Identification of the membrane-spanning domain of glycoprotein 45 in bovine immunodeficiency virus.

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.

The bovine immunodeficiency virus Rev protein: identification of a novel nuclear import pathway and nuclear export signal among retroviral Rev/Rev-like proteins.

The Rev protein is essential for the replication of lentiviruses. Rev is a shuttling protein that transports unspliced and partially spliced lentiviral RNAs from the nucleus to the cytoplasm via the nucleopore. To transport these RNAs, the human immunodeficiency virus type 1 (HIV-1) Rev uses the karyopherin ß family importin ß and CRM1 proteins that interact with the Rev nuclear localization signal (NLS) and nuclear exportation signal (NES), respectively. Recently, we reported the presence of new types of bipartite NLS and nucleolar localization signal (NoLS) in the bovine immunodeficiency virus (BIV) Rev protein. Here we report the characterization of the nuclear import and export pathways of BIV Rev. By using an in vitro nuclear import assay, we showed that BIV Rev is transported into the nucleus by a cytosolic and energy-dependent importin α/ß classical pathway. Results from glutathione S-transferase (GST) pulldown assays that showed the binding of BIV Rev with importins α3 and α5 were in agreement with those from the nuclear import assay. We also identified a leptomycin B-sensitive NES in BIV Rev, which indicates that the protein is exported via CRM1 like HIV-1 Rev. Mutagenesis experiments showed that the BIV Rev NES maps between amino acids 109 to 121 of the protein. Remarkably, the BIV Rev NES was found to be of the cyclic AMP (cAMP)-dependent protein kinase inhibitor (PKI) type instead of the HIV-1 Rev type. In summary, our data showed that the nuclear import mechanism of BIV Rev is novel among Rev proteins characterized so far in lentiviruses.

Bovine HEXIM1 inhibits bovine immunodeficiency virus replication through regulating BTat-mediated transactivation.

The bovine immunodeficiency virus (BIV) transactivator (BTat) recruits the bovine cyclin T1 (B-cyclin T1) to the LTR to facilitate the transcription of BIV. Here, we demonstrate that bovine hexamethylene bisacetamide (HMBA)-induced protein 1 (BHEXIM1) inhibits BTat-mediated BIV LTR transcription. The results of in vivo and in vitro assays show direct binding of BHEXIM1 to the B-cyclin T1. These results suggest that the repression arises from BHEXIM1-BTat competition for B-cyclin T1, which allows BHEXIM1 to displace BTat from B-cyclin T1. Furthermore, we found that the C-terminal region and the centrally located region of BHEXIM1 are required for BHEXIM1 to associate with B-cyclin T1. Knockdown of BHEXIM1 enhances BIV replication. Taken together, our study provides the first clear evidence that BHEXIM1 is involved in BIV replication through regulating BTat-mediated transactivation.

Sequence analysis of mRNA transcripts encoding Jembrana disease virus Tat-1 in vivo.

Jembrana disease virus (JDV) is a lentivirus which replicates to very high titres in vivo and its Tat-1 protein has been shown to be a potent transactivator in vitro. Analysis of tat mRNA transcripts produced early in infection studies identified four predominant species which were generated by multiple splicing events. The use of a splice donor downstream of tat-1 was common indicating that a Tat-1 protein of 97 amino acids is expressed during the acute phase of Jembrana disease. The presence of an in-frame stop codon between tat-1 and tat-2 was identified in transcripts from three different strains of JDV confirming that expression of a single exon Tat-1 correlates with high viral titres in vivo. Sequence conservation in the regions of tat-1 that are critical for RNA binding and transcription activation in three different virus strains was high and the tat-2 sequences were completely conserved.

How Tat targets TAR: structure of the BIV peptide-RNA complex.

Two new NMR structures describing the complex formed by a peptide from the BIV Tat protein with the TAR RNA provide a significant advance in our understanding of the ways in which peptides interact with specific sites in the major groove of their RNA targets.

Anchoring an extended HTLV-1 Rex peptide within an RNA major groove containing junctional base triples.

BACKGROUND: The Rex protein of the human T cell leukemia virus type 1 (HTLV-1) belongs to a family of proteins that use arginine-rich motifs (ARMs) to recognize their RNA targets. Previously, an in vitro selected RNA aptamer sequence was identified that mediates mRNA transport in vivo when placed in the primary binding site on stem-loop IID of the Rex response element. We present the solution structure of the HTLV-1 arginine-rich Rex peptide bound to its RNA aptamer target determined by multidimensional heteronuclear NMR spectroscopy. RESULTS: The Rex peptide in a predominantly extended conformation threads through a channel formed by the shallow and widened RNA major groove and a looped out guanine. The RNA aptamer contains three stems separated by a pair of two-base bulges, and adopts an unanticipated fold in which both junctional sites are anchored through base triple formation. Binding specificity is associated with intermolecular hydrogen bonding between guanidinium groups of three non-adjacent arginines and the guanine base edges of three adjacent G.C pairs. CONCLUSIONS: The extended S-shaped conformation of the Rex peptide, together with previous demonstrations of a beta-hairpin conformation for the bovine immunodeficiency virus (BIV) Tat peptide and an alpha-helical conformation for the human immunodeficiency virus (HIV) Rev peptide in complex with their respective RNA targets, expands our understanding of the strategies employed by ARMs for adaptive recognition and highlights the importance of RNA tertiary structure in accommodating minimalist elements of protein secondary structure.

New features in RNA recognition: a Tat-TAR complex.

The structure of a peptide from the BIV Tat protein bound to its cognate TAR RNA has been solved. This structure reveals a beta-hairpin motif for the protein, bound at a widened bulge site in the major groove of the RNA.

Construction and characterization of a chimeric virus (BIV/HIV-1) carrying the bovine immunodeficiency virus gag-pol gene.

HIV-1(HXB2) 5'LTR region, most of BIV(R29) gag-pol segment and HIV-1(HXB2) pol IN-3'LTR region were respectively amplified. A chimeric clone, designated as pHBIV(3753), was constructed by cloning three fragments sequentially into pUC18. MT4 cells were transfected with pHBIV3753. The replication and expressions of the chimeric virus (HBIV3753) were monitored by RT activity and IFA. The results firstly demonstrated that it is possible to generate a new type of the BIV/HIV-1 chimeric virus containing BIV gag-pol gene.

Use of bacterial trpE fusion vectors to express and characterize the bovine immunodeficiency-like virus core protein.

The gag coding region from Bovine Immunodeficiency-like Virus (BIV) was cloned into E. coli and expressed as a bacterial fusion protein. Six different clones spanning various regions of the gag open reading frame were generated. The resulting fusion proteins were expressed at high concentrations and readily purified. A panel of bovine immune sera specifically recognized the recombinant Gag proteins, as did immune sera from animals infected or immunized with lentiviruses related to BIV, such as Equine Infectious Anemia Virus (EIAV) and Human Immunodeficiency Virus (HIV). Analysis of the deletion clones, using the bovine immune sera panel, enabled us to identify at least one major epitope which was specifically recognized by all bovine sera examined. The ease of expression, purification, and specificity of these fusion proteins should enable a thorough study of the epidemiology of BIV infection.

Thermodynamics and solvation dynamics of BIV TAR RNA-Tat peptide interaction.

The interaction of the trans-activation responsive (TAR) region of bovine immunodeficiency virus (BIV) RNA with the Tat peptide is known to play important role in viral replication. Despite being thoroughly studied through a structural point of view, the nature of binding between BIV TAR RNA and the BIV Tat peptide requires information related to its thermodynamics and the nature of hydration around the TAR-Tat complex. In this context, we carried out the thermodynamic study of binding of the Tat peptide to the BIV TAR RNA hairpin through different calorimetric and spectroscopic measurements. Fluorescence titration of 2-aminopurine tagged BIV TAR RNA with the Tat peptide gives their binding affinity. The isothermal titration calorimetric experiment reveals the enthalpy of binding between BIV TAR RNA and the Tat peptide to be largely exothermic with the value of -11.7 (SEM 0.2) kcal mol(-1). Solvation dynamics measurements of BIV TAR RNA having 2-AP located at the bulge region have been carried out in the absence and presence of the BIV Tat peptide using the time correlated single photon counting technique. The solvent cage around the Tat binding site of RNA appears to be more rigid in the presence of the Tat peptide as compared to the free RNA. The displacement of solvent and ions on RNA due to peptide binding influences the entropic contributions to the total binding energy.

Substrate variations that affect the nucleic acid clamp activity of reverse transcriptases.

We have recently shown that reverse transcriptases (RTs) perform template switches when there is a very short (two-nucleotide) complementarity between the 3' ends of the primer (donor) strand and the DNA or RNA template acceptor strands [Oz-Gleenberg et al. (2011) Nucleic Acids Res 39, 1042-1053]. These dinucleotide pairs are stabilized by RTs that are capable of 'clamping' together the otherwise unstable duplexes. This RT-driven stabilization of the micro-homology sequence promotes efficient DNA synthesis. In the present study, we have examined several factors associated with the sequence and structure of the DNA substrate that are critical for the clamp activity of RTs from human immunodeficiency virus type 1 (HIV-1), murine leukemia virus (MLV), bovine immunodeficiency virus (BIV) and the long terminal repeat retrotransposon Tf1. The parameters studied were the minimal complementarity length between the primer and functional template termini that sustains stable clamps, the effects of gaps between the two template strands on the clamp activity of the tested RTs, the effects of template end phosphorylations on the RT-associated clamp activities, and clamp activity with a long 'hairpin' double-stranded primer comprising both the primer and the complementary non-functional template strands. The results show that the substrate conditions for clamp activity of HIV-1 and MLV RTs are more stringent, while Tf1 and BIV RTs show clamp activity under less rigorous substrate conditions. These differences shed light on the dissimilarities in catalytic activities of RTs, and suggest that clamp activity may be a potential new target for anti-retroviral drugs.

Molecular and biological aspects of the bovine immunodeficiency virus.

The bovine immunodeficiency virus (BIV) was isolated in 1969 from a cow, R-29, with a wasting syndrome suggesting bovine leucosis. The virus, first designated bovine visna-like virus, remained unstudied until HIV was discovered in 1983. Then, it was demonstrated in 1987 that the bovine R-29 isolate was a lentivirus with striking similarity to the human immunodeficiency virus (HIV). Moreover, BIV has the most complex genomic structure among all identified lentiviruses shown by several regulatory/accessory genes encoding proteins, some of which are involved in the regulation of virus gene expression. This manuscript aims to review biological and molecular aspects of BIV, with emphasis on regulatory/accessory viral genes/proteins which are involved in virus expression.

Molecular basis of the internalization of bovine immunodeficiency virus Tat protein.

Bovine Immunodeficiency Virus (BIV) is a nonacute, pathogenic, and horizontally transmitted lentivirus. It shares the parallel properties in morphology and genetics with human immunodeficiency virus type 1 and other lentiviruses. BIV encodes its own transactivator (BTat), which transactivates its cognate long terminal repeat (LTR). However, the mechanism involved in the transactivation is different from that in HIV and other lentiviruses. We determined the mechanisms of BTat internalization by cells and the effect of BTat on neighboring cells. The green fluorescent protein fusion analysis indicated that the internalization of extracellular BTat was a time and dose-dependent, but endocytosis and energy-independent manner. Arginine residues in the arginine-rich motif (ARM) of BTat were definitively responsible for the internalization. Internalized BTat is predominantly present in the nucleus, resulting in LTR activation and NF-kappaB induction. These results propose that the secretion and internalization of BTat facilitates BIV in influencing neighboring cells and makes the cellular environment propitious to viral replication.

A comparative binding study of BIV Tat peptide against its TAR RNA duplex, RNA-DNA heteroduplex and DNA duplex.

Association between RNA and DNA strands to form RNA-DNA heteroduplex is important in many biological processes such as transcription, DNA replication and reverse transcription. Herein, binding affinities of a 17-mer BIV Tat peptide is compared with TAR DNA duplex, TAR RNA-DNA heteroduplex and TAR RNA duplex. It was observed that binding affinities of Tat peptide is comparable against DNA-RNA heteroduplex and RNA duplex, whereas DNA duplex binding is decidedly poor.

A single intermolecular contact mediates intramolecular stabilization of both RNA and protein.

An arginine-rich peptide from the Jembrana disease virus (JDV) Tat protein is a structural "chameleon" that binds bovine immunodeficiency virus (BIV) or HIV TAR RNAs in two different binding modes, with an affinity for BIV TAR even higher than the cognate BIV peptide. We determined the NMR structure of the JDV Tat-BIV TAR high-affinity complex and found that the C-terminal tyrosine in JDV Tat forms a network of inter- and intramolecular hydrogen bonding and stacking interactions that simultaneously stabilize the beta-hairpin conformation of the peptide and a base triple in the RNA. A neighboring histidine also appears to help stabilize the peptide conformation. Induced fit binding is recurrent in protein-protein and protein-nucleic acid interactions, and the JDV Tat complex demonstrates how high affinity can be achieved not only by optimization of the binding interface but also by inducing new intramolecular contacts that stabilize each binding partner. Comparison to the cognate BIV Tat peptide-TAR complex shows how such a costabilization mechanism can evolve with only small changes to the peptide sequence. In addition, the bound structure of BIV TAR in the chameleon peptide complex is strikingly similar to the bound conformation of HIV TAR, suggesting new strategies for the development of HIV TAR binding molecules.

The bovine immunodeficiency virus: cloning of a tat/rev cDNA encoding a novel Tat protein with enhanced transactivation activity.

Previous studies have shown that BIV may encode two types of Tat proteins of 103 and 108 amino acids, respectively. Here, we report the characterization of a new BIV Tat protein (Tat236) derived from a tat/rev cDNA. The tat/rev cDNA was obtained by reverse transcription-PCR from RNA extracted from cells infected with BIV. BIV was rescued by cell co-cultivation from the spleen of rabbits exposed for 3 years to the R29 isolate of BIV. Sequence analysis indicated that BIV Tat236 contains the first 98 amino acids of Tat103 and the 3' end 138 amino acids of Rev. Reporter gene assays indicated that transactivation of BIV long terminal repeat (LTR) by Tat236 is higher than by the original BIV Tat proteins in several cell types. By using overlapping deletion mutants, evidence was given that the predicted basic domain of Rev within Tat236 plays a major role in the observed enhanced transactivation activity of the protein. However, the intact functional domain of the original BIV Tat is required for efficient transactivation. This is the first report of a hybrid Tat protein from BIV or any lentiviruses that shows higher transactivation than the original transactivator Tat proteins.

Expression of bovine immunodeficiency-like virus envelope glycoproteins by a recombinant baculovirus in insect cells.

The bovine immunodeficiency-like virus (BIV) env open reading frame (ORF) contains both sequences encoding env and sequences for exon 1 of the putative rev gene. Recombinant baculoviruses incorporating BIV env ORF sequences were constructed to characterize the expression, processing, and immunogenicity of products of the BIV env ORF in insect cells and to develop reagents to study native BIV Env glycoproteins. A recombinant baculovirus containing the entire env ORF synthesized a nonglycosylated, 20-kDa, BIV-specific protein, apparently unrelated to native BIV Env proteins. In contrast, a recombinant baculovirus containing a truncated env ORF in which the coding sequences for rev exon 1 were deleted synthesized three size classes of glycosylated proteins in insect cells related to the BIV Env precursor (gp145), surface (gp100), and transmembrane (gp45) glycoproteins observed in BIV-infected mammalian cells. Oligomers of recombinant BIV Env proteins also formed in these baculovirus-infected insect cells. Immunofluorescence staining of intact insect cells infected by the baculovirus expressing BIV Env with BIV-specific serum demonstrated that the recombinant Env glycoproteins were expressed on the cell surface. Antisera raised to recombinant Env glycoproteins immunoprecipitated native gp145, gp100, and gp45 in BIV-infected bovine cells similar to sera from animals naturally or experimentally infected with BIV.

Functional differences between human and bovine immunodeficiency virus Tat transcription factors.

Transcriptional transactivation of the human immunodeficiency virus type 1 (HIV-1) long terminal repeat (LTR) promoter element by the essential viral Tat protein requires recruitment of positive transcription elongation factor b (P-TEFb) to the viral TAR RNA target. The recruitment of P-TEFb, which has been proposed to be necessary and sufficient for activation of viral gene expression, is mediated by the highly cooperative interaction of Tat and cyclin T1, an essential component of P-TEFb, with the HIV-1 TAR element. Species, such as rodents, that encode cyclin T1 variants that are unable to support TAR binding by the Tat-cyclin T1 heterodimer are also unable to support HIV-1 Tat function. In contrast, we here demonstrate that the bovine immunodeficiency virus (BIV) Tat protein is fully able to bind to BIV TAR both in vivo and in vitro in the absence of any cellular cofactor. Nevertheless, BIV Tat can specifically recruit cyclin T1 to the BIV TAR element, and this recruitment is as essential for BIV Tat function as it is for HIV-1 Tat activity. However, because the cyclin T1 protein does not contribute to TAR binding, BIV Tat is able to function effectively in cells from several species that do not support HIV-1 Tat function. Thus, BIV Tat, while apparently dependent on the same cellular cofactor as the Tat proteins encoded by other lentiviruses, is nevertheless unique in terms of the mechanism used to recruit the BIV Tat-cyclin T1 complex to the viral LTR promoter.

RNA bulge entropies in the unbound state correlate with peptide binding strengths for HIV-1 and BIV TAR RNA because of improved conformational access.

For the binding of peptides to wild-type HIV-1 and BIV TAR RNA and to mutants with bulges of various sizes, changes in the DeltaDelta G values of binding were determined from experimental K d values. The corresponding entropies of these bulges are estimated by enumerating all possible RNA bulge conformations on a lattice and then applying the Boltzmann relationship. Independent calculations of entropies from fluctuations are also carried out using the Gaussian network model (GNM) recently introduced for analyzing folded structures. Strong correlations are seen between the changes in free energy determined for binding and the two different unbound entropy calculations. The fact that the calculated entropy increase with larger bulge size is correlated with the enhanced experimental binding free energy is unusual. This system exhibits a dependence on the entropy of the unbound form that is opposite to usual binding models. Instead of a large initial entropy being unfavorable since it would be reduced upon binding, here the larger entropies actually favor binding. Several interpretations are possible: (i) the higher conformational freedom implies a higher competence for binding with a minimal strain, by suitable selection amongst the set of already accessible conformations; (ii) larger bulge entropies enhance the probability of the specific favorable conformation of the bound state; (iii) the increased freedom of the larger bulges contri-butes more to the bound state than to the unbound state; (iv) indirectly the large entropy of the bound state might have an unfavorable effect on the solvent structure. Nonetheless, this unusual effect is interesting.

Important role for the CA-NC spacer region in the assembly of bovine immunodeficiency virus Gag protein.

Lentiviral Gag proteins contain a short spacer sequence that separates the capsid (CA) from the downstream nucleocapsid (NC) domain. This short spacer has been shown to play an important role in the assembly of human immunodeficiency virus type 1 (HIV-1). We have now extended this finding to the CA-NC spacer motif within the Gag protein of bovine immunodeficiency virus (BIV). Mutation of this latter spacer sequence led to dramatic reductions in virus production, which was mainly attributed to the severely disrupted association of the mutated Gag with the plasma membrane, as shown by the results of membrane flotation assays and confocal microscopy. Detailed mutagenesis analysis of the BIV CA-NC spacer region for virus assembly determinants led to the identification of two key residues, L368 and M372, which are separated by three amino acids, 369-VAA-371. Incidentally, the same two residues are present within the HIV-1 CA-NC spacer region at positions 364 and 368 and have also been shown to be crucial for HIV-1 assembly. Regardless of this conservation between these two viruses, the BIV CA-NC spacer could not be replaced by its HIV-1 counterpart without decreasing virus production, as opposed to its successful replacement by the CA-NC spacer sequences from the nonprimate lentiviruses such as feline immunodeficiency virus (FIV), equine infectious anemia virus and visna virus, with the sequence from FIV showing the highest effectiveness in this regard. Taken together, these data suggest a pivotal role for the CA-NC spacer region in the assembly of BIV Gag; however, the mechanism involved therein may differ from that for the HIV-1 CA-NC spacer.