Characterization and sequence variation of the virulence-associated proteins of different tissue culture isolates of African Horsesickness virus serotype 4

African horsesickness, a disease of equines caused by African horsesickness virus (AHSV), is often fatal, although the pathogenic effect in different animals is variable. Current AHSV vaccines are live attenuated viruses generated by serial passage in cell culture. This process affects virus plaque size, which has been considered an indicator of AHSV virulence (Erasmus, 1966; Coetzer and Guthrie, 2004). The most likely AHSV proteins to be involved in viral virulence and attenuation are the outer capsid proteins, VP2 and VP5, due to their role in attachment of viral particles to cells and early stages of viral replication. Nonstructural protein NS3 may play an equally important role due to its function in release of viral particles from cells. Two viruses were obtained for this study, AHSV-4(1) and AHSV-4(13). The thirteenth passage virus, AHSV-4(13), originated from the primary isolate AHSV-4(1). The three most variable AHSV proteins are VP2, VP5 and NS3. The question of sequence variation of these proteins between AHSV-4(1) and AHSV-4(13) arising during the attenuation process was addressed. The subject of plaque size variation between these viruses was also investigated. Some of the sequence variation observed in NS3, VP2 and VP5, between AHSV-4(1) and AHSV-4(13), occurred in protein regions that may be involved in virus entry into and exit from cells. The sequence information also indicated that AHSV-4(1) and AHSV-4(13) consist of genetically heterogeneous viral pools. The plaque size of AHSV-4(1) was variable, with small to relatively large plaques, whereas the plaques of AHSV-4(13) were mostly large. During serial plaque purification of AHSV-4(1) plaque size increased and became homogenous in size. No sequence variation in NS3 or VP5 of any of the plaque variants could be linked to variation or change in plaque size. NS3 and VP5 have a possible role in the AHSV virulence phenotype, and exhibit cytotoxic properties in bacterial and insect cells. As these proteins have not been studied in mammalian cells, an aim of this study was to express them in Vero cells and investigate their cytotoxic and membrane permeabilization properties within these cells. The NS3 and VP5 genes of AHSV-4(1) and AHSV-4(13) were successfully inserted into a mammalian expression vector and transiently expressed in Vero cells. The transfection procedure was optimized using eGFP, but expression levels were still low. When NS3 and VP5 were expressed, no obvious signs of cytotoxicity were observed. Cell viability and membrane integrity assays were performed and expression of NS3 and VP5 in Vero cells had no detectable effect on cell viability or membrane integrity. Low expression levels may have resulted in protein levels too low to cause membrane damage or affect cell viability. As Vero cells support AHSV replication, low levels of NS3 and VP5 may not be cytotoxic in these cells. NS3 was further investigated by expressing an NS3-eGFP fusion protein in Vero cells. Putative localization with membranous components and possible perinuclear localization of the fusion protein was observed. These observations may be confirmed with more sensitive microscopic techniques for a better assessment of the localization.Dissertation (MSc (Genetics))--University of Pretoria, 2009.Geneticsunrestricte

mRNA Poly(A) tail: a 3\u27 Enhancer of Translational Initiation: a Thesis

Most eukaryotic mRNAs have a sequence of polyadenylic acid [poly(A)] at their 3\u27-termini. Although it has been almost two decades since the discovery of these poly(A) tracts, their function(s) have yet to be clarified. Earlier results from our laboratory led us to propose that poly(A) has a role in translation. More specifically, we proposed that an interaction of the cytoplasmic poly(A)-binding protein (PABP) with a critical minimum length of poly(A) facilitates the initiation of translation of poly(A)+, but not poly(A)-, mRNAs. The results of several different experimental approaches have provided evidence which indirectly supports this hypothesis. These results include: 1) the correlation of specific changes in mRNA poly(A) tail length with translational efficiency in vivo and in vitro; 2) correlations between the abundance and stability of PABPs and the rate of translational initiation in vivo and in vitro; and 3) the demonstration that exogenous poly(A) is a potent and specific inhibitor of the in vitro translation of poly(A)+, but not poly(A)-mRNAs. To evaluate the hypothesis that the 3\u27-poly(A) tract of mRNA plays a role in translational initiation, we have constructed derivatives of pSP65 which direct the in vitro synthesis of mRNAs with different poly(A) tail lengths and compared, in reticulocyte extracts, the relative efficiencies with which such mRNAs are translated, degraded, recruited into polysomes, and assembled into mRNPs or intermediates in the translational initiation pathway. Relative to mRNAs which are polyadenylated, we find that poly(A)- mRNAs have a reduced translational capacity which is not due to an increase in their decay rates, but is attributable to a reduction in their efficiency of recruitment into polysomes. The defect in poly(A)- mRNAs affects a late step in translational initiation, is distinct from the phenotype associated with cap-deficient mRNAs, and results in a reduced ability to form 80S initiation complexes. Moreover, poly(A) added in trans inhibits translation from capped poly(A)+ mRNAs, but stimulates translation from capped poly(A)- mRNAs. We suggest that poly(A) is the formal equivalent of a transcriptional enhancer, i.e., that poly(A)-binding protein (PABP) bound at the 3\u27-end of mRNA may facilitate the binding of an initiation factor or ribosomal subunit at the mRNA 5\u27-end

Functional Study of the Structural VP6 Protein of Bluetongue Virus

This study was undertaken to investigate the structure-function relationship of VP6 protein of bluetongue virus (BTV) using molecular cloning techniques. VP6 is present in small quantities in BTV and its enzymatic activity and role in the viral replication cycle have not been studied. Since the availability of large amounts of purified VP6 is essential for the analysis of VP6, a BTV -11 S3 gene was cloned into a prokaryotic protein expression system. VP6 protein was expressed in large amounts and purified to near homogeneity. A series of C-terminal and internal deletion mutants of S3 gene was constructed and the truncated VP6 proteins were expressed and purified. The nucleic acid binding activities of the VP6 protein towards dsRNA, dsDNA, and ssRNA were confirmed and a new ssDNA binding activity was also determined. The binding activities of VP6 were concentration-dependent. The sites responsible for the binding activities were mapped using the truncated proteins and synthetic sequence-specific oligopeptides. Two domains of VP6 were responsible for the nucleic acid binding activities and have been mapped within 28 amino acids near the middle and 11 residues near the carboxyl terminus of VP6. The binding affinities of the middle domain of VP6 towards single-stranded and double-stranded nucleic acid were slightly different. Three synthetic oligopeptides corresponding to these domains exhibited concentration-dependent nucleic acid binding activities. Based on these results I suggest that synthetic oligopeptides might be useful to screen nucleic acid binding activities and domains responsible for these activities. Expressed VP6 was used to produce polyclonal and monoclonal antibodies. Oligoclonal antibodies were raised by synthetic oligopeptides. Ten epitopes of VP6 were mapped and characterized. The amino acid sequences and sizes of six linear epitopes identified by oligoclonal antibodies were determined, and their locations were mapped and confirmed by deletion mutant analyses. These linear epitopes were surface-accessible except one. Based on these results I suggest that synthetic sequence-specific oligopeptides could mimic major components of antigenic determinants. Four epitopes recognized by four monoclonal antibodies were mapped and characterized. Three determinants were surface-accessible and three were conformational epitopes. These four determinants were distinct and different from the six linear epitopes determined using oligoclonal antibodies

Structure and Replication of Alphavirus RNAs

Both ends of the alphavirus genomic RNA are potentially important in its replication. The region preceding and including the 5'-end of the subgenomic 26S RNA in genomic RNA might also be involved in 26S RNA transcription. Sequences of these regions of up to 10 alphaviruses were determined by using strategies including enzymatic, chain-termination and cDNA sequencing methods. Comparison of the nucleotide sequences reveals three highly conserved sequences. The first conserved sequence is 19 nucleotides in length and is located at the extreme 3'-end next to the poly(A) tail. The second conserved sequence, which is 21 nucleotides in length, precedes the 5'-end of 26S RNA and includes the first two nucleotides of it. The third conserved sequence is 51 nucleotides in length and is located at a position of about 130 to 150 nucleotides from the 5'-end, depending on the virus. The last conserved sequence in all alphaviruses examined is capable of forming two stable hairpin structures and could also base-pair stably with the 3'-terminal sequences to cyclize genomic RNAs. Besides these three conserved sequences, a highly conserved stem and loop structure could also be formed at the extreme 5'-end of genomic RNA. Defective interfering (DI) RNAs of alphaviruses are mutated genomic RNAs which often contain deleted, repeated and translocated sequences, but yet retain all elements essential for their replication. By studying the sequence organization of alphavirus DI RNAs, and the 3'-terminal sequences of the genomic RNAs of two alphavirus variants and their replication, the importance of these conserved sequences and secondary structures in alphavirus replication are discussed. Both the 3'- and 5'-terminal sequences of several alphavirus 26S RNAs were also determined. Results show that 26S and genomic RNAs are coterminal. Together with the results previously published, the total length of the 26S RNAs of two alphaviruses, Sindbis virus and Semliki Forest virus, were determined to be 4102 and 4074 nucleotides, respectively. The NH2- and COOH-terminal sequences of the precursors of nonstructural proteins (translated from genomic RNA) and structural proteins (translated from 26S RNA) of several alphaviruses were deduced from the nucleotide sequences determined. The initiation codons of most alphavirus genomic and 26S RNAs are preceded by the sequence CANN. To determine the importance of these tetranucleotides, their sequences in 65 eucaryotic mRNAs were surveyed. Results show that the sequence distribution of these tetranucleotides are non-random and they might be involved in initiation of translation. The 3'-noncoding regions of alphavirus genomic RNAs contain AU rich sequences. Sequence organization in the 3'-noncoding regions is similar to those in alphavirus DI RNAs. Mechanisms for the generation of these sequence rearrangements are discussed.</p

The stability, movement and expression of natural and synthetic mRNAs injected into 'Xenopus' oocytes

The stability and movement of several natural and synthetic mRNAs in Xenonus oocytes was examined. Although the movement of injected mRNAs has important implications for experiments in oocytes, this aspect of mRNA behaviour has never been examined before. At least 50% of the injected natural poly(A)+ mRNAs (9S rabbit globin, chicken ovalbumin and lysozyme mRNAs) remained stable over a 48h period, irrespective of the amount injected. 50% of the natural poly(A) reovirus mRNA was degraded within 24h of injection, irrespective of the amount injected, and no further degradation occurred over the next 24h. Synthetic mRNAs coding for chicken lysozyme, calf preprochymosin and Xenonus β globin protein were transcribed in vitro using Sp6 RNA polymerase. Capping and polyadenylation increased the stability of the synthetic mRNAs with at least 42% of capped, poly(A) transcripts remaining 48h after injection into oocytes. Capping and polyadenylation also increased the translational efficiency of most of the synthetic mRNAs. The exception was one Xenopus β globin transcript with an unusual 3' end of 20 As and 30 Cs, where further polyadenylation decreased translational efficiency. The movement of all the natural poly(A)+ mRNAs injected into oocytes was very slow. Little movement of RNA from the animal to the vegetal half was observed, even 48h after injection. In contrast, similar amounts of mRNA were present in both halves 48h after vegetal pole injection. Similar results were obtained with poly(A) reovirus mRNAs. The capped poly(A) synthetic mRNAs moved more rapidly in oocytes than either capped poly(A) synthetic mRNAs or naturally occurring mRNAs. However equilibration of the injected RNA still did not occur even 24h after injection. The movement of the proteins encoded by the natural poly(A)+ mRNA was examined in the 6h period after injection, when little mRNA movement had occurred. The sequestered secretory proteins ovalbumin and lysozyme moved much more slowly than the cytosolic protein globin in the same oocytes

FUNCTIONAL STUDIES OF INFECTIOUS PANCREATIC NECROSIS VIRUS PROTEINS AND MECHANISM OF VIRUS-INDUCED APOPTOSIS

Infectious pancreatic necrosis virus (IPNV) encodes a 12 or 15-kDa nonstructural protein, known as VP5. To study the function of VP5, we generated three recombinant viruses rNVI15, rNVI15-15K, and rNVI15-&amp;#916;VP5, which could encode either 12-kDa VP5, 15-kDa VP5 or be deficient in VP5, respectively. VP5 was detected in rNVI15 and rNVI-15K infected cells but not in the cells infected with rNVI15-&amp;#916;VP5. However, the opal stop codon at nucleotide position 427 in rNVI15 virus was read-through, giving rise to a 15-kDa VP5 that is expressed poorly than rNVI15-15K virus-infected cells. All three recombinant viruses show similar replication kinetics in both Chinook salmon embryo (CHSE-214) and rainbow trout gonad (RTG-2) cells. Moreover, in Sp strains, IPNV segment A could encode a novel, putative 25-kDa protein from another ORF. This 25-kDa protein could not be detected in virus-infected cells, however, we could recover a mutant virus lacking this ORF, indicating that it is not essential for virus replication. To assess the molecular basis of virus adaptation in the cell culture, virulent rNVI15 was serially passaged in CHSE cells nine times to obtain a tissue-culture adapted virus, rNVI15TC. Comparison of the deduced amino acid sequences showed only one amino acid substitution at position 221 (Ala &amp;#8594; Thr) in VP2. However, this adaptation mutation is only acquired in CHSE cells but not in RTG-2 cells. Two chimeric viruses, rNVI15&amp;#916;VP2 and rNVI15-15K&amp;#916;VP2 were also generated, in which the residues at positions 217 and 247 in VP2 of the rNVI15 and rNVI15-15K viruses were replaced by the corresponding residues of an attenuated strain, Sp103. These two viruses have similar replication kinetics as Sp103, which replicates faster than rNVI15 in vitro, indicating that residues at positions 217 and 247 of VP2 may be the important markers for virus adaptation and attenuation in vitro. By generating a reassortant virus between rNVI15-15K and Sp103, we also demonstrate that VP1 is not involved in virus cell adaptation. The signal pathways and nature of IPNV-induced apoptosis were investigated in RTG-2 cells. IPNV-induced apoptosis occurs at the late stage of viral life cycle. Caspase-3 is activated during virus infection, and inhibition of caspase-3 could partially inhibit virus-induced apoptosis. Moreover, NF-&amp;#954;B activation is essential for IPNV-induced apoptosis, and it is involved in interferon-induced antiviral state. Both the NF-&amp;#954;B inhibitor and the antioxidant could inhibit NF-&amp;#954;B activity and apoptosis induced by IPNV infection, but they do not affect viral replication