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Computationally designed icosahedral protein-based assemblies can protect their genetic material and evolve in biochemical environments, suggesting a route to the custom design of synthetic nanomaterials for non-viral drug delivery. Synthetic protein shells Viruses use capsids, or protein shells, to envelop and protect their genomes. This tricks the cell's machinery into using their genetic material for their own replication. Several viruses have been used for gene therapy. In this work, David Baker and colleagues design and build icosahedral protein-based assemblies which protect their genetic material and can evolve in biochemical environments, like a synthetic virus capsid. These assemblies are termed synthetic nucleocapsids. Evolution of the nucleocapsid designs improved both their mRNA packaging efficiency, which competes with commonly used viral vectors for gene therapy, and their circulation stability in vivo . This work raises the possibility of designing custom synthetic nucleocapsids for various uses and RNA cargoes, including for drug delivery. 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 1 and to display proteins or peptides 2 , 3 , 4 , 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 5 , 6 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 7 , 8 . 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 1 , 9 , 10 ; 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.

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.

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.

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.

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.

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.

We have performed molecular dynamics (MD) simulations, with particle-mesh Ewald, explicit waters, and counterions, and binding specificity analyses using combined molecular mechanics and continuum solvent (MM-PBSA) on the bovine immunodeficiency virus (BIV) Tat peptide-TAR RNA complex. The solution structure for the complex was solved independently by Patel and co-workers and Puglisi and co-workers. We investigated the differences in both structures and trajectories, particularly in the formation of the U-A-U base triple, the dynamic flexibility of the Tat peptide, and the interactions at the binding interface. We observed a decrease in RMSD in comparing the final average RNA structures and initial RNA structures of both trajectories, which suggests the convergence of the RNA structures to a MD equilibrated RNA structure. We also calculated the relative binding of different Tat peptide mutants to TAR RNA and found qualitative agreement with experimental studies.

Bovine immunodeficiency virus (BIV) was first isolated in 1969 from a cow, R-29, with a wasting syndrome. The virus isolated induced the formation of syncytia in cell cultures and was structurally similar to maedi-visna virus. Twenty years later, it was demonstrated that the bovine R-29 isolate was indeed a lentivirus with striking similarity to the human immunodeficiency virus. Like other lentiviruses, BIV has a complex genomic structure characterized by the presence of several regulatory/accessory genes that encode proteins, some of which are involved in the regulation of virus gene expression. This manuscript aims to review biological and, more particularly, molecular aspects of BIV, with emphasis on regulatory/accessory viral genes/proteins, in comparison with those of other lentiviruses.

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.

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-κB 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.