Inhibition of Chikungunya virus genome replication by targeting essential RNA structures within the virus genome

Chikungunya virus (CHIKV) is a pathogenic arbovirus spread by Aedes spp. mosquitos. CHIKV has a wide global prevalence and represents a significant health burden in affected populations. Symptoms of CHIKV infection include fever, rashes and debilitating joint and muscle pain, which can persist for several months to years in some patients. To date, there remains no vaccine or specific antiviral therapy against this important human pathogen. Based on our previously published structural and phenotypic analysis of the 5′ region of the CHIKV genome, we designed a panel of locked nucleic acid oligonucleotides to bind structured RNA replication elements within the virus genome, which are essential for efficient CHIKV replication. Using electromobility shift assays, we confirmed the relative binding efficiencies of each LNA to target CHIKV genomic RNA. We then went on to demonstrate, using both sub-genomic replicon and infectious virus systems, that targeting individual RNA replication elements inhibits CHIKV genome replication and production of infectious virus. Time course assays demonstrated that LNAs can access the CHIKV replication complex and virus genome, during active virus replication. For the first time, these findings show that functional RNA elements can be specifically targeted during the CHIKV lifecycle and consequently represent potential novel antiviral targets

The Oxysterol 25-Hydroxycholesterol Inhibits Replication of Murine Norovirus

Cholesterol, an essential component of mammalian cells, is also an important factor in the replicative-cycles of several human and animal viruses. The oxysterol, 25-hydroxycholesterol, is produced from cholesterol by the enzyme, cholesterol 25-hydroxylase. 25-hydroxycholesterol (25-HC) has been shown to have anti-viral activities against a wide range of viruses, including a range of positive-sense RNA viruses. In this study, we have investigated the role of 25-HC in norovirus replication using murine norovirus (MNV) as a model system. As a control, we employed herpes simplex virus-1 (HSV-1), a pathogen previously shown to be inhibited by 25-HC. Consistent with previous studies, 25-HC inhibited HSV-1 replication in the MNV-susceptible cell line, RAW264.7. Treating RAW264.7 cells with sub-cytotoxic concentrations of 25-HC reduced the MNV titers. However, other sterols such as cholesterol or the oxysterol, 22-S-hydroxycholesterol (22-S-HC), did not inhibit MNV replication. Moreover, treating MNV-infected RAW264.7 cells with 25-HC-stimulated caspase 3/7 activity, which leads to enhanced apoptosis and increased cell death. Our study adds noroviruses to the list of viruses inhibited by 25-HC and begins to offer insights into the mechanism behind this inhibition

Photodynamic inactivation of bacteriophage MS2: the A- protein is the target of virus inactivation

Singlet oxygen mediated oxidation has been shown to be responsible for photodynamic inactivation (PDI) of viruses in solution with photosensitisers such as 5, 10, 15, 20-tetrakis (1-methyl-4-pyridinio) porphyrin tetra p-toluenesulfonate (TMPyP). The capsids of non-enveloped viruses, such as bacteriophage MS2, are possible targets for viral inactivation by singlet oxygen oxidation. Within the capsid (predominantly composed of coat protein), the A-protein acts as the host recognition and attachment protein. The A-protein has two domains; an α-helix domain and a β-sheet domain. The α-helix domain is attached to the viral RNA genome inside the capsid while the β-sheet domain, which is on the surface of the capsid, is believed to be the site for attachment to the host bacteria pilus during infection. In this study, 4 sequence-specific antibodies were raised against 4 sites on the A-protein. Changes induced by the oxidation of singlet oxygen were compared to the rate of PDI of the virus. Using these antibodies, our results suggest that the rate of PDI is relative to loss of antigenicity of two sites on the A-protein. Our data further showed that PDI caused aggregation of MS2 particles and crosslinking of MS2 coat protein. However, these inter- and intra-capsid changes did not correlate to the rate of PDI we observed in MS2. Possible modes of action are discussed as a means to gaining insight to the targets and mechanisms of PDI of viruses

Potent antiviral agents fail to elicit genetically-stable resistance mutations in either enterovirus 71 or Coxsackievirus A16

Enterovirus 71 (EV71) and Coxsackievirus A16 (CVA16) are the two major causative agents 13 of hand, foot and mouth disease (HFMD), for which there are currently no licenced 14 treatments. Here, the acquisition of resistance towards two novel capsid-binding compounds, 15 NLD and ALD, was studied and compared to the analogous compound GPP3. During serial 16 passage, EV71 rapidly became resistant to each compound and mutations at residues I113 17 and V123 in VP1 were identified. A mutation at residue 113 was also identified in CVA16 18 after passage with GPP3. The mutations were associated with reduced thermostability and 19 were rapidly lost in the absence of inhibitors. In silico modelling suggested that the mutations 20 prevented the compounds from binding the VP1 pocket in the capsid. Although both viruses 21 developed resistance to these potent pocket-binding compounds, the acquired mutations were 22 associated with large fitness costs and reverted to WT phenotype and sequence rapidly in the 23 absence of inhibitors. The most effective inhibitor, NLD, had a very large selectivity index, 24 showing interesting pharmacological properties as a novel anti-EV71 agent

Both cis and trans Activities of Foot-and-Mouth Disease Virus 3D Polymerase Are Essential for Viral RNA Replication

The Picornaviridae is a large family of positive-sense RNA viruses that contains numerous human and animal pathogens, including foot-and-mouth disease virus (FMDV). The picornavirus replication complex comprises a co-ordinated network of protein-protein and protein-RNA interactions involving multiple viral and host-cellular factors. Many of the proteins within the complex possess multiple roles in viral RNA replication, some of which can be provided in trans (i.e. via expression from a separate RNA molecule), whilst other are required in cis (i.e. expressed from the template RNA molecule). In vitro studies have suggested that multiple copies of the RNA-dependent RNA-polymerase (RdRp), 3D, are involved in the viral replication complex. However, it is not clear whether all these molecules are catalytically active or what other function(s) they provide. In this study, we aimed to distinguish between catalytically-active 3D molecules and those which build a replication complex. We report a novel non-enzymatic cis-acting function of 3D that is essential for viral genome replication. Using a FMDV replicon in complementation experiments, our data demonstrate that this cis-acting role of 3D is distinct from the catalytic activity, which is predominantly trans-acting. Immunofluorescence studies suggest that both cis- and trans acting 3D molecules localise to the same cellular compartment. However, our genetic and structural data suggest that 3D interacts in cis with RNA stem-loops that are essential for viral RNA replication. Together, this study identifies a previously undescribed aspect of picornavirus replication complex structure-function and an important methodology for probing such interactions further

Photodynamic inactivation of non-enveloped RNA viruses

We recently reported the photodynamic inactivation (PDI) of bacteriophage MS2 with a photosensitiser- 5, 10, 15, 20-tetrakis (1-methyl-4-pyridinio) porphyrin- tetra- p-toluene sulfonate (TMPyP) in solution and concluded that the A-protein of the virus is the main target of inactivation. Here, we have extended these studies and carried out PDI of bacteriophage Qβ, bovine enterovirus 2 (BEV-2) and type 1 murine norovirus (MNV-1). The rate of inactivation observed was in the order MS2 > Qβ > MNV-1 > BEV-2. Data suggested that TMPyP-treatment could also target the viral genome as well as result in disintegration/disassembly of viral particles. Although emergence of viral drug resistance is a well-documented phenomenon, it was not possible to generate PDI-resistant MS2. However, emergence of a mutation in the lysis protein was detected after serial exposure to PDI

Development of an ELISA to distinguish between foot-and-mouth disease virus infected and vaccinated animals utilising the viral non-structural protein 3ABC

Introduction. Foot-and-mouth disease (FMD) is a highly contagious and economically devastating viral disease of livestock and is endemic in much of Asia, including Pakistan. Vaccination is used to control disease outbreaks and sensitive diagnostic methods which can differentiate infected animals from vaccinated animals (DIVA) are essential for monitoring the effectiveness of disease control programmes. Tests based on the detection of the non-structural protein (NSP) 3ABC are reliable indicators of virus replication in infected and vaccinated populations. Hypothesis/Gap statement. Diagnosis of FMD is expensive using commercial ELISA kits, yet is essential for controlling this economically-important disease. Aim. The development of a low-cost diagnostic ELISA, using protein made in Escherichia coli . Methodology. In this study, the viral precursor protein 3ABC (r3ABC) was expressed in E. coli , solubilised using detergent and purified using nickel affinity chromatography. The fusion protein contained an attenuating mutation in the protease and a SUMO tag. It was characterised by immunoblotting and immunoprecipitation, which revealed antigenicity against virus-specific polyclonal sera. Using r3ABC, an indirect ELISA was developed and evaluated using field sera from healthy/naïve, vaccinated and infected animals. Results. The diagnostic sensitivity and specificity of the r3ABC in-house ELISA were 95.3 and 96.3% respectively. The ELISA was validated through comparison with the commercially available ID Screen FMD NSP competition kit. Results indicated good concordance rates on tested samples and high agreement between the two tests. Conclusion. The ELISA described here can effectively differentiate between infected and vaccinated animals and represents an important low cost tool for sero-surveillance and control of FMD in endemic settings

Membrane Interactions and Uncoating of Aichi Virus, a Picornavirus That Lacks a VP4.

Kobuviruses are an unusual and poorly characterized genus within the picornavirus family and can cause gastrointestinal enteric disease in humans, livestock, and pets. The human kobuvirus Aichi virus (AiV) can cause severe gastroenteritis and deaths in children below the age of 5 years; however, this is a very rare occurrence. During the assembly of most picornaviruses (e.g., poliovirus, rhinovirus, and foot-and-mouth disease virus), the capsid precursor protein VP0 is cleaved into VP4 and VP2. However, kobuviruses retain an uncleaved VP0. From studies with other picornaviruses, it is known that VP4 performs the essential function of pore formation in membranes, which facilitates transfer of the viral genome across the endosomal membrane and into the cytoplasm for replication. Here, we employ genome exposure and membrane interaction assays to demonstrate that pH plays a critical role in AiV uncoating and membrane interactions. We demonstrate that incubation at low pH alters the exposure of hydrophobic residues within the capsid, enhances genome exposure, and enhances permeabilization of model membranes. Furthermore, using peptides we demonstrate that the N terminus of VP0 mediates membrane pore formation in model membranes, indicating that this plays an analogous function to VP4. IMPORTANCE To initiate infection, viruses must enter a host cell and deliver their genome into the appropriate location. The picornavirus family of small nonenveloped RNA viruses includes significant human and animal pathogens and is also a model to understand the process of cell entry. Most picornavirus capsids contain the internal protein VP4, generated from cleavage of a VP0 precursor. During entry, VP4 is released from the capsid. In enteroviruses this forms a membrane pore, which facilitates genome release into the cytoplasm. Due to high levels of sequence similarity, it is expected to play the same role for other picornaviruses. Some picornaviruses, such as Aichi virus, retain an intact VP0, and it is unknown how these viruses rearrange their capsids and induce membrane permeability in the absence of VP4. Here, we have used Aichi virus as a model VP0 virus to test for conservation of function between VP0 and VP4. This could enhance understanding of pore function and lead to development of novel therapeutic agents that block entry

Recombinant expression of tandem-HBc virus-like particles (VLPs)

The hepatitis B virus (HBV) core protein (HBc) has formed the building block for virus-like particle (VLP) production for more than 30 years. The ease of production of the protein, the robust ability of the core monomers to dimerize and assemble into intact core particles, and the strong immune responses they elicit when presenting antigenic epitopes all demonstrate its promise for vaccine development (reviewed in Pumpens and Grens (Intervirology 44: 98–114, 2001)). HBc has been modified in a number of ways in attempts to expand its potential as a novel vaccine platform. The HBc protein is predominantly α-helical in structure and folds to form an L-shaped molecule. The structural subunit of the HBc particle is a dimer of monomeric HBc proteins which together form an inverted T-shaped structure. In the assembled HBc particle the four-helix bundle formed at each dimer interface appears at the surface as a prominent “spike.” The tips of the “spikes” are the preferred sites for the insertion of foreign sequences for vaccine purposes as they are the most highly exposed regions of the assembled particles. In the tandem-core modification two copies of the HBc protein are covalently linked by a flexible amino acid sequence which allows the fused dimer to fold correctly and assemble into HBc particles. The advantage of the modified structure is that the assembly of the dimeric subunits is defined and not formed by random association. This facilitates the introduction of single, larger sequences at the tip of each surface “spike,” thus overcoming the conformational clashes contingent on insertion of large structures into monomeric HBc proteins. Differences in inserted sequences influence the assembly characteristics of the modified proteins, and it is important to optimize the design of each novel construct to maximize efficiency of assembly into regular VLPs. In addition to optimization of the construct, the expression system used can also influence the ability of recombinant structures to assemble into regular isometric particles. Here, we describe the production of recombinant tandem-core particles in bacterial, yeast and plant expression systems

Structure–function analysis of the equine hepacivirus 5′ untranslated region highlights the conservation of translational mechanisms across the hepaciviruses

Equine hepacivirus (EHcV) (now also classified as hepacivirus A) is the closest genetic relative to hepatitis C virus (HCV) and is proposed to have diverged from HCV within the last 1000years. The 5′ untranslated regions (UTRs) of both HCV and EHcV exhibit internal ribosome entry site (IRES) activity, allowing cap-independent translational initiation, yet only the HCV 5′UTR has been systematically analysed. Here, we report a detailed structural and functional analysis of the EHcV 5′UTR. The secondary structure was determined using selective 2′ hydroxyl acylation analysed by primer extension (SHAPE), revealing four stem–loops, termed SLI, SLIA, SLII and SLIII, by analogy to HCV. This guided a mutational analysis of the EHcV 5′UTR, allowing us to investigate the roles of the stem–loops in IRES function. This approach revealed that SLI was not required for EHcV IRES-mediated translation. Conversely, SLIII was essential, specifically SLIIIb, SLIIId and a GGG motif that is conserved across the Hepaciviridae. Further SHAPE analysis provided evidence that this GGG motif mediated interaction with the 40S ribosomal subunit, whilst a CUU sequence in the apical loop of SLIIIb mediated an interaction with eIF3. In addition, we showed that a microRNA122 target sequence located between SLIA and SLII mediated an enhancement of translation in the context of a subgenomic replicon. Taken together, these results highlight the conservation of hepaciviral translation mechanisms, despite divergent primary sequences