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Bovine viral immunodeficiency (BIV), often known as bovine lentivirus, is a disease affecting cattle and buffaloes worldwide. It is caused by the bovine immunodeficiency virus, a member of the Retroviridae family. Once BIV infects animals, it integrates the genome of the host cells and causes a progressive lifelong infection. Despite the record of the virus in various countries, BIV screening in Egyptian animals has never been investigated yet. Therefore, this study aimed to determine whether the BIV provirus was present in blood samples taken from dairy and beef cattle from six provinces in Egypt's northern, central, and southern regions. A total of 310 DNA samples extracted from cattle blood were investigated by the quantitative real-time polymerase chain reaction (qRT-PCR) technique using specific pol gene primers. The findings indicated the absence of BIV provirus in the analyzed samples. The cycle threshold (Ct) values of the tested samples range from 30 - 36.5, while the Ct values of negative control were 29.7 - 36.8. Because of the lack of positive control, UV light visualization of the target amplicon (385 bp) was conducted and revealed no evidence of BIV provirus in the tested samples. Although the investigated animals were grouped according to location, age, sex, breed, production type, and farming system, we could not analyze the potential risk factors because of the negative results in all tested samples. In conclusion, this study presented the first molecular screening of BIV provirus in Egyptian cattle. Additionally, a future large-scale survey on BIV in Egyptian cattle and buffaloes is recommended. [ABSTRACT FROM AUTHOR]

The membrane-spanning domain (MSD) of the transmembrane subunit (TM) anchors the envelope glycoprotein (Env) on the lipid bilayer of the host cell membrane and virions. Its functions include membrane fusion efficiency and intracellular trafficking of the lentivirus envelope protein. Our study aimed to determine the MSD of bovine immunodeficiency virus (BIV) glycoprotein 45 (gp45) and reveal structural characteristics of the BIV Env protein. We have predicted the region of the BIV MSD and obtained the sequence using bioinformatics software. Various kinds of assays, including analogy analysis, fluorescence microscopy, and dye-transfer-based assays, were carried out to validate the prediction. The results, for the first time, show that the BIV MSD is located at the D170 to M191 amino acids of gp45, and the identified MSD divides gp45 into the extracellular domain (ED), MSD and cytoplasmic domain (CT). We further found that the BIV MSD had a similar structure and function as the HIV MSD using amino acid sequence alignment and fluorescence microscopy. Additionally, the dye-transfer-based assay demonstrates that deletion of the BIV MSD efficiently decreases cell-cell fusion. Based on the identification of the MSD, a "snorkeling" model, in which the flanking charged amino acid residues are buried in the lipid bilayer while their side chains interact with polar head groups, was proposed for the BIV MSD. Ultimately, we further improved the primary structure of the BIV envelope glycoprotein. [ABSTRACT FROM AUTHOR]

The bovine immunodeficiency virus (BIV) Rev shuttling protein contains nuclear/nucleolar localization signals and nuclear import/export mechanisms that are novel among lentivirus Rev proteins. Several viral proteins localize to the nucleolus, which may play a role in processes that are essential to the outcome of viral replication. Although BIV Rev localizes to the nucleoli of transfected/infected cells and colocalizes with one of its major proteins, nucleophosmin (NPM1, also known as B23), the role of the nucleolus and B23 in BIV replication remains to be determined. Here, we demonstrate for the first time that BIV Rev interacts with nucleolar phosphoprotein B23 in cells. Using small interfering RNA (siRNA) technology, we show that depletion of B23 expression inhibits virus production by BIV-infected cells, indicating that B23 plays an important role in BIV replication. The interaction between Rev and B23 may represent a potential new target for the development of antiviral drugs against lentiviruses. [ABSTRACT FROM AUTHOR]

This study was stimulated by our previous research of the dUTPase-related protein from bovine immunodeficiency virus (BIV) (Voronin et al., 2014). Despite the lack of detectable enzymatic BIV dUTPase activity (both of the recombinant protein and in virions), mutating the dUTPase gene was deleterious to viral production. However, cDNA synthesis and integration were apparently unaffected. Consequently, we have studied here two important issues. First, we showed that in cDNA produced by the dUTPase-mutated virions, the incidence of mutations was not higher than that found in wild-type BIV-infected cells. Second, single mutations, introduced in preserved dUTPase residues Asp48 and Asn57 (in the putative dUTPase active site or close to it), have led to abortive BIV infections (except for the conservative Asp48Glu mutation). Therefore, we postulate that the BIV dUTPase-related protein has a critical role in retroviral replication at steps that take place after viral cDNA synthesis and integration. [ABSTRACT FROM AUTHOR]

Bovine immunodeficiency virus is a Lentivirus from the large family of retroviridae. Different opinions have been submitted about the possibility of varieties of infectious complications due to Bovine immunodeficiency virus and induced losses to dairy industry. Considering the worldwide distribution of the BIV infection in cattle herds and correlation of the infection with different kinds of clinical disorders and consequent severe economic losses to dairy cattle industry and the fact that only few surveys about the status of the BIV infection have been performed in Iran as well as the probability of increasing infection rate with aging and therefore increasing the culling rate, this study was done to identify the BIV infection rate in aged Holstein cattle and evaluation of their associated clinical disorders in some dairy herds around Tabriz. In this study which was performed in some industrial or traditional especially the small dairy farms around Tabriz, 50 Holstein dairy cattle with the age of higher than 6 years old were selected and their clinical characteristics recorded. Blood samples were taken to detect BIV genome. According to the results of the Nested-PCR method in this study, 2% (1/50) of the aged cattle were infected with BIV. There wasn’t any significant correlation between BIV infection rate and age. Also there weren’t any significant correlations between BIV infection rate and BCS, milk production rate, any clinical problems, enlarged lymph nodes and haemal nodes. The results showed that the BIV infection rate among the aged cattle was relatively low and in contrast to some other studies, aging does not affect the sensitivity to infection. Decreasing the population in the livestock units and farms, could be helpful in prevention and control of BIV infection in cattle.

Bovine immunodeficiency is a chronic progressive disease caused by a lentivirus that affects cattle and buffaloes. Although the infection has been described in cattle in some countries, including in Brazil, there are only two reports of infection in buffaloes: one in Pakistan and one in Cambodia. The aim of the present study was to survey the occurrence of bovine immunodeficiency virus (BIV) in water buffaloes from the Amazon region, Pará state, Brazil. BIV proviral DNA was surveyed in 607 whole blood samples of water buffaloes from 10 farms located in the state of Pará using semi-nested polymerase chain reaction (PCR) (PCR-SN) to amplify the pol region of the viral genome. Of the 607 samples tested, 27 (4.4 %) were positive for BIV proviral DNA. The amplified fragments were confirmed by sequence analysis after cloning and nucleotide sequencing. The sequence obtained had 99 % similarity to the reference strain (R-29). The present study provides important epidemiological data because BIV was detected for the first time in water buffaloes in Brazil. Further, the results suggest the possibility of the virus being a risk factor for herd health because it may be a potential causal agent of chronic disease and, also may be associated to other infectious diseases. [ABSTRACT FROM AUTHOR]

The bovine immunodeficiency virus (BIV) is a lentivirus which is known to infect cattle worldwide. Though serological and genomic evidence of BIV in cattle has been found throughout the world, isolation of the virus has been reported only from few places. Very little is known about its impact on animal health status, pathogenesis and mode of transmission. BIV is considered generally non-pathogenic and is not known to cause any serious disease in cattle. BIV is genetically and antigenically related to Jembrana disease virus (JDV), the cause of an acute disease in Bali cattle ( Bos javanicus) and human immunodeficiency virus, the cause of acquired immunodeficiency syndrome in human. Therefore, it is important to monitor the presence of BIV in cattle to keep vigil over its possible evolution in its natural host to emerge as pathogenic lentivirus like JDV. Differentiation of BIV infection in cattle from the acutely pathogenic JDV is important for diagnosis of the latter. Currently, BIV is considered as a safe model for understanding the complex genome of lentiviruses. Further research on BIV is indeed needed to elucidate its possible role in animal health as well as for insight into the molecular mechanisms adopted by related lentiviruses. [ABSTRACT FROM AUTHOR]

Bovine immunodeficiency virus (BIV), a member of the lentivirus subfamily of retroviridae, was originally isolated from a dairy cow with persistent lymphocytosis, lymphoid hyperplasia, and prevascular cuffing in the brain. Serological studies indicated that BIV has worldwide distribution, but its prevalence in Iran is not clearly known. In this investigation, we report the detection of proviral DNA sequence of BIV in 300 blood samples of cattle and 150 blood samples of sheep by polymerase chain reaction (PCR) using oligonucleotiode primers specific for the gag gene region of the virus. Blood samples of cattle and sheep were taken from Chaharmahal Bakhtiary province, Iran. According to the PCR results, infection rate in the cattle and sheep population were 60% and 30%, respectively. This is the first report for the presence of BIV in cattle and sheep population of Chaharmahal Bakhtiary province. [ABSTRACT FROM AUTHOR]

Abstract: In cattle the interaction between the two genetically and antigenically related bovine lentiviruses, the acutely pathogenic Jembrana disease virus (JDV) and the non-pathogenic Bovine immunodeficiency virus (BIV) has not been reported although both JDV and a BIV-like virus have been reported in the Bali cattle (Bos javanicus) population in Indonesia. The outcome of infection of Bali cattle with the R29 strain of BIV prior to superinfection 42days later with JDVTAB/87 was determined. All BIV-inoculated cattle were successfully infected and developed an antibody response to the TM and CA proteins. BIV infection did not prevent subsequent infection with JDV or ameliorate the clinical signs of Jembrana disease in the infected cattle. It did, however, modify the dynamics of the JDV infection with an earlier onset and end of the acute disease process, and a reduction in the duration of viremia that exceeded 106 genome copies/ml of plasma. [Copyright &y& Elsevier]

Integrase plays a critical role in the recombination of viral DNA into the host genome. Therefore, over the past decade, it has been a hot target of drug design in the fight against type 1 human immunodeficiency virus (HIV-1). Bovine immunodeficiency virus (BIV) integrase has the same function as HIV-1 integrase. We have determined crystal structures of the BIV integrase catalytic core domain (CCD) in two different crystal forms at a resolution of 2.45 Å and 2.2 Å, respectively. In crystal form I, BIV integrase CCD forms a back-to-back dimer, in which the two active sites are on opposite sides. This has also been seen in many of the CCD structures of HIV-1 integrase that were determined previously. However, in crystal form II, BIV integrase CCD forms a novel face-to-face dimer in which the two active sites are close to each other. Strikingly, the distance separating the two active sites is approximately 20 Å, a distance that perfectly matches a 5-base pair interval. Based on these data, we propose a model for the interaction of integrase with its target DNA, which is also supported by many published biochemical data. Our results provide important clues for designing new inhibitors against HIV-1. [ABSTRACT FROM AUTHOR]

Abstract: Infection of Bali cattle (Bos javanicus) in Indonesia with a non-pathogenic bovine lentivirus similar to Bovine immunodeficiency virus (BIV) is suspected but efforts to detect the virus have been unsuccessful. To define the kinetics of BIV infection in Bali cattle, 13 were infected with the R-29 strain of BIV and monitored for 60 days. No clinical effects were detected. Proviral DNA was detected in peripheral blood mononuclear cells from 4 to 60 days with peak titres 20 days post-infection (dpi). There was a transient viraemia from 4 to 14dpi with a maximum titre of 1×104 genome copies/ml plasma. An antibody response to the transmembrane (TM) glycoprotein commenced 12dpi but an antibody response to the capsid (CA) protein was detected in one animal only and not until 34dpi. The results indicated that detection of BIV in infected Bali cattle would have a greater chance of success soon after infection and prior to the onset of a CA antibody response. [Copyright &y& Elsevier]

Abstract: Indicator cell lines are useful biological tools for monitoring virus infection. In order to monitor infection with bovine immunodeficiency virus (BIV) in vitro, an indicator cell line derived from baby hamster kidney cells which contains integrated copies of an enhanced green fluorescent protein gene driven by the BIV long terminal repeat was constructed. The BIV indicator cell line, designated BIVE, can detect BIV infection more easily and effectively than the established method, which involves the observation of cell cytopathic effects. Furthermore, viral titration using an assay based on the indicator cells is 100 times more sensitive than the assay based on cytopathic effect. The finding that BIV can infect the hamster cell line expands the known host range of BIV in vitro. The BIV indicator cell line could also be used for the evaluation of the inhibitory effect of antiviral agents. The fusion inhibition effect of the heptad repeat 2 region of the BIV envelope protein could also be quantified. [Copyright &y& Elsevier]

The clinical and haematological findings in dairy cattle with naturally infected bovine immunodeficiency virus (BIV) infection were evaluated. Thirty-seven (12.3%) out of 300 cattle that had previously been found positive for BIV infection were monitored. Thirty-seven BIV-free cattle selected from BIV-positive herds were used as a control group. Routine clinical and haematological parameters were recorded 6 times, at 1-month intervals. Mastitis (n = 18), metritis (n = 9), respiratory system diseases (n = 8), retained placenta (n = 7), and regional lymphadenopathy (n = 7) were predominantly diagnosed during the monitoring period in BIV-infected cattle, and mastitis (n = 1) and metabolic disturbance (n = 1) in the control animals. Heart and respiratory rates were significantly higher (P < 0.01) in BIV-infected cattle than in the control group. White blood cell (WBC) count and lymphocyte rate were lower (P < 0.01) in BIV-infected cattle, but the neutrophil rate was higher (P < 0.05) than those of the control group. There were no significant differences in erythrocyte or platelet indices within or between the groups during the study. These findings suggest that the presence of BIV infection should be considered a health risk to cattle populations, and may have a role in changing WBC and differential cell counts in the host. [ABSTRACT FROM AUTHOR]

Abstract: The non-toxic enzymic A subunit of Shiga toxin 1 (StxA1) reduces expression and replication of the bovine retroviruses, bovine leukemia virus and bovine immunodeficiency virus (BIV). Here, the impact of StxA1 on representative positive and negative stranded RNA viruses was compared. BIV and equine infectious anemia virus were sensitive to picomolar concentrations of StxA1 while poliovirus, rhinovirus, and vesicular stomatitis virus were only marginally sensitive to nanomolar concentrations of toxin. Thus, the length of the reproductive cycle and/or other factors, but not viral encapsulation may play a role in determining sensitivity to StxA1. The effects of StxA1 at concentrations from 0.01 to 10μg/ml on the most sensitive virus (BIV-infected cultures of fetal bovine lung cells) were analyzed by electron microscopy 48h post challenge. Cells treated with 0.1μg StxA1/ml or higher toxin concentrations were similar in appearance and showed progressively fewer viral factories with increasing toxin concentration. However, cells treated with 0.01μg/ml StxA1 had a radically different appearance, exhibiting smooth cell membranes and high vacuolization. These results showed that complex retroviruses were more sensitive to StxA1 than single-stranded RNA viruses and that StxA1 interfered with retroviral replication in a concentration-dependent manner. [Copyright &y& Elsevier]

Abstract: Shiga toxins are ribosome-inactivating proteins many of which are antiviral. Shiga toxin-producing Escherichia coli (STEC) may be pathogenic to humans, but are carried without ill effects by ruminants. We hypothesize that STEC have antiviral activity in ruminants, and showed previously that the non-toxic subunit A of Shiga toxin 1 (StxA1) acts selectively on cells infected with bovine leukemia virus, without harming normal cells, and that the numbers of intestinal STEC are inversely correlated with viral load in bovine leukemia virus-infected sheep. The purpose of the present study was to characterize StxA1 activity against a second bovine retrovirus, bovine immunodeficiency virus (BIV). Flow cytometry showed that StxA1 treatment induced apoptosis in BIV-infected cells but not in uninfected cells and immunoblot analysis showed that StxA1 curtailed synthesis of Gag p26 protein. A systematic electron microscopy description of BIV infection in fetal bovine lung fibroblasts showed an orderly sequence of changes in cell membrane, endoplasmic reticulum, Golgi, nucleus, and mitochondria, and suggested that the infected cells produce the virus within multivesicular bodies (MVBs). StxA1 interfered with all manifestations of BIV-induced transformation of infected cells into BIV-producing units. BIV-infected cells provided a suitable experimental system for investigation of the mechanism of Stx-antiviral activity. [Copyright &y& Elsevier]

Abstract: Bovine immunodeficiency virus (BIV) is a lentivirus with no proven pathogenesis in infected cattle. Yet, in experimentally infected rabbits, it causes an AIDS-like disease. Consequently, we expressed two recombinant isoforms of BIV reverse transcriptase (RT), which differ in their C-termini, and studied their catalytic properties. Both isoforms prefer Mg+2 over Mn+2 with most DNA polymerase and ribonuclease-H substrates. The processivity of DNA synthesis by the BIV RTs is higher than that of HIV-1 RT, whereas the fidelity of synthesis is even lower than that of the HIV-1 enzyme. The ribonuclease-H cleavage pattern suggests that the spatial distance between the polymerase and ribonuclease-H active sites of the two BIV RT isoforms equals 20 nt, unlike the 17 nt distance observed in almost all other RTs. The longer BIV RT version is somewhat less active than the shorter version, suggesting that the extra 74 residues (with homology to dUTPases) might obstruct efficient catalysis. [Copyright &y& Elsevier]

Abstract: Entry of lentiviruses, such as human immunodeficiency virus type 1 (HIV-1) and simian immunodeficiency virus (SIV), requires folding of two heptad repeat regions (HR1 and HR2) of gp41 into a trimer-of-hairpins, which subsequently brings virus and cell membrane into fusion. This motif is a generalized feature of viral fusion proteins and has been exploited in generating antiviral fusion agents. In the present paper, we report structural characters of Env protein from another lentivirus, bovine immunodeficiency virus (BIV), which contributes to a good animal model of HIV. BIV HR1 and HR2 regions are predicted by two different programs and expressed separately or conjointly in Escherichia coli. Biochemical and biophysical analyses show that the predicted HRs of BIV Env can form a stable trimer-of-hairpins or six-helix bundle just like that formed by feline immunodeficiency virus Env. Cell fusion assay demonstrates that the HR2 peptide of BIV can efficiently inhibit the virus-mediated cell fusion. [Copyright &y& Elsevier]

Abstract: In Indonesia, it is suspected that there are two bovine lentiviruses circulating in the cattle population: a pathogenic Jembrana disease virus (JDV), and a nonpathogenic bovine immunodeficiency-like virus (BIV). Both viruses cross-react antigenically and cannot be differentiated by current serological tests using JDV antigens. To identify possible type-specific epitopes, a series of recombinant protein constructs including the matrix, capsid and nucleocapsid proteins were produced from JDV gag and the expressed proteins were tested by Western blot using JDV and BIV hyperimmune sera. JDV matrix and truncated capsid proteins were recognised by both JDV and BIV hyperimmune sera indicating that there were multiple cross-reactive epitopes present in JDV gag. At least three epitopic regions were identified in these constructs, including the major homology region, by monoclonal antibody binding studies. JDV nucleocapsid recombinant protein was not recognised by either JDV or BIV hyperimmune sera and none of the recombinant gag proteins were able to differentiate between JDV positive sera from Jembrana disease endemic and Jembrana disease-free areas. Additionally, a 40 amino acid recombinant subunit protein encompassing the region recently found to contain an epitope unique to BIV [Zheng, L., Zhang, S., Wood, C., Kapil, S., Wilcox, G.E., Loughin, T.A., Minocha, H.C., 2001. Differentiation of two bovine lentiviruses by a monoclonal antibody on the basis of epitope specificity. Clin. Diagn. Lab. Immunol. 8, 283–287] was tested but was not recognised by either JDV positive sera from Jembrana disease-endemic or Jembrana disease-free areas. [Copyright &y& Elsevier]

Sensitive assays are required to detect bovine retroviruses in donor cattle used for the in vivo preparation of Australian tick fever vaccines. 5′ Taq nuclease assays using 3′ minor groove binder DNA probes (TaqMan®MGB) were developed and compared to conventional PCR assays for the sensitive detection of bovine syncytial virus (BSV) and bovine immunodeficiency virus (BIV). Seven beef and dairy herds were screened to evaluate these tests. Comparative sensitivities of PCR tests were determined by testing log10 dilutions of plasmids with inserts containing corresponding provirus sequences. Published PCR assays targeting BIV env sequences did not adequately amplify Australian BIV sequences. Pol sequences from Australian strains of BIV and BSV were used to design TaqMan®MGB assays, which improved sensitivity 10-fold (BIV) and 100-fold (BSV), respectively, over conventional PCR tests. This is the first report of Australian sequences of BIV and BSV and the first 5′ Taq nuclease assays described to detect these viruses. These methods could be applied to future studies requiring sensitive detection of these two bovine retroviruses. [Copyright &y& Elsevier]

The validation of assays for bovine immunodeficiency virus (BIV) in cattle is hampered by the absence of a gold standard. Two tests that often are used to detect BIV are the indirect fluorescent-antibody assay (IFA) and the nested-set polymerase chain-reaction assay (PCR). IFA detects an antibody response whereas PCR detects the provirus in white blood cells.Using Bayesian techniques performed simultaneously on animals from two different dairy herds, we estimated the performance of the IFA and PCR assays and infection prevalence. Bayesian techniques also were used to derive posterior distributions of sensitivities, specificities, and prevalences. The Bayesian estimates were IFA sensitivity=60%, IFA specificity=88%, PCR sensitivity=80%, PCR specificity=86%, Herd A prevalence=20%, and Herd B prevalence=71%. Although PCR was the more sensitive assay, substantial misclassification of infection would be expected in epidemiological studies of BIV regardless of which assay was used. [Copyright &y& Elsevier]

Gene transfer systems based on lentiviruses have emerged as promising gene delivery vehicles for human gene therapy due to their ability to efficiently transduce nondividing target cells. Both primate and nonprimate lentiviruses have been used for construction of lentiviral vectors. An early generation of gene transfer system based on bovine immunodeficiency virus (BIV) has been developed (R. D. Berkowitz, H. Ilves, W. Y. Lin, K. Eckert, A. Coward, S. Tamaki, G. Veres, and I. Plavec, 2001, J. Virol. 75, 3371–3382). In this study, we mapped the BIV Rev response element (RRE) to 312 bp of the Env coding region. Furthermore, we compared transduction efficiencies of vectors containing different portions of the BIV Gag coding region and found that the first 104 bp of gag contains a functional part of the BIV packaging signal. These findings enabled the generation of a minimal BIV-based lentiviral vector. The minimal transfer vector construct consists of a self-inactivating long terminal repeats (LTR), minimal packaging sequence, putative central polypurine tract, minimal RRE, an internal promoter driving the gene of interest, and a woodchuck hepatitis posttranscriptional regulatory element. In addition, we constructed a BIV packaging construct containing gag/pol, minimal Rev/RRE, and the accessory gene vpy. The regulatory gene tat and the accessory genes vif and vpw have been inactivated or truncated. The current system has significantly reduced regions of homologies between the transfer vector and the packaging constructs. The vectors generated from this system achieved a titer of greater than 1 × 106 transducing units per milliliter and are fully functional as indicated by their ability to efficiently transduce both dividing and nondividing cells. These modifications should provide improved safety features for the BIV-based gene transfer system. [Copyright &y& Elsevier]

The inhibition of HIV expression in vitro by a cocktail of the β-chemokines MIP-1α, MIP-1β and RANTES provided the initial evidence that HIV utilizes chemokine receptors as co-receptors for infection of cells. Bovine immunodeficiency virus (BIV), a lentivirus, infects a wide variety of leukocyte populations, but the cellular receptor(s) utilized by this virus for infection of cells is not known. The purpose of this study was to determine whether MIP-1α, MIP-1β and RANTES affect BIV expression in vitro, as a prelude to identifying the cellular receptors utilized by this virus. Fetal bovine lung (FBL) cells were pretreated with serial dilutions of a cocktail of the chemokines, and then the cells were infected with BIV. Virus expression in these cells was determined by counting the syncytia that had developed in the cultures by five days after infection. A significant decrease in syncytium formation, corresponding to increasing concentrations of the chemokines, was the result. Reacting the chemokines with chemokine-specific neutralizing antibodies prior to treatment of the cells neutralized the effect of the chemokines on virus replication in a dose-dependent manner, restoring viral expression to a level similar to that of untreated cells. The presence of a CCR5 homologue on the surface of FBL cells was confirmed using an anti-CCR5 monoclonal antibody and FACS analysis. Collectively, these data provide preliminary evidence that BIV may utilize the CCR5 receptor for infection of cells in vitro, but additional studies are necessary to confirm this. [ABSTRACT FROM AUTHOR]

The association of bovine immunodeficiency virus (BIV) with embryos derived by in vitro fertilization from oocytes of experimentally infected heifers or oocytes/embryos exposed to the virus in vitro was investigated. Using a nested-PCR assay, proviral DNA of BIV was not detected in follicular fluid or in embryos derived from BIV-infected donors. In vitro exposure of oocytes to BIV during maturation or insemination with BIV-infected semen resulted in zona pellucida-intact embryos testing negative for BIV provirus. However, exposure of zona pellucida-free day-7 embryos to the virus resulted in a positive BIV assay for 28% of the batches of embryos, suggesting that the zona pellucida has a role in protecting against BIV infection. The presence of BIV in the IVF system had no apparent effect on the development of bovine embryos to the blastocyst stage. [ABSTRACT FROM AUTHOR]

The arginine-rich RNA-binding domain of bovine immunodeficiency virus (BIV) Tat adopts a ß-hairpin conformation upon binding to the major groove of BIV TAR. Based on its NMR structure, we modeled dimeric arrangements in which two adjacent TAR sites might be recognized with high affinity by a dimeric peptide. Some dimeric RNAs efficiently bound two unlinked BIV Tat peptides in vitro, but could not bind even one monomeric peptide in vivo, as monitored by transcriptional activation of human immunodeficiency virus long terminal repeat reporters. Results with additional reporters suggest that extending the RNA helix in the dimeric arrangements inhibits peptide binding by decreasing major groove accessibility. In contrast, a dimeric peptide efficiently bound an optimally arranged dimeric TAR in vivo, and bound with an affinity at least 10-fold higher than the monomeric peptide in vitro. Mutating specific nucleotides in each RNA 'half-site' or specific amino acids in each ß-hairpin of the dimeric peptide substantially decreased binding affinity, providing evidence for the modeled dimer-dimer interaction. These studies provide a starting point for identifying dimeric RNA-protein interactions with even higher binding affinities and specificities. [ABSTRACT FROM AUTHOR]

Abstract: To study the functions of RNA-binding proteins independent of their RNA-binding activity, tethering methods have been developed, based on the use of the RNA-binding domain of a well-characterized RNA-binding protein and its target RNA. Two bacteriophage proteins have mainly been used as tethers: the MS2 coat protein and the lambda N protein. Here we report an alternative system using the Tat (trans-activator) peptide from the bovine immunodeficiency virus (BIV), which binds to BIV–TAR (trans-activation response) RNA. We demonstrate the usefulness of this system by applying it to the analysis of the TNRC6B protein, a component of the microRNA-induced silencing complex. [Copyright &y& Elsevier]