):oligo(dT) as a template, PF-8 stimulated the production of elongated DNA products by Pol-8 in a dose-dependent manner. Functional domains of PF-8 were determined using PF-8 truncation mutants. The carboxyl-terminal 95 amino acids (aa) of PF-8 were dispensable for all three functions of PF-8: enhancing processivity of Pol-8, binding dsDNA, and binding Pol-8. Residues 10 to 27 and 279 to 301 were identified as regions critical for the processivity function of PF-8. Interestingly, aa 10 to 27 were also essential for binding Pol-8, whereas aa 1 to 62 and aa 279 to 301 were involved in binding dsDNA, suggesting that the processivity function of PF-8 is correlated with both the Pol-8-binding and the dsDNA-binding activities of PF-8.

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