Jembrana disease virus (JDV) is an acutely pathogenic lentivirus affecting Bali cattle in Indonesia. The inability to propagate the virus in vitro has made it difficult to quantitate JDV and determine the kinetics of virus replication during the acute disease process. Reported for the first time are 2 techniques that enable quantification of the virus, and the use of these techniques to quantify the virus load in plasma of cattle during the acute disease process. The 2 techniques were a qualitative one-step JDV real-time reverse-transcription polymerase chain reaction (qRT-PCR) assay for the detection and quantification of JDV RNA, and a JDV p26 capture ELISA for the detection and quantification of JDV capsid protein. The limit of detection of the qRT-PCR was 9.8 x 102 JDV viral RNA copies over 35 cycles, equivalent to 4.2 x 104 JDV genome copies/ml, and a peak virus load of 1.6 x 1012 JDV genome copies /ml during the acute febrile period. Viral RNA and JDV p26 levels were correlated in 48 plasma samples obtained from experimentally infected cattle. A significant positive correlation (R = 0.835 and r2 = 0.697) was observed between the 2 techniques within the range of their detection limits, providing a solid basis for the use of the economical capture ELISA to quantify JDV load when real-time PCR capability is not available. The detection of JDV p26 by capture ELISA was, however, much less sensitive than the real-time RTPCR with a detection limit equating to approximately 1 x 108 JDV genome copies/ml. The transcriptional pattern of JDV during the acute phase of infection was studied by RT-PCR, sequencing and northern blot analysis. Analysis revealed a complex pattern of transcription with the identification of 14 transcripts, which confirmed 6 predicted splice sites and the identified 7 splice sites not reported previously. A small 78 bp putative non-coding exon was identified that shared the same splice acceptor as vif and was associated with the alternative transcripts of tat, rev and env. Four tat, 3 rev and 2 env transcripts were identified. The rev and env transcripts were demonstrated to use the same splice site. The study confirmed that the production of a tmx transcript, a unique gene identified in the two bovine lentiviruses JDV and Bovine immunodeficiency virus (BIV). Northern blot analysis identified 11 of the 14 transcripts identified by RT-PCR, including a 7.8 kb gag/pol primary transcript and singly spliced transcripts. The complexity of the transcript map produced suggested that JDV replication is a highly regulated process. One of the aims of this thesis was to determine the functional role of the Tmx and Vif accessory proteins of the bovine lentiviruses. Although this aim was not achieved, molecular reagents were produced that will allow these investigations to proceed. The Vif and Tmx proteins of both JDV and BIV were successfully expressed as C-terminal fusions with glutathione S-transferase (GST) using the pGEX-6P-1 bacterial expression system. The recombinant proteins were purified and were recognised by both BIV and JDV antisera from Bos taurus and Bos javanicus respectively, and by antibody in sera from cattle that had been vaccinated with a tissue-derived JDV vaccine and also those that had been naturally infected with JDV. The Vif, Tmx and Rev proteins of JDV and vif BIV were successfully expressed in a Rev-independent manner in COS7 and bovine macrophage cells using a pcDNA3.1® mammalian expression system. Cellular localisation of the recombinant viral proteins varied in the 2 cell types: in COS7 cells, both JDV and BIV Vif were detected predominantly in the nucleus, whereas in bovine macrophage cells BIV Vif localised in the cytoplasm and JDV Vif localised in the cytoplasm and nuclear membrane. JDV Tmx localised in the cytoplasm of COS7 cells but the nuclear membrane of bovine macrophage cells, and BIV Tmx localised in the nucleus and nuclear membrane in both cell types and appeared to affect the morphology of the nucleus. Mutations of vif and tmx were also successfully engineered into an infectious clone of BIV and these mutated clones will provide a valuable resource for further investigation of the role of Vif and Tmx in replication of the bovine lentiviruses.

Dans la cellule, les ARN pré messagers contenant des introns sont normalement retenus au noyau par leur interaction avec des facteurs d’épissage. Cependant, les ARN partiellement et non épissés des rétrovirus doivent entrer dans le cytoplasme pour servir de matrice pour la synthèse de certaines protéines telles que Env, Gag et Gag-Pol ainsi que d’ARN génomique qui sera empaqueté dans les nouveaux virions. Un mécanisme post-transcriptionnel utilisé par les lentivirus pour éviter la séquestration nucléaire de ces ARNm dépend d’une protéine virale appelée Rev. Pour assurer sa fonction d’exportation, Rev doit transiter entre le noyau et le cytoplasme et doit aussi pouvoir former des multimères. Par conséquent, Rev est dotée de domaines fonctionnels lui procurant ces habiletés. On retrouve le domaine riche en arginines qui contient le domaine de liaison à l’ARN et le signal de localisation nucléaire (NLS), un second domaine, riche en leucines, porte le signal d’exportation nucléaire (NES) et finalement le domaine de multimérisation. Bien que les protéines Rev du virus de l’immunodéficience humaine de type 1 (VIH-1) et bovine (VIB) aient été caractérisées, aucune étude n’a été réalisée pour la protéine Rev du virus de la maladie de Jembrana (JDV) et très peu sur le virus de l’immunodéficience féline (VIF). Comme les domaines fonctionnels et la voie d’importation des protéines Rev déjà caractérisées sont différents, nous supposons que chaque protéine Rev possède une organisation qui lui est propre et que les mécanismes de transport nucléo-cytoplasmique diffèrent entre les virus. Ce projet a pour objectif de caractériser ces domaines pour la protéine Rev du JDV et ceux du VIF ainsi que les mécanismes permettant leur transport nucléaire. L’utilisation de mutants de la protéine Rev de ces virus couplés à la protéine de fluorescente verte (EGFP) exprimés dans des cellules appropriées et observés par microscopie a permis d’identifier des séquences NLS et NES différentes de celles déjà caractérisées. Le NLS de la protéine Rev du JDV a été identifié et est composé des résidus arginines de la séquence 76-RRPARRPPIRR-87 avec un NoLS composé des mêmes résidus en plus des arginines R74, R103 et R104. Son NES est composé des résidus hydrophobes de la séquence 116-MAELEERFEDLAL-128 et est du type de l’inhibiteur de la protéine kinase (PKI pour « protéine kinase inhibitor »). Pour la protéine Rev du VIF, son NLS est composé des résidus basiques de la séquence 84-KKKRQRRRRKKKAFKK-99. Le NoLS est composé des mêmes acides aminés en plus du résidu K82. De plus, les essais d’importation nucléaires et d’interaction semblent indiquer que les voies d’importation utilisées diffèrent entre les virus et que plusieurs voies peuvent être utilisées. Ces travaux pourront éventuellement servir de base pour identifier de nouvelles cibles thérapeutiques contre les lentivirus. / In the cell, pre-messenger RNAs containing introns are normally retained in the nucleus by their interaction with splicing factors. However, the partially and unspliced ​​RNAs of retroviruses must enter the cytoplasm to serve as a template for the synthesis of certain proteins such as Env, Gag and Gag-Pol as well as genomic RNA to be packaged in the new virions. A post-transcriptional mechanism used by lentiviruses to prevent nuclear sequestration of these mRNAs depends on a trans-activator, the viral protein Rev. To ensure its export function, Rev must be able to shuttle between the nucleus and the cytoplasm and to form multimers. As a result, Rev has functional domains that provide these abilities: the arginine-rich domain, which contains the RNA binding domain and the nuclear localization signal (NLS), a second domain, rich in leucine, corresponding to the nuclear export signal (NES) and finally the multimerization domain. Although the Rev proteins of the human and bovine immunodeficiency virus (HIV-1 and BIV respectively) have been characterized, no studies have been performed for the Jembrana disease virus (JDV) Rev protein and very little on the feline immunodeficiency virus (FIV). Since the functional domains and import pathway of the already characterized Rev proteins are different, we assume that each Rev protein has its own organization and that the nucleo-cytoplasmic transport mechanisms differ between viruses. The goal of this project is to characterize these domains for the JDV and FIV Rev proteins as well as to elucidate mechanisms for their nuclear transport. The use of Rev mutants fused to the EGFP expressed in appropriate cells and observed by microscopy has identified NLS and NES sequences that differ from those already characterized. JDV Rev NLS is composed of arginine residues in the 76-RRPARRPPIRR-87 sequence with a NoLS composed of the same residues with the addition of arginine R74, R103 and R104. JDV Rev NES is composed of hydrophobic residues in the 116-MAELEERFEDLAL-128 sequence and is of the protein kinase inhibitor type (PKI). For the FIV Rev protein, its NLS is composed of basic residues in the 84-KKKRQRRRRKKKAFKK-99 sequence. FIV Rev NoLS is composed of the same residues with the addition of the lysine at position 82. In addition, the nuclear import and interaction tests suggest that the import routes used by Rev differ between the different viruses studied and that more than one import pathway may be used. This work could serve as a basis for identifying new therapeutic targets against lentiviruses.