In Vitro Evolution of Bovine Foamy Virus Variants with Enhanced Cell-Free Virus Titers and Transmission

ABSTRACT Viruses are obligate intracellular parasites that depend on cellular machinery for their efficient transcription and replication. In a previous study we reported that bovine foamy virus (BFV) is able to activate the nuclear factor κB (NF-κB) pathway through the action of its transactivator BTas to enhance viral transcription. However, the mechanism used by NF-κB to enhance BFV transcription remains elusive. To address this question, we employed a yeast two-hybrid assay to screen for BTas-interacting proteins. We found that RelB, a member of NF-κB protein family, interacts with BTas. We confirmed the putative RelB-BTas interaction in vitro and in vivo and identified the protein regions responsible for the RelB-BTas interaction. Using a luciferase reporter assay, we next showed that RelB enhances BFV transcription (BTas-induced long terminal repeat [LTR] transactivation) and that this process requires both the localization of the RelB-BTas interaction in the nucleus and the Rel homology domain of RelB. The knockdown of the cellular endogenous RelB protein using small interfering RNA (siRNA) significantly attenuated BTas-induced LTR transcription. The results of chromatin immunoprecipitation (ChIP) analysis showed that endogenous RelB binds to the viral LTR in BFV-infected cells. Together, these results suggest that BFV engages the RelB protein as a cotransactivator of BTas to enhance viral transcription. In addition, our findings indicate that BFV infection upregulates cellular RelB expression through BTas-induced NF-κB activation. Thus, this study demonstrates the existence of a positive-feedback circuit in which BFV utilizes the host's NF-κB pathway through the RelB protein for efficient viral transcription.

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Functional Analyses of Bovine Foamy Virus-Encoded miRNAs Reveal the Importance of a Defined miRNA for Virus Replication and Host–Virus Interaction

Viruses ◽

10.3390/v12111250 ◽

2020 ◽

Vol 12 (11) ◽

pp. 1250

Author(s):

Wenhu Cao ◽

Erik Stricker ◽

Agnes Hotz-Wagenblatt ◽

Anke Heit-Mondrzyk ◽

Georgios Pougialis ◽

...

Keyword(s):

Target Genes ◽

Foamy Virus ◽

Rna Polymerase Iii ◽

Long Terminal Repeats ◽

Bovine Foamy Virus ◽

Host Interactions ◽

Micro Rnas ◽

Efficient Replication ◽

Control Virus

In addition to regulatory or accessory proteins, some complex retroviruses gain a repertoire of micro-RNAs (miRNAs) to regulate and control virus–host interactions for efficient replication and spread. In particular, bovine and simian foamy viruses (BFV and SFV) have recently been shown to express a diverse set of RNA polymerase III-directed miRNAs, some with a unique primary miRNA double-hairpin, dumbbell-shaped structure not known in other viruses or organisms. While the mechanisms of expression and structural requirements have been studied, the functional importance of these miRNAs is still far from understood. Here, we describe the in silico identification of BFV miRNA targets and the subsequent experimental validation of bovine Ankyrin Repeat Domain 17 (ANKRD17) and Bax-interacting factor 1 (Bif1) target genes in vitro and, finally, the suppression of ANKRD17 downstream genes in the affected pathway. Deletion of the entire miRNA cassette in the non-coding part of the U3 region of the long terminal repeats attenuated replication of corresponding BFV mutants in bovine cells. This repression can be almost completely trans-complemented by the most abundant miRNA BF2-5p having the best scores for predicted and validated BFV miRNA target genes. Deletion of the miRNA cassette does not grossly affect particle release and overall particle composition.

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Bovine Foamy Virus: Shared and Unique Molecular Features In Vitro and In Vivo

Viruses ◽

10.3390/v11121084 ◽

2019 ◽

Vol 11 (12) ◽

pp. 1084 ◽

Cited By ~ 2

Author(s):

Magdalena Materniak-Kornas ◽

Juan Tan ◽

Anke Heit-Mondrzyk ◽

Agnes Hotz-Wagenblatt ◽

Martin Löchelt

Keyword(s):

Animal Studies ◽

Foamy Virus ◽

Companion Animals ◽

Population Level ◽

Medical Problems ◽

Bovine Foamy Virus ◽

Molecular Features ◽

Unclear Etiology

The retroviral subfamily of Spumaretrovirinae consists of five genera of foamy (spuma) viruses (FVs) that are endemic in some mammalian hosts. Closely related species may be susceptible to the same or highly related FVs. FVs are not known to induce overt disease and thus do not pose medical problems to humans and livestock or companion animals. A robust lab animal model is not available or is a lab animal a natural host of a FV. Due to this, research is limited and often focused on the simian FVs with their well-established zoonotic potential. The authors of this review and their groups have conducted several studies on bovine FV (BFV) in the past with the intention of (i) exploring the risk of zoonotic infection via beef and raw cattle products, (ii) studying a co-factorial role of BFV in different cattle diseases with unclear etiology, (iii) exploring unique features of FV molecular biology and replication strategies in non-simian FVs, and (iv) conducting animal studies and functional virology in BFV-infected calves as a model for corresponding studies in primates or small lab animals. These studies gained new insights into FV-host interactions, mechanisms of gene expression, and transcriptional regulation, including miRNA biology, host-directed restriction of FV replication, spread and distribution in the infected animal, and at the population level. The current review attempts to summarize these findings in BFV and tries to connect them to findings from other FVs.

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The Influence of Envelope C-Terminus Amino Acid Composition on the Ratio of Cell-Free to Cell-Cell Transmission for Bovine Foamy Virus

Viruses ◽

10.3390/v11020130 ◽

2019 ◽

Vol 11 (2) ◽

pp. 130 ◽

Cited By ~ 3

Author(s):

Suzhen Zhang ◽

Xiaojuan Liu ◽

Zhibin Liang ◽

Tiejun Bing ◽

Wentao Qiao ◽

...

Keyword(s):

High Titer ◽

Foamy Virus ◽

Evolution System ◽

Bovine Foamy Virus ◽

C Terminus ◽

Release Capacity ◽

Cell Transmission ◽

Extensive Cell ◽

Original Cell

Foamy viruses (FVs) have extensive cell tropism in vitro, special replication features, and no clinical pathogenicity in naturally or experimentally infected animals, which distinguish them from orthoretroviruses. Among FVs, bovine foamy virus (BFV) has undetectable or extremely low levels of cell-free transmission in the supernatants of infected cells and mainly spreads by cell-to-cell transmission, which deters its use as a gene transfer vector. Here, using an in vitro virus evolution system, we successfully isolated high-titer cell-free BFV strains from the original cell-to-cell transmissible BFV3026 strain and further constructed an infectious cell-free BFV clone called pBS-BFV-Z1. Following sequence alignment with a cell-associated clone pBS-BFV-B, we identified a number of changes in the genome of pBS-BFV-Z1. Extensive mutagenesis analysis revealed that the C-terminus of envelope protein, especially the K898 residue, controls BFV cell-free transmission by enhancing cell-free virus entry but not the virus release capacity. Taken together, our data show the genetic determinants that regulate cell-to-cell and cell-free transmission of BFV.

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Faculty Opinions recommendation of In vitro evolution suggests multiple origins for the hammerhead ribozyme.

Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature ◽

10.3410/f.1002070.21655 ◽

2001 ◽

Author(s):

Dipankar Sen

Keyword(s):

Hammerhead Ribozyme ◽

In Vitro Evolution ◽

Multiple Origins

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Faculty Opinions recommendation of Limits of neutral drift: lessons from the in vitro evolution of two ribozymes.

Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature ◽

10.3410/f.718545195.793499713 ◽

2014 ◽

Author(s):

Scott Silverman

Keyword(s):

In Vitro Evolution ◽

Neutral Drift

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Optimization of Heterologous Gene Expression for In Vitro Evolution

BioTechniques ◽

10.2144/01303bm01 ◽

2001 ◽

Vol 30 (3) ◽

pp. 474-476 ◽

Cited By ~ 5

Author(s):

Ichiro Matsumura ◽

Mark J. Olsen ◽

Andrew D. Ellington

Keyword(s):

Gene Expression ◽

Heterologous Gene Expression ◽

Heterologous Gene ◽

In Vitro Evolution

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Molecular Epidemiology and Whole-Genome Analysis of Bovine Foamy Virus in Japan

Viruses ◽

10.3390/v13061017 ◽

2021 ◽

Vol 13 (6) ◽

pp. 1017

Author(s):

Hirohisa Mekata ◽

Tomohiro Okagawa ◽

Satoru Konnai ◽

Takayuki Miyazawa

Keyword(s):

Phylogenetic Analyses ◽

Foamy Virus ◽

Pcr Analysis ◽

Whole Genome ◽

Genome Sequences ◽

Whole Genome Analysis ◽

Virus Family ◽

Bovine Foamy Virus ◽

Novel Genotype

Bovine foamy virus (BFV) is a member of the foamy virus family in cattle. Information on the epidemiology, transmission routes, and whole-genome sequences of BFV is still limited. To understand the characteristics of BFV, this study included a molecular survey in Japan and the determination of the whole-genome sequences of 30 BFV isolates. A total of 30 (3.4%, 30/884) cattle were infected with BFV according to PCR analysis. Cattle less than 48 months old were scarcely infected with this virus, and older animals had a significantly higher rate of infection. To reveal the possibility of vertical transmission, we additionally surveyed 77 pairs of dams and 3-month-old calves in a farm already confirmed to have BFV. We confirmed that one of the calves born from a dam with BFV was infected. Phylogenetic analyses revealed that a novel genotype was spread in Japan. In conclusion, the prevalence of BFV in Japan is relatively low and three genotypes, including a novel genotype, are spread in Japan.

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