DNA-Binding Properties of African Swine Fever Virus pA104R, a Histone-Like Protein Involved in Viral Replication and Transcription

Abstract

African swine fever virus (ASFV) codes for a putative histone-like protein (pA104R) with extensive sequence homology to bacterial proteins that are implicated in genome replication and packaging. Functional characterization of purified recombinant pA104R revealed that it binds to single-stranded DNA (ssDNA) and double-stranded DNA (dsDNA) over a wide range of temperatures, pH values, and salt concentrations and in an ATP-independent manner, with an estimated binding site size of about 14 to 16 nucleotides. Using site-directed mutagenesis, the arginine located in pA104R's DNA-binding domain, at position 69, was found to be relevant for efficient DNA-binding activity. Together, pA104R and ASFV topoisomerase II (pP1192R) display DNA-supercoiling activity, although none of the proteins by themselves do, indicating that the two cooperate in this process. In ASFV-infected cells, A104R transcripts were detected from 2 h postinfection (hpi) onward, reaching a maximum concentration around 16 hpi. pA104R was detected from 12 hpi onward, localizing with viral DNA replication sites and being found exclusively in the Tritoninsoluble fraction. Small interfering RNA (siRNA) knockdown experiments revealed that pA104R plays a critical role in viral DNA replication and gene expression, with transfected cells showing lower viral progeny numbers (up to a reduction of 82.0%), lower copy numbers of viral genomes (Ϫ78.3%), and reduced transcription of a late viral gene (Ϫ47.6%). Taken together, our results strongly suggest that pA104R participates in the modulation of viral DNA topology, probably being involved in viral DNA replication, transcription, and packaging, emphasizing that ASFV mutants lacking the A104R gene could be used as a strategy to develop a vaccine against ASFV.IMPORTANCE Recently reintroduced in Europe, African swine fever virus (ASFV) causes a fatal disease in domestic pigs, causing high economic losses in affected countries, as no vaccine or treatment is currently available. Remarkably, ASFV is the only known mammalian virus that putatively codes for a histone-like protein (pA104R) that shares extensive sequence homology with bacterial histone-like proteins. In this study, we characterized the DNA-binding properties of pA104R, analyzed the functional importance of two conserved residues, and showed that pA104R and ASFV topoisomerase II cooperate and display DNA-supercoiling activity. Moreover, pA104R is expressed during the late phase of infection and accumulates in viral DNA replication sites, and its downregulation revealed that pA104R is required for viral DNA replication and transcription. These results suggest that pA104R participates in the modulation of viral DNA topology and genome packaging, indicating that A104R deletion mutants may be a good strategy for vaccine development against ASFV.

Role of Condensing Particles in Polymer Confinement: A Model for Virus-Packed “Minichromosomes”

“…The largest, most complex members of the lineage form the recently proposed Megavirales order (66), which includes viruses of ecological (amphibian ranaviruses), economical (fish iridoviruses and poxviruses and African swine fever virus), and biomedical (human poxviruses) importance. There is a strong indication from gene sequencing, as well as molecular and structural analyses, that the presence of DNA-condensing proteins is likely to be a common trait in this growing virus family (8)(9)(10)(11)(12)(13)(14)(15)(16)67). Assembly of most of these viruses is poorly characterized, and even less work has been done to understand DNA packing within their capsids.…”

A Proteomic Atlas of the African Swine Fever Virus Particle

“…3, antibodies against p10 strongly labeled the electron-dense DNA-containing nucleoid of mature ASFV particles, which is consistent with a hypothetical structural role in this viral domain that deserves further research. The second structural DNA-binding protein, pA104R, is a histone-like protein, also located at the virus nucleoid (34), that, on the basis of knockdown experiments with small interfering RNAs, has been involved in viral transcription, DNA replication, and genome packaging (39). Thus, proteins p10 and pA104R are ideal targets to study unexplored ASFV processes relating to nucleoid formation and genome encapsidation.…”

African swine fever virus-like integrated elements in a soft tick genome – an ancient virus vector arms race?

“…The possible role of EVEs in the antiviral response of ticks might also explain why some EVEs have been protected from accumulating mutations. However, it remains unclear why for the highly identical ASFLI-A104R protein, whose viral counterpart is crucial for virus replication (49), that is one of the most abundant ASFV proteins in infected wild boar lung cells and infected Vero cells (50) and is highly conserved between ASFV isolates (49), no siRNA or piRNA could be detected. While our data might lead to the conclusion that the ASFLI-A104R protein is not translated, the amount of protein might just have been too low for detection or cellular machinery might degrade the protein.…”