Dataset Information

In silico studies of the African swine fever virus DNA polymerase X support an induced-fit mechanism.

ABSTRACT: The African swine fever virus DNA polymerase X (pol X), a member of the X family of DNA polymerases, is thought to be involved in base excision repair. Kinetics data indicate that pol X catalyzes DNA polymerization with low fidelity, suggesting a role in viral mutagenesis. Though pol X lacks the fingers domain that binds the DNA in other members of the X family, it binds DNA tightly. To help interpret details of this interaction, molecular dynamics simulations of free pol X at different salt concentrations and of pol X bound to gapped DNA, in the presence and in the absence of the incoming nucleotide, are performed. Anchors for the simulations are two NMR structures of pol X without DNA and a model of one NMR structure plus DNA and incoming nucleotide. Our results show that, in its free form, pol X can exist in two stable conformations that interconvert to one another depending on the salt concentration. When gapped double stranded DNA is introduced near the active site, pol X prefers an open conformation, regardless of the salt concentration. Finally, under physiological conditions, in the presence of both gapped DNA and correct incoming nucleotide, and two divalent ions, the thumb subdomain of pol X undergoes a large conformational change, closing upon the DNA. These results predict for pol X a substrate-induced conformational change triggered by the presence of DNA and the correct incoming nucleotide in the active site, as in DNA polymerase beta. The simulations also suggest specific experiments (e.g., for mutants Phe-102Ala, Val-120Gly, and Lys-85Val that may reveal crucial DNA binding and active-site organization roles) to further elucidate the fidelity mechanism of pol X.

SUBMITTER: Sampoli Benitez BA

PROVIDER: S-EPMC1367036 | biostudies-literature | 2006 Jan

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

In silico studies of the African swine fever virus DNA polymerase X support an induced-fit mechanism.

Sampoli Benítez Benedetta A BA Arora Karunesh K Schlick Tamar T

Biophysical journal 20051007 1

The African swine fever virus DNA polymerase X (pol X), a member of the X family of DNA polymerases, is thought to be involved in base excision repair. Kinetics data indicate that pol X catalyzes DNA polymerization with low fidelity, suggesting a role in viral mutagenesis. Though pol X lacks the fingers domain that binds the DNA in other members of the X family, it binds DNA tightly. To help interpret details of this interaction, molecular dynamics simulations of free pol X at different salt con ...[more]

PMID: 16214865

Similar Datasets

Project description:Recently moderate-virulence classical swine fever virus (CSFV) strains have been proven capable of generating postnatal persistent infection (PI), defined by the maintenance of viremia and the inability to generate CSFV-specific immune responses in animals. These animals also showed a type I interferon blockade in the absence of clinical signs. In this study, we assessed the infection generated in 7-week-old CSFV PI wild boars after infection with the African swine fever virus (ASFV). The wild boars were divided in two groups and were infected with ASFV. Group A comprised boars who were CSFV PI in a subclinical form and Group B comprised pestivirus-free wild boars. Some relevant parameters related to CSFV replication and the immune response of CSFV PI animals were studied. Additionally, serum soluble factors such as IFN-α, TNF-α, IL-6, IL-10, IFN-γ and sCD163 were analysed before and after ASFV infection to assess their role in disease progression.After ASFV infection, only the CSFV PI wild boars showed progressive acute haemorrhagic disease; however, the survival rates following ASFV infection was similar in both experimental groups. Notwithstanding, the CSFV RNA load of CSFV PI animals remained unaltered over the study; likewise, the ASFV DNA load detected after infection was similar between groups. Interestingly, systemic type I FN-α and IL-10 levels in sera were almost undetectable in CSFV PI animals, yet detectable in Group B, while detectable levels of IFN-γ were found in both groups. Finally, the flow cytometry analysis showed an increase in myelomonocytic cells (CD172a+) and a decrease in CD4+ T cells in the PBMCs from CSFV PI animals after ASFV infection.Our results showed that the immune response plays a role in the progression of disease in CSFV subclinically infected wild boars after ASFV infection, and the immune response comprised the systemic type I interferon blockade. ASFV does not produce any interference with CSFV replication, or vice versa. ASFV infection could be a trigger factor for the disease progression in CSFV PI animals, as their survival after ASFV was similar to that of the pestivirus-free ASFV-infected group. This fact suggests a high resistance in CSFV PI animals even against a virus like ASFV; this may mean that there are relevant implications for CSF control in endemic countries. The diagnosis of ASFV and CSFV co-infection in endemic countries cannot be ruled out and need to be studied in greater depth.

Project description:African swine fever virus (ASFV) causes hemorrhagic fever in domestic pigs, presenting the biggest global threat to animal farming in recorded history. Despite the importance of ASFV, little is known about the mechanisms and regulation of ASFV transcription. Using RNA sequencing methods, we have determined total RNA abundance, transcription start sites, and transcription termination sites at single-nucleotide resolution. This allowed us to characterize DNA consensus motifs of early and late ASFV core promoters, as well as a polythymidylate sequence determinant for transcription termination. Our results demonstrate that ASFV utilizes alternative transcription start sites between early and late stages of infection and that ASFV RNA polymerase (RNAP) undergoes promoter-proximal transcript slippage at 5' ends of transcription units, adding quasitemplated AU- and AUAU-5' extensions to mRNAs. Here, we present the first much-needed genome-wide transcriptome study that provides unique insight into ASFV transcription and serves as a resource to aid future functional analyses of ASFV genes which are essential to combat this devastating disease.IMPORTANCE African swine fever virus (ASFV) causes incurable and often lethal hemorrhagic fever in domestic pigs. In 2020, ASF presents an acute and global animal health emergency that has the potential to devastate entire national economies as effective vaccines or antiviral drugs are not currently available (according to the Food and Agriculture Organization of the United Nations). With major outbreaks ongoing in Eastern Europe and Asia, urgent action is needed to advance our knowledge about the fundamental biology of ASFV, including the mechanisms and temporal control of gene expression. A thorough understanding of RNAP and transcription factor function, and of the sequence context of their promoter motifs, as well as accurate knowledge of which genes are expressed when and the amino acid sequence of the encoded proteins, is direly needed for the development of antiviral drugs and vaccines.

Dataset Information

In silico studies of the African swine fever virus DNA polymerase X support an induced-fit mechanism.

Publications

In silico studies of the African swine fever virus DNA polymerase X support an induced-fit mechanism.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure