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Projeto de investigação
Targeted research effort on African swine fever
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Spatiotemporal study of morpho-functional modifications on cell nucleus during African swine fever virus infection
Publication . Simões, Margarida Pires; Ferreira, Fernando António da Costa; Martins, Carlos Manuel Lopes Vieira
Studies on virus-host interactions are decisive to enhance our understanding on how African swine fever virus (ASFV) subverts cellular mechanisms, and also to better characterize host nucleus changes enabling this infection. Immunofluorescence studies and immunoblotting analysis of ASFV-infected cells, allowed us to identify the Ataxia telangiectasia mutated and Rad3-related (ATR) pathway as the specific DNA damage response (DDR) mechanism activated by ASFV infection. Additionally, the use of ATR kinase-dead cells confirmed that ATR has an essential role for the infection success.
The viral intranuclear replication was then pursued using BrdU-pulse experiments, supported on previous reports about ASFV genome presence inside the host nucleus and the proven ATR activation. BrdU-labelled DNA molecules confirmed the active viral replication at early infection times, exclusively within the cell nucleus. Related spatial and morphological nuclear changes during ASFV infection were further addressed, particularly on subnuclear domains and host chromatin epigenetic signatures. Promyelocytic leukaemia nuclear bodies (PML-NBs), nuclear speckles and Cajal bodies displayed major alterations, accompanied by a repressive nuclear environment. PML knockdown revealed an essential proviral activity for ASFV successful infection. Herein, suggestions on how this work may help in the development of therapeutic strategies against ASFV infections can be found.
Functional characterization of unassigned african swine fever virus proteins putatively involved in transcription and replication towards an efficient vaccine design
Publication . Freitas, Ferdinando; Ferreira, Fernando António da Costa; Martins, Carlos Manuel Lopes Vieira
African swine fever (ASF) is an infectious disease of domestic pigs and wild boars with mortality rates reaching up to 100% and is endemic in most of the Sub-Saharan countries. In 2007 it was introduced in Georgia and spread to neighbouring countries, reaching the Russian Federation, several European countries and, more recently, China and Vietnam (February 2019). Currently, there is neither a vaccine nor a treatment against ASF and the control of the disease depends strictly on sanitary measures, including stamping out and trade bans of animals and pork products leading to devastating socio-economic losses to affected countries. The etiologic agent of the disease is African swine fever virus (ASFV), a large (approx. 190 nm) double-stranded DNA (170 to 193 kbp) enveloped virus. ASFV genome encloses more than 150 open reading frames (ORFs) and to this date most of them lack any known or predictable function. ASFV is quite independent from cellular machinery encoding enzymes required for replication, transcription and virion assembly, including the putative I215L E2 Ubiquitin-conjugating enzyme, QP509L, Q706L RNA Helicases and the P1192R type II topoisomerase. The E2 ubiquitin-conjugating enzymes are part of the essential cellular post-transcriptional regulation ubiquitin-proteasome pathway. In this study, the pI215L binding activity was characterized as being mono and poly-ubiquitinated in the Cys85 at different temperatures and pH values. Moreover, I215L gene is transcribed from 2 hours post infection (hpi), and immunoblot analysis confirmed that pI215L is expressed from 4 hpi being detected all over the cell specially in the viral factories from 8 hpi. Downregulation assays by siRNA suggested that pI215L plays a critical role in the transcription of late viral genes and in viral DNA replication. RNA helicases are described as essential for infections, modulating RNA-RNA and RNA-protein interactions, gene expression, viral egress and host antiviral responses. In the present work, we found that QP509L, Q706L are conserved between ASFV virulent and non-virulent isolates. Furthermore, ASFV-QP509L and Q706L are actively transcribed from 2 hours post infection, and both proteins are localized in the viral factories at 12 hours post infection. However, QP509L was also detected in the cell nucleus. Transcript downregulation uncovered the essential role of these proteins during viral cycle progression, in particular for the late transcription. Type II topoisomerases are involved in resolving DNA tangles and supercoils by cutting the duplex and allowing the DNA replication to proceed. In this study, we report that P1192R is actively transcribed throughout infection, being detected from 2 hpi and reaching a maximum concentration around 16 hpi. P1192R knockdown experiments revealed its critical role for viral infection, given by a reduction in viral transcripts, cytopathic effect, the number of viral factories per cell, and virus yields. We also demonstrated that enrofloxacin exposure during the late phase of infection induces viral genomes fragmentation, whereas, when added at early phase of infection completely abolishes replication. The data obtained from I215L, QP509L. Q706L and P1192R characterization studies opens new venues to the rational design of a mutant virus lacking these genes, and also points new pathways to be targeted by antiviral drugs.
Molecular characterization and functional analysis of ORF P1192R from African swine fever virus
Publication . Coelho, João Nuno Santos; Leitão, José Alexandre da Costa Perdigão e Cameira; Ferreira, Fernando António da Costa
African swine fever virus (ASFV) is a nucleo-cytoplasmic large DNA arbovirus and the
single member of the family Asfarviridae. It infects soft ticks of the genus Ornithodoros as
well as all members of the family Suidae, representing a global threat for pig husbandry for
which there is currently no effective vaccine or treatment. Since the ASFV viral cycle is
mainly cytoplasmic, it has been found/predicted to code for many components of the
replicative and transcriptional machineries. Of these, and based in sequence homologies, a
putative type II DNA topoisomerase-coding ORF (P1192R) was identified in the ASFV
genome. DNA topoisomerases are enzymes that modulate the topological state of DNA
molecules. They are ubiquitous and essential, participating in processes such as DNA
replication, recombination and repair and also in transcription. Since ASFV has a large linear
genome, with 170 to 190 kbp depending on the isolate, containing terminal inverted repeats
and covalently closed ends, a type II topoisomerase may be indispensable for viral replication
and/or transcriptional events. The main objectives of this work were to deepen the study on
ORF P1192R and determine if it indeed codes for a type II DNA topoisomerase and, if so, to
characterize its activity. Bioinformatics and phylogenetic analyses showed that ORF P1192R
is highly conserved among the fourteen ASFV isolates analyzed and, although its amino acid
sequence clearly diverges from other type II topoisomerases, the structural organization is
preserved and conserved motifs and domains essential for activity are present. Transient
expression of GFP-pP1192R in COS-7 cells revealed an exclusively cytoplasmic distribution
of the protein, which remained unaltered by treatment with leptomycin B. Using Vero cells or
swine macrophages infected with ASFV isolate Ba71V or L60, respectively, expression of
pP1192R was observed in the late phase of infection, co-localizing with the viral factories,
where the bulk of viral replication and transcription occurs. Heterologous expression of
pP1192R in Saccharomyces cerevisiae demonstrated that it functionally complements a top2
thermo-sensitive mutation and that it exhibits ATP-dependent decatenation activity. The
purified recombinant pP1192R was found to efficiently decatenate kDNA and to processively
relax supercoiled plasmid DNA, which are characteristics of a type II topoisomerase. The
optimal requirements in terms of pH, temperature and salt, divalent ions and ATP
concentrations for pP1192R activity in vitro were determined and its sensitivity to a panel of
topoisomerase poisons and inhibitors was tested. Our results indicate that P1192R may be a
target for studying, and possibly controlling, ASFV transcription and replication.
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Entidade financiadora
European Commission
Programa de financiamento
FP7
Número da atribuição
311931