Mapping codon usage of the translation initiation region in porcine reproductive and respiratory syndrome virus genome

<p>Abstract</p> <p>Background</p> <p>Porcine reproductive and respitatory syndrome virus (PRRSV) is a recently emerged pathogen and severely affects swine populations worldwide. The replication of PRRSV is tightly controlled by viral gene expression and the codon usage of translation initiation region within each gene could potentially regulate the translation rate. Therefore, a better understanding of the codon usage pattern of the initiation translation region would shed light on the regulation of PRRSV gene expression.</p> <p>Results</p> <p>In this study, the codon usage in the translation initiation region and in the whole coding sequence was compared in PRRSV ORF1a and ORFs2-7. To investigate the potential role of codon usage in affecting the translation initiation rate, we established a codon usage model for PRRSV translation initiation region. We observed that some non-preferential codons are preferentially used in the translation initiation region in particular ORFs. Although some positions vary with codons, they intend to use codons with negative CUB. Furthermore, our model of codon usage showed that the conserved pattern of CUB is not directly consensus with the conserved sequence, but shaped under the translation selection.</p> <p>Conclusions</p> <p>The non-variation pattern with negative CUB in the PRRSV translation initiation region scanned by ribosomes is considered the rate-limiting step in the translation process.</p

Development of a foot-and-mouth disease virus replicon system for the study of RNA replication

Foot-and-mouth disease (FMD) is a highly infectious disease of wild and domestic cloven–hoofed animals such as cattle, swine and deer. It is caused by one of the most contagious animal diseases known; FMD virus (FMDV). Since the disease is endemic in many countries, transmission by international travel/trade presents an on-going potential threat to the UK. Very little is known at the molecular level about how FMDV replicates within host cells. In this study, FMDV replicons have been used to investigate FMDV RNA replication and to improve our understanding of the viral life cycle: a process which will aid in the production of a new generation of live-attenuated vaccine candidate strains. Sequences encoding the capsid coding region of the genome have been replaced with green fluorescent protein (GFP) such that replication can be monitored in live cells via GFP fluorescence. This provides a rapid, non-invasive screen for replicative fitness that can be used outwith high disease security facilities. Differences between replicating and non-replicating forms could easily be distinguished, highlighting the potential of this system to screen for attenuated genomes. The FMDV replicon system was improved through a series of construct modifications until an optimal system was produced. A range of different methods were used to attenuate the replication of these genomes. Of major significance is the finding that increasing dinucleotide frequencies were shown to decrease growth kinetics of Echovirus 7 – as opposed to altering the codon-pair bias - and the application of this finding to construction of further replicon systems (and RNA viruses in general) is described

Bioinformatics in New Generation Flavivirus Vaccines

Flavivirus infections are the most prevalent arthropod-borne infections world wide, often causing severe disease especially among children, the elderly, and the immunocompromised. In the absence of effective antiviral treatment, prevention through vaccination would greatly reduce morbidity and mortality associated with flavivirus infections. Despite the success of the empirically developed vaccines against yellow fever virus, Japanese encephalitis virus and tick-borne encephalitis virus, there is an increasing need for a more rational design and development of safe and effective vaccines. Several bioinformatic tools are available to support such rational vaccine design. In doing so, several parameters have to be taken into account, such as safety for the target population, overall immunogenicity of the candidate vaccine, and efficacy and longevity of the immune responses triggered. Examples of how bio-informatics is applied to assist in the rational design and improvements of vaccines, particularly flavivirus vaccines, are presented and discussed

Modifications to the foot-and-mouth disease virus 2A peptide; influence on polyprotein processing and virus replication

ABSTRACT Foot-and-mouth disease virus (FMDV) has a positive-sense single-stranded RNA (ssRNA) genome that includes a single, large open reading frame encoding a polyprotein. The cotranslational “cleavage” of this polyprotein at the 2A/2B junction is mediated by the 2A peptide (18 residues in length) using a nonproteolytic mechanism termed “ribosome skipping” or “StopGo.” Multiple variants of the 2A polypeptide with this property among the picornaviruses share a conserved C-terminal motif [D(V/I)E(S/T)NPG↓P]. The impact of 2A modifications within this motif on FMDV protein synthesis, polyprotein processing, and virus viability were investigated. Amino acid substitutions are tolerated at residues E 14 , S 15 , and N 16 within the 2A sequences of infectious FMDVs despite their reported “cleavage” efficiencies at the 2A/2B junction of only ca. 30 to 50% compared to that of the wild type (wt). In contrast, no viruses containing substitutions at residue P 17 , G 18 , or P 19 , which displayed little or no “cleavage” activity in vitro , were rescued, but wt revertants were obtained. The 2A substitutions impaired the replication of an FMDV replicon. Using transient-expression assays, it was shown that certain amino acid substitutions at residues E 14 , S 15 , N 16 , and P 19 resulted in partial “cleavage” of a protease-free polyprotein, indicating that these specific residues are not essential for cotranslational “cleavage.” Immunofluorescence studies, using full-length FMDV RNA transcripts encoding mutant 2A peptides, indicated that the 2A peptide remained attached to adjacent proteins, presumably 2B. These results show that efficient “cleavage” at the 2A/2B junction is required for optimal virus replication. However, maximal StopGo activity does not appear to be essential for the viability of FMDV. IMPORTANCE Foot-and-mouth disease virus (FMDV) causes one of the most economically important diseases of farm animals. Cotranslational “cleavage” of the FMDV polyprotein precursor at the 2A/2B junction, termed StopGo, is mediated by the short 2A peptide through a nonproteolytic mechanism which leads to release of the nascent protein and continued translation of the downstream sequence. Improved understanding of this process will not only give a better insight into how this peptide influences the FMDV replication cycle but may also assist the application of this sequence in biotechnology for the production of multiple proteins from a single mRNA. Our data show that single amino acid substitutions in the 2A peptide can have a major influence on viral protein synthesis, virus viability, and polyprotein processing. They also indicate that efficient “cleavage” at the 2A/2B junction is required for optimal virus replication. However, maximal StopGo activity is not essential for the viability of FMDV. </jats:p

Optimization of Bacterial Luciferase for Expression in Mammalian Cells

This study describes the optimization of expression of the bacterial luciferase enzyme in mammalian cells. Previous attempts to express this heterodimeric enzyme complex in mammalian cells have been met with only modest success. In this research effort, several vector formats were evaluated to fully determine the optimal format for the expression of these genes. It was determined that the bacterial luciferase enzyme produced optimal bioluminescence in mammalian cells when the genes were cloned and expressed as a bicistronic transcript fused with an internal ribosomal entry site (IRES). To optimize the enzyme expression further, a novel approach to codon optimize the genes was performed. To accomplish this task, completely synthetic versions of the codon optimized sequences were generated. This codon optimization, led to an increase in bioluminescence levels greater than two orders of magnitude versus the wild type genes. Additionally, the availability of the FMNH2 substrate was evaluated and determined to be a limiting substrate for the reaction. In an attempt to alleviate this limitation, a flavin oxidoreductase gene (frp) from Vibrio harveyi was cloned and expressed along with the codon optimized luxA and luxB genes. Although the expression of this enzyme enhanced the bioluminescence significantly, FMNH2 remains the limiting substrate for optimal bioluminescence. To produce a usable reporter cell line, the reporter must remain stable within the cells for long periods of time. The overall stability of the engineered cells was assessed to determine the persistence of the reporter for long-term monitoring applications. These data revealed that the luciferase genes were stable in HEK293 cells for more than forty passages (five months) in culture in the absence of antibiotic, indicating that these cell lines would be stable enough for relatively long term monitoring projects and applications

The preparation of antigen for the generation of polyclonal antibodies against the capsid subunit, VP1, and the viral protease, 3Cpro, of Theiler's murine encephalomyelitis virus (TMEV)

The Picornaviridae is a family of viruses of economic importance that have a major impact on human and animal health. Some of the major genera found in the Picornaviridae family are Enterovirus which includes Poliovirus (PV) and Human Rhinovirus (HRV), Cardiovirus which includes Theiler’s murine encephalomyelitis virus (TMEV) and Saffold virus (SAFV), Aphthovirus of which the Foot and Mouth disease virus (FMDV) is a member and Hepatovirus which includes Hepatitis A virus (HAV). Picornaviruses have a single stranded, positive sense RNA genome which is approximately 7.5-8.4 kb pairs in size. The picornavirus genome is translated into a large polyprotein and is proteolytically cleaved by viral proteases namely 2Apro, 3Cpro and 3CDpro into mature viral structural and non-structural polypeptides encoded by the P1, P2 and P3 domains. Picornaviruses utilise host cell machinery and cellular pathways for entry and uncoating, genome replication and capsid assembly. In our laboratory, we are studying the mechanisms by which TMEV interacts with host cell components and our recent research shows that molecular chaperones are required for a production infection. To follow up on this observation, the overall aim of this study was to prepare antigen for the generation of polyclonal antibodies against the TMEV VP1 and 3Cpro proteins. To this end, the TMEV VP1 and 3Cpro amino acid sequences were analysed to identify hydrophobic, hydrophilic and antigenic regions. Homology modelling was performed in order to predict linear B cell epitopes exposed on the surface of the protein structures. The full length coding sequences of VP1 and 3Cpro were selected for amplification by the PCR and cloning into pQE-80L for expression in a bacterial system. Time course induction studies of recombinant VP1 and 3Cpro showed that the proteins were maximally expressed at 6 hrs and 4 hrs respectively. Recombinant VP1 was solubilised using the detergent, Sarcosyl and purified by Nickel affinity chromatography under native conditions. Because recombinant VP1 co-purified with an unidentified protein, the pET expression system was used. Although no protein of the estimated size was observed by SDS-PAGE analysis in the time course induction study, Western analysis using anti-His6 (2) antibodies detected a signal of ~35 kDa. Solubility studies resulted in the presence of two protein bands in the insoluble fraction resolved between 35 and 40 kDa. Recombinant 3Cpro expressed in a bacterial system was predominantly present in the insoluble fraction. Treatment with Sarcosyl had no effect on the solubility of the recombinant protein and it was therefore purified under denaturing conditions using 8M urea. Following dialysis, 3Cpro was used for immunisation of rabbits. Crude anti-TMEV 3Cpro antibodies were able to detect as little as 107 ng of bacterially expressed antigen at a dilution of 1:100 000 by Western analysis. The presence of contaminating proteins was reduced using pre-cleared anti-TMEV 3Cpro antibodies. The antibodies were unable to detect virally expressed 3Cpro in BHK-21 cell lysate supernatant. In an attempt to determine whether TMEV 3Cpro is present in the insoluble fraction, anti-TMEV 3Cpro antibodies were tested using total protein prepared from infected and mock-infected cell lysates. Once again, no protein band the size of 3Cpro was detected. The antibodies were further tested for detection of 3Cpro in TMEV-infected cells by indirect immunofluorescence and confocal microscopy. A diffuse cytoplasmic and perinuclear distribution, as well as nuclear staining, was observed in infected BHK-21 cells. This staining pattern resembled that observed for the HRV, FMDV and EMCV 3Cpro in similar experiments. Further experiments are required to confirm specificity of these antibodies for virally-expressed 3Cpro by Western analysis and indirect immunofluorescence

Characterization of 5 UTR splicing and the CGI in HIV-2 RNA

HIV-2 tightly regulates several steps of its replication cycle via regulatory elements found within the 5 untranslated region of HIV-2 genomic RNA. Two elements of interest are the 5 UTR intron and the proposed long distance base pairing interaction between the C-box and G-box termed the CGI. This research focuses on the effects of 5 UTR splicing and the CGI in HIV-2 translation and replication. The central hypothesis is that both 5 UTR splicing and the CGI modulate HIV-2 translation and replication. This hypothesis was tested and supported by employing in vitro translation assays, SELEX, and cell culture studies. Results of these experiments demonstrate that splicing of the 5 UTR intron produces an isoform of gag mRNA that is specialized for high translational efficiency. Further, evidence is provided for a novel branched secondary structure within the CGI that is important for HIV-2 replication. In addition, we show that mutation of the C-box alone can enhance RNA encapsidation and mutation of the G-box can alter levels of Gag protein isoforms. These studies suggest coordinated regulation of RNA translation, dimerization, and encapsidation during HIV-2 replication. This research provides new insight into HIV translational mechanisms, in turn identifying potential antiviral targets that may be exploited for antiviral therapeutic strategies

The polyclonal antibody response to FMDV in cattle and African buffalo

Protection against the highly contagious foot and mouth disease virus (FMDV) coincides with neutralising antibody titres. Infection in cattle is characterised by 100% morbidity of an acute vesicular disease whilst infection of their closest relative, the African buffalo, is sub-clinical despite having diverged only 5.7 – 9.3 million years ago (Glanzmann et al., 2016; 1). The germline and antibody repertoire in African buffalo has not previously been characterised and so the cause of their differential disease response may be the production of a more specific and / or avid antibody response to FMDV than cattle. The cattle and African buffalo antibody germline was sought to characterise the recombinatorial potential of the antibody loci and their subsequent primary antibody repertoire. Expression of the antibody heavy chain (IGH) and antibody lambda light chain (IGL) was investigated with qPCR and RNA-seq. The antibody repertoire in response to FMDV infection was interrogated in African buffalo infected with SAT1 FMDV and compared to the cattle IGH repertoire inoculated with highly purified SAT1 FMDV antigen. The recombinatorial potential of the cattle and African buffalo IGH and IGL is severely limited compared to other species such as mice and human. The characterisation of the cattle IGH and IGL is the most accurate to date and reveals internal duplications of the IGH, disrupting the expected IGHV-IGHD-IGHJ-IGHC ordering seen in mammalian immune loci and resulting in four IGHD regions, containing long and ultra-long IGHD. These IGHD provide a novel diversification mechanism that can compensate for limited germline diversity by forming long and ultra-long CDR3H loops that are highly diverse in their length and amino acid composition. The African buffalo antibody repertoire also forms highly diverse long and ultra-long CDR3H, despite lack of evidence for the existence of the duplications in their IGH. Limited variability is seen in the length and amino acid composition of the IGL in both species, suggesting they are playing a structural role to support these unusual long and ultra-long CDR3H. In response to FMDV infection in African buffalo, a dramatic increase in specific long and ultra-long CDR3H sequence abundance occurs but this change in frequency of specific transcripts is absent in cattle. The differential antibody response may account for the protection of African buffalo against FMD