The Foot-and-Mouth Disease Virus Replication Complex: Dissecting the Role of the Viral Polymerase(3Dpol) and Investigating Interactions with Phosphatidylinositol-4-kinase (PI4K)

Replication of many positive-strand RNA viruses have been shown to occur within intracellular membrane-associated compartments termed replication complexes. Replication of viral RNA occurs within these intracellular compartments as a way for the virus to concentrate the structural and non-structural components into a small area to facilitate replication as well as protecting the virus components from host cell pathogen recognition and innate immune responses. Using immunofluorescent confocal and electron microscopy, foot-and-mouth disease virus (FMDV) has been shown to dysregulate Golgi and ER-derived membranes, but to date, no distinct membrane-bound replication complex comprised of viral RNA, structural and nonstructural proteins, and host-cell proteins have yet to be identified for FMDV. The FMDV RNA-dependent RNA polymerase, 3Dpol, is the primary protein involved in virus genome replication and has been previously shown to form higher-order fibril like structures in vitro in the presence of RNA. These 3Dpol fibril structures could act to ‘scaffold’ replication complex formation. Here, several mutations were made in 3Dpol to assess their role in higher-order complex formation. The ability for the different 3Dpol mutations to function was assessed biochemically, structurally and in cell culture. The results point towards the necessity for a fully functional (catalytically active) polymerase in the formation of the higher-order structures. Furthermore, complementation studies indicate that 3Dpol has two distinct functions necessary for replication within cells. Additionally, it was pertinent to investigate the role of membrane-associated kinases,such as PI4K, as a number of related viruses utilise this cellular pathway to form an optimal environment within which viral replication can occur by upregulating the formation of lipids used in the building of intracellular membranes. Investigation of translation and replication of FMDV RNA within cells show that FMDV does not appear to utilise the PI4K pathway. These results highlight differences between FMDV and other related picornaviruses and provide a basis to investigate alternative methods for replication complex formation

Foot-and-mouth disease virus genome replication is unaffected by inhibition of type III phosphatidylinositol-4-kinases

Foot-and-mouth disease virus (FMDV) causes economically-damaging infections of cloven-hooved animals, with outbreaks resulting in large financial losses to the agricultural industry. Due to the highly contagious nature of FMDV, research with infectious virus is restricted to a limited number of key facilities worldwide. FMDV subgenomic replicons are therefore important tools for the study of viral translation and genome replication. The type III phosphatidylinositol-4-kinases (PI4K) are a family of enzymes that play a key role in the production of replication complexes (viral factories) of a number of positive-sense RNA viruses and represents a potential target for novel pan-viral therapeutics. Here, we have investigated whether type III PI4Ks also play a role in the FMDV lifecycle, using a combination of FMDV subgenomic replicons and bicistronic IRES-containing reporter plasmids. We have demonstrated that replication of the FMDV replicon was unaffected by inhibitors of either PI4KIIIα or PI4KIIIβ. However, PIK93, an inhibitor previously demonstrated to target PI4KIIIβ, did inhibit IRES-mediated protein translation. Consistent with this, cells transfected with FMDV replicons did not exhibit elevated levels of PI4P lipids. These results are therefore supportive of the hypothesis that FMDV genome replication does not require type III PI4K activity and does not activate these kinases

The effect of temperature on the stability of African swine fever virus BA71V isolate in environmental water samples

African swine fever virus (ASFV) is known to be very stable and can remain infectious over long periods of time especially at low temperatures and within different matrices, particularly those containing animal-derived organic material. However, there are some gaps in our knowledge pertaining to the survivability and infectivity of ASFV in groundwater. This study aims to determine the stability and infectivity of the cell culture-adapted ASFV strain BA71V by plaque assay after incubation of the virus within river water samples at three different environmentally relevant temperatures (4 °C, 15 °C, and 21 °C) over the course of 42 days. The results from this study indicate that ASFV can remain stable and infectious when maintained at 4 °C in river water for more than 42 days, but as incubation temperatures are increased, the stability is reduced, and the virus is no longer able to form plaques after 28 days and 14 days, respectively, when stored at 15 °C and 21 °C. Characterizing the survivability of ASFV in groundwater can allow us to develop more appropriate inactivation and disinfection methods to support disease control and mitigate ASFV outbreaks

Employing transposon mutagenesis to investigate foot-and-mouth disease virus replication

Probing the molecular interactions within the foot-and-mouth disease virus (FMDV) RNA replication complex has been restricted in part to the lack of suitable reagents. Random insertional mutagenesis has proven an excellent method to reveal domains of proteins essential for viral replication as well as locations that can tolerate small genetic insertions. Such insertion sites can be subsequently adapted by the incorporation of commonly used epitope tags and so facilitate their detection with commercial available reagents. In this study, we use random transposon-mediated mutagenesis to produce a library of 15 nucleotide insertions in the FMDV nonstructural polyprotein. Using a replicon-based assay we isolated multiple replication-competent as well as replication-defective insertions. We have adapted the replication competent insertion sites for the successful incorporation of epitope tags within FMDV non-structural proteins, for the use in a variety of downstream assays. Additionally, we show that replication of some of the replication-defective insertion mutants can be rescued by co-transfection of a 'helper' replicon, demonstrating a novel use of random mutagenesis to identify inter-genomic trans-complementation. Both the epitope tags and replication-defective insertions identified here will be valuable tools for probing interactions within picornaviral replication complexes

Site-directed M2 proton channel inhibitors enable synergistic combination therapy for rimantadine-resistant pandemic influenza.

Pandemic influenza A virus (IAV) remains a significant threat to global health. Preparedness relies primarily upon a single class of neuraminidase (NA) targeted antivirals, against which resistance is steadily growing. The M2 proton channel is an alternative clinically proven antiviral target, yet a near-ubiquitous S31N polymorphism in M2 evokes resistance to licensed adamantane drugs. Hence, inhibitors capable of targeting N31 containing M2 (M2-N31) are highly desirable. Rational in silico design and in vitro screens delineated compounds favouring either lumenal or peripheral M2 binding, yielding effective M2-N31 inhibitors in both cases. Hits included adamantanes as well as novel compounds, with some showing low micromolar potency versus pandemic "swine" H1N1 influenza (Eng195) in culture. Interestingly, a published adamantane-based M2-N31 inhibitor rapidly selected a resistant V27A polymorphism (M2-A27/N31), whereas this was not the case for non-adamantane compounds. Nevertheless, combinations of adamantanes and novel compounds achieved synergistic antiviral effects, and the latter synergised with the neuraminidase inhibitor (NAi), Zanamivir. Thus, site-directed drug combinations show potential to rejuvenate M2 as an antiviral target whilst reducing the risk of drug resistance