Evolution of a designed protein assembly encapsulating its own RNA genome.

The challenges of evolution in a complex biochemical environment, coupling genotype to phenotype and protecting the genetic material, are solved elegantly in biological systems by the encapsulation of nucleic acids. In the simplest examples, viruses use capsids to surround their genomes. Although these naturally occurring systems have been modified to change their tropism and to display proteins or peptides, billions of years of evolution have favoured efficiency at the expense of modularity, making viral capsids difficult to engineer. Synthetic systems composed of non-viral proteins could provide a 'blank slate' to evolve desired properties for drug delivery and other biomedical applications, while avoiding the safety risks and engineering challenges associated with viruses. Here we create synthetic nucleocapsids, which are computationally designed icosahedral protein assemblies with positively charged inner surfaces that can package their own full-length mRNA genomes. We explore the ability of these nucleocapsids to evolve virus-like properties by generating diversified populations using Escherichia coli as an expression host. Several generations of evolution resulted in markedly improved genome packaging (more than 133-fold), stability in blood (from less than 3.7% to 71% of packaged RNA protected after 6 hours of treatment), and in vivo circulation time (from less than 5 minutes to approximately 4.5 hours). The resulting synthetic nucleocapsids package one full-length RNA genome for every 11 icosahedral assemblies, similar to the best recombinant adeno-associated virus vectors. Our results show that there are simple evolutionary paths through which protein assemblies can acquire virus-like genome packaging and protection. Considerable effort has been directed at 'top-down' modification of viruses to be safe and effective for drug delivery and vaccine applications; the ability to design synthetic nanomaterials computationally and to optimize them through evolution now enables a complementary 'bottom-up' approach with considerable advantages in programmability and control.

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.

Multicentric lymphoma in buffaloes in the Amazon region, Brazil.

BACKGROUND: The presence of lymphoma in buffaloes was first reported in India in the 1960s. The disease is similar to Enzootic Bovine Leucosis (EBL) caused by Bovine leukemia virus (BLV) in cattle; however, according to our results and those of other studies, the etiology of these lymphomas in buffalo do not appear to be associated with BLV. The objectives of this study are to describe four cases of the disease in buffaloes belonging to the same herd in the Amazon region of Brazil and to perform a clinical-anatomopathological, immunohistochemical, and etiological study of the lymphomas. RESULTS: Over a period of ten years, four buffaloes were observed presenting progressive weight loss, swelling of peripheral lymph nodes, and nodules in the subcutaneous tissue. Upon necropsy, whitish-colored tumor masses were observed in the form of nodules in the subcutaneous tissue, along with miliary nodules on the serosal surfaces of abdominal and thoracic organs and tumors in lymph nodes and other organs. Neoplastic lymphocyte proliferation was observed through histopathology. An immunohistochemical study revealed that the neoplasias were formed by proliferation of predominantly B lymphocytes. The presence of BLV genome was not detected in the lymphomas when using the real-time PCR technique, nor was it detected through immunohistochemical staining using monoclonal antibodies against two viral proteins. Bovine herpesvirus 6 was not detected in the tumors. However, Bovine immunodeficiency virus (BIV) was detected in samples of lymphoma and in the lymph nodes and kidneys of one of the animals. CONCLUSIONS: The occurrence of lymphoma in buffaloes is reported for the first time in Brazil and is characterized by B-cell multicentric lymphoma. The etiology of the disease does not appear to be associated with BLV; however, the detection of BIV in samples of lymphoma from one sick animal deserves further study, considering the oncogenic potential of this virus.

Molecular detection of bovine immunodeficiency virus in water buffaloes (Bubalus bubalis) from the Amazon region, Brazil.

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.

The dUTPase-related gene of bovine immunodeficiency virus is critical for viral replication, despite the lack of dUTPase activity of the encoded protein.

BACKGROUND: Deoxyuridine 5'-triphosphate nucleotide-hydrolases (dUTPases) are essential for maintaining low intra-cellular dUTP/dTTP ratios. Therefore, many viruses encode this enzyme to prevent dUTP incorporation into their genomes instead of dTTP. Among the lentiviruses, the non-primate viruses express dUTPases. In bovine immunodeficiency virus (BIV), the putative dUTPase protein is only 74 residues-long, compared to ~130 residues in other lentiviruses. RESULTS: In this study, the recombinant BIV dUTPase, as well as infectious wild-type (WT) BIV virions, were shown to lack any detectable dUTPase activity. Controls of recombinant dUTPase from equine infectious anemia virus (EIAV) or of EIAV virions showed substantial dUTPase activities. To assess the importance of the dUTPase to BIV replication, we have generated virions of WT BIV or BIV with mutations in the dUTPase gene. The two mutant viral dUTPases were the double mutant D48E/N57S (in the putative enzyme active site and its vicinity) and a deletion of 36 residues. In dividing Cf2Th cells and under conditions where the WT virus was infectious and generated progeny virions, both mutant viruses were defective, as no progeny viruses were generated. Analyses of the integrated viral cDNA showed that cells infected with the mutant virions carry in their genomic DNA levels of integrated BIV DNA that are comparable to those in WT BIV-infected cells. CONCLUSIONS: The herby presented results show that the two BIV mutants with the modified dUTPase gene could infect cells, as viral cDNA was synthesized and integrated into the host cell DNA. However, no virions were generated by cells infected by these mutants. The most likely explanation is that either the integrated cDNA of the mutants is defective (due to potential multiple mutations, introduced during reverse-transcription) or that the original dUTPase mutations have led to severe blocks in viral replication at steps post integration. These results emphasize the importance of the dUTPase-related sequence to BIV replication, despite the lack of any detectable catalytic activity.

Tethering of proteins to RNAs using the bovine immunodeficiency virus-Tat peptide and BIV-TAR RNA.

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.

Evidence of bovine immunodeficiency virus: A molecular survey in water buffalo populations of Iran.

BACKGROUND: Bovine immunodeficiency virus (BIV) is a member of the Retroviridae family causing a progressive lifelong infection in cattle and buffaloes. OBJECTIVE: Despite the worldwide distribution of the virus, the studies concerning the prevalence of BIV in buffalo populations have not been conducted in Iran as yet. METHODS: The BIV proviral DNA was surveyed in 120 whole blood samples of water buffaloes in southwestern Iran. Nested PCR was employed to amplify a 298-bp fragment of the pol gene. The BIV Pol sequence was detected in 9.1% of the samples. Among PCR-positive samples, two amplified fragments were confirmed by nucleotide sequencing. RESULTS AND CONCLUSIONS: The studied sequences were completely identical to each other and had more than 98%-99% nucleotide homology to R-29 and HXB3 sequences previously deposited in GenBank. Some point mutations that caused coding substitutions were observed in the studied isolates, compared to other strains. A phylogenetic tree was generated based on the BIV Pol nucleotide sequences reported from other countries. All the BIV strains originated from a unique main cluster and then separated from each other over time. This is the first report on the molecular detection of BIV infections in water buffalo populations in Iran. The wide distribution of BIV in different countries including Iran indicates the importance of the infection as it relates to animal health. Although buffaloes show greater resistance to diseases, they should be considered a health risk to cattle. Furthermore, BIV has negative effects on buffalo milk production and can predispose them to secondary infections. Hence, the findings of this study can advance our understanding of the occurrence of BIV infection in Iran, which can play an important role in the distribution of the disease worldwide.

Bovine ISG15: an antiviral and inducible protein in BIV infected fetal bovine lung cells.

Bovine ISG15 (bISG15) is an interferon inducible ubiquitin-like protein that is responsible for the establishment of early pregnancy in ruminant, understanding the properties of bISG15 capable of being inducible in fetal bovine lung (FBL) cells upon infection of bovine immunodeficiency virus (BIV) is of significant importance. In this study, we investigated the expression of bISG15 in poly I:C treated FBL cells. The increased expression of bISG15 was observed, and the inhibition of BIV replication was also detected in FBL cells. Elimination of bISG15 expression by small interfering RNA reversed the bISG15 mediated inhibition of BIV replication. These findings demonstrate that bISG15 plays an important role in inhibition of the BIV replication in FBL cells. Furthermore, real-time PCR and western blot assay revealed that bISG15's expression can also be induced in BIV infected FBL cells. Taken together, bISG15 is an antiviral and inducible protein in BIV infected FBL cells.

[Polypurine/polypyrimidine sequences with potential of forming triplexes in the proviral DNA of bovine retroviruses].

Perfect interstranded triplexes that can potentially arise in the proviral DNA of wide-spread bovine retroviruses like as bovine leukemia virus (BLV) and bovine immunodeficiency virus (BIV) have been determined. In the BLV and BIV genomes 2 and 5 fragments respectively were found to form triple helixes under acidic conditions. One of those fragments that is localized on the BLV gag gene can exist as cruciform structure too. Experimentally the existence of triplexes is confirmed by atomic force microscopic visualization of supercoiled pGEMEX DNA for which genome 6 fragments are found with mirror symmetry that is necessary for intramolecular triplex formation. The diagrams of triplexes (one of the elements of signaling genome function) localization on the genome of bovine retroviruses are obtained.

Bovine immunodeficiency virus: identification of a long terminal repeat sequence with enhanced promoter activity.

We previously identified a new bovine immunodeficiency virus (BIV) trans-activator factor of transcription (Tat236) that was derived from a variant of BIV. Here, we report a new BIV long terminal repeat (LTR) sequence (LTRn) that was obtained by PCR from the DNA of cells infected with the BIV variant mentioned above. Sequence analysis indicated that the LTRn U3 region harbors three nucleic acid mutations at residue positions -194, -135 and -114 when compared to the original (wild-type) LTR sequence. Reporter gene assays indicated that LTRn promotes basal and Tat-mediated transactivation activity to levels significantly higher than those obtained with the wild-type LTR. Restoration experiments to the wild-type genotype indicated that both the -135 and -114 nucleic acid substitutions were responsible for the enhanced promoter activity of BIV LTRn.

[Distribution of potentially hairpin-loop structures in the genome of bovine retroviruses].

Inverted repeats which can form hairpin-loop structures in the genomic RNA and cruciform structures in the proviral DNA of bovine leukemia virus (BLV) and bovine immunodeficiency virus (BIV) have been determined. Localization diagrams have been made up for hairpins (one of the elements of signaling genome function). The retroviruses BLV and BIV, about 8.5 kbp in length, are characterized by the varying quantitative and qualitative composition of hairpin-loop structures. The BLV and BIV genomes have been found to have 7 and 18 hairpins with energy (-deltaG) of more than 10 kcal/mol, respectively. Furthermore, in the BIV genome, there are 3 thermodynamically stable (i.e. detectable on model systems in vitro) hairpins (with the loop up to 6 nucleotides), two of them are perfect. But thermodynamically stable hairpins have not been found in the BLV genome.

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.

B23/nucleophosmin interacts with bovine immunodeficiency virus Rev protein and facilitates viral replication.

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.

Getting into the major groove. Protein-RNA interactions.

The solution structure of the complex between a peptide derived from the bovine immunodeficiency virus tat protein and the TAR RNA to which it binds reveals a new motif in protein-RNA interactions.

Adaptive recognition in RNA complexes with peptides and protein modules.

Recently, progress has been made towards the structural characterization of the novel folds of RNA-bound arginine-rich peptides and the architecture of their peptide-binding RNA pockets in viral and phage systems. These studies are based on an approach whereby the peptide and RNA components are minimalist modular domains that undergo adaptive structural transitions upon complex formation. Such complexes are characterized by recognition alignments in which the tertiary fold of the RNA generates binding pockets with the potential to envelop minimal elements of protein secondary structure. Strikingly, the peptides fold as isolated alpha-helical or beta-hairpin folds within their RNA major-groove targets, without the necessity of additional appendages for anchorage within the binding pocket. The RNA peptide-binding pocket architectures are sculptured through precisely positioned mismatches, triples and looped-out bases, which accommodate amino acid sidechains through hydrophobic, hydrogen bonding and ionic intermolecular contacts. By contrast, protein modules associated with the HIV-1 nucleocapsid and MS2 phage coat target their RNA binding sites through the insertion of specificity-determining RNA base residues within conserved hydrophobic pockets and crevices on the protein surface, with the bases anchored through hydrogen bonding interactions. These alternative strategies of RNA recognition at the peptide and protein module level provide novel insights into the principles, patterns and diversity of the adaptive transitions associated with the recognition process.

[Amplification of proviral DNA of bovine immunodeficiency virus in experimentally infected animals].

The primer set for detection of bovine immunodeficiency virus (BIV) proviral DNA, retrovirus of unknown pathology, by standard polymerase chain reaction was developed. Amplicon of the expected size was detected in DNA from peripheral blood mononuclear cells of seropositive experimentally BIV infected sheep and cattle. Primers targeted the short fragment of BIV pol gene. Sequences of BIV proviral DNA extracted from BIV infected cell culture as well as from experimentally infected cows were taken for targeted pol BI BPX gene locus. Problems of BIV detection from the clinical material of the experimentally and naturally infected animals are discussed.

Insights into the molecular and biological features of the dUTPase-related gene of bovine immunodeficiency virus.

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.

Preparation of quadri-subtype influenza virus-like particles using bovine immunodeficiency virus gag protein.

Influenza VLPs comprised of hemagglutinin (HA), neuraminidase (NA), and matrix (M1) proteins have been previously used for immunological and virological studies. Here we demonstrated that influenza VLPs can be made in Sf9 cells by using the bovine immunodeficiency virus gag (Bgag) protein in place of M1. We showed that Bgag can be used to prepare VLPs for several influenza subtypes including H1N1 and H10N8. Furthermore, by using Bgag, we prepared quadri-subtype VLPs, which co-expressed within the VLP the four HA subtypes derived from avian-origin H5N1, H7N9, H9N2 and H10N8 viruses. VLPs showed hemagglutination and neuraminidase activities and reacted with specific antisera. The content and co-localization of each HA subtype within the quadri-subtype VLP were evaluated. Electron microscopy showed that Bgag-based VLPs resembled influenza virions with the diameter of 150-200nm. This is the first report of quadri-subtype design for influenza VLP and the use of Bgag for influenza VLP preparation.

Cloning, expression, purification, crystallization and preliminary crystallographic study of the protein module (BIV2-Helix) in the fusion core of bovine immunodeficiency-like virus (BIV) gp40.

The fusion core of bovine immunodeficiency virus (BIV) gp40 is proposed to be involved in membrane fusion. However, no crystal structures are yet available. A predicted protein module BIV2-Helix of BIVgp40 has been expressed in Escherichia coli and purified by chromatography. Recombinant BIV2-Helix was crystallized using the hanging-drop vapour-diffusion technique at 291 K. The crystals were grown in MPD and belonged to the primitive rhombohedral space group R3, with unit-cell parameters a = 39.17, b = 39.17, c = 295.05 A and two molecules per asymmetric unit. X-ray diffraction data were collected to 1.76 A in the home laboratory from a single crystal.

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

AIM: To explore the possibility of the replacement of the gag gene between human immunodeficiency virus and bovine immunodeficiency virus, to achieve chimeric virions, and thereby gain a new kind of AIDS vaccine based on BHIV chimeric viruses. METHODS: A series of chimeric BHIV proviral DNAs differing in the replacement regions in gag gene were constructed, and then were transfected into 293T cells. The expression of chimeric viral genes was detected at the RNA and protein level. The supernatant of 293T cell was ultra centrifuged to detect the probable chimeric virion. Once the chimeric virion was detected, its biological activities were also assayed by infecting HIV-sensitive MT4 cells. RESULTS: Four chimeric BHIV proviral DNAs were constructed. Genes in chimeric viruses expressed correctly in transfected 293T cells. All four constructs assembled chimeric virions with different degrees of efficiency. These virions had complete structures common to retroviruses and packaged genomic RNAs, but the cleavages of the precursor Gag proteins were abnormal to some extent. Three of these virions tested could attach and enter into MT4 cells, and one of them could complete the course of reverse transcription. Yet none of them could replicate in MT4 cells. CONCLUSION: The replacement of partial gag gene of HIV with BIV gag gene is feasible. Genes in chimeric BHIVs are accurately expressed, and virions are assembled. These chimeric BHIVs (proviral DNA together with virus particles) have the potential to become a new kind of HIV/AIDS vaccine.