HIV-1 Tat commandeers nuclear export of Rev-viral RNA complex by controlling hnRNPA2-mediated splicing.

IMPORTANCE: HIV-infected host cells impose varied degrees of regulation on viral replication, from very high to abortive. Proliferation of HIV in astrocytes is limited when compared to immune cells, such as CD4+ T lymphocytes. Understanding such differential regulation is one of the key questions in the field as these cells permit HIV persistence and rebound viremia, challenging HIV treatment and clinical cure. This study focuses on understanding the molecular mechanism behind such cell-specific disparities. We show that one of the key mechanisms is the regulation of heterogenous nuclear ribonucleoprotein A2, a host factor involved in alternative splicing and RNA processing, by HIV-1 Tat in CD4+ T lymphocytes, not observed in astrocytes. This regulation causes an increase in the levels of unspliced/partially spliced viral RNA and nuclear export of Rev-RNA complexes which results in high viral propagation in CD4+ T lymphocytes. The study reveals a new mechanism imposed by HIV on host cells that determines the fate of infection.

HIV-1 Rev-RRE functional activity in primary isolates is highly dependent on minimal context-dependent changes in Rev.

During HIV infection, intron-containing viral mRNAs are exported from the cell nucleus to the cytoplasm to complete the replication cycle. Cellular restrictions on the export of incompletely spliced transcripts are overcome by a viral protein, Rev, and an RNA structure found in all unspliced and incompletely spliced viral mRNAs, the Rev Response Element (RRE). Primary HIV isolates display substantial variation in the sequence and functional activity of Rev proteins. We analyzed Rev from two primary isolates with disparate activity that resulted in differences in in vitro fitness of replication-competent viral constructs. The results showed that amino acid differences within the oligomerization domain, but not the arginine-rich motif or the nuclear export signal, determined the level of Rev activity. Two specific amino acid substitutions were sufficient to alter the low-activity Rev to a high-activity phenotype. Other mutations in Rev sequences had unpredictable effects on activity that differed between the two Rev backbones. The sensitivity of Rev function level to small sequence changes likely permits modulation of Rev-RRE activity during HIV infection, which may play a role in pathogenesis. The functional consequences of Rev mutations differed between primary isolates, highlighting the challenge of generalizing studies of Rev conducted using laboratory HIV strains.

Identification of a Novel Post-transcriptional Transactivator from the Equine Infectious Anemia Virus.

All lentiviruses encode a post-transcriptional transactivator, Rev, which mediates the export of viral mRNA from the nucleus to the cytoplasm and which is required for viral gene expression and viral replication. In the current study, we demonstrate that equine infectious anemia virus (EIAV), an equine lentivirus, encodes a second post-transcriptional transactivator that we designate Grev. Grev is encoded by a novel transcript with a single splicing event that was identified using reverse transcription-PCR (RT-PCR) and RNA-seq in EIAV-infected horse tissues and cells. Grev is about 18 kDa in size, comprises the first 18 amino acids (aa) of Gag protein together with the last 82 aa of Rev, and was detected in EIAV-infected cells. Similar to Rev, Grev is localized to the nucleus, and both are able to mediate the expression of Mat (a recently identified viral protein of unknown function from EIAV), but Rev can mediate the expression of EIAV Gag/Pol, while Grev cannot. We also demonstrate that Grev, similar to Rev, specifically binds to rev-responsive element 2 (RRE-2, located in the first exon of mat mRNAs) to promote nuclear export of mat mRNA via the chromosome region maintenance 1 (CRM1) pathway. However, unlike Rev, whose function depends on its multimerization, we could not detect multimerization of Grev using coimmunoprecipitation (co-IP) or bimolecular fluorescence complementation (BiFC) assays. Together, these data suggest that EIAV encodes two post-transcriptional transactivators, Rev and Grev, with similar, but not identical, functions. IMPORTANCE Nuclear export of viral transcripts is a crucial step for viral gene expression and viral replication in lentiviruses, and this export is regulated by a post-transcriptional transactivator, Rev, that is shared by all lentiviruses. Here, we report that the equine infectious anemia virus (EIAV) encodes a novel viral protein, Grev, and demonstrated that Grev, like Rev, mediates the expression of the viral protein Mat by binding to the first exon of mat mRNAs via the chromosome region maintenance 1 (CRM1) pathway. Grev is encoded by a single-spliced transcript containing two exons, whereas Rev is encoded by a multiple-spliced transcript containing four exons. Moreover, Rev is able to mediate EIAV Gag/Pol expression by binding to rev-responsive element (RRE) located within the Env-coding region, while Grev cannot. Therefore, the present study demonstrates that EIAV encodes two post-transcriptional regulators, Grev and Rev, suggesting that post-transcriptional regulation patterns in lentivirus are diverse and complex.

IL-6 deletion decreased REV-ERBα protein and influenced autophagy and mitochondrial markers in the skeletal muscle after acute exercise.

Interleukin 6 (IL-6) acts as a pro and anti-inflammatory cytokine, has an intense correlation with exercise intensity, and activates various pathways such as autophagy and mitochondrial unfolded protein response. Also, IL-6 is interconnected to circadian clock-related inflammation and can be suppressed by the nuclear receptor subfamily 1, group D, member 1 (Nr1d1, protein product REV-ERBα). Since IL-6 is linked to physical exercise-modulated metabolic pathways such as autophagy and mitochondrial metabolism, we investigated the relationship of IL-6 with REV-ERBα in the adaptations of these molecular pathways in response to acute intense physical exercise in skeletal muscle. The present study was divided into three experiments. In the first one, wild-type (WT) and IL-6 knockout (IL-6 KO) mice were divided into three groups: Basal time (Basal; sacrificed before the acute exercise), 1 hour (1hr post-Ex; sacrificed 1 hour after the acute exercise), and 3 hours (3hr post-Ex; sacrificed 3 hours after the acute exercise). In the second experiment, C2C12 cells received IL-6 physiological concentrations or REV-ERBα agonist, SR9009. In the last experiment, WT mice received SR9009 injections. After the protocols, the gastrocnemius muscle or the cells were collected for reverse transcription-quantitative polymerase chain reaction (RTq-PCR) and immunoblotting techniques. In summary, the downregulation of REV-ERBα, autophagic flux, and most mitochondrial genes was verified in the IL-6 KO mice independent of exercise. The WT and IL-6 KO treated with SR9009 showed an upregulation of autophagic genes. C2C12 cells receiving IL-6 did not modulate the Nr1d1 mRNA levels but upregulated the expression of some mitochondrial genes. However, when treated with SR9009, IL-6 and mitochondrial gene expression were upregulated in C2C12 cells. The autophagic flux in C2C12 suggest the participation of REV-ERBα protein in the IL-6-induced autophagy. In conclusion, the present study verified that the adaptations required through physical exercise (increases in mitochondrial content and improvement of autophagy machinery) might be intermediated by an interaction between IL-6 and REVERBα.

Cell-based and cell-free firefly luciferase complementation assay to quantify Human Immunodeficiency Virus type 1 Rev-Rev interaction.

Rev is an essential regulatory protein of Human Immunodeficiency Virus type 1 (HIV) that is found in the nucleus of infected cells. Rev multimerizes on the Rev-response element (RRE) of HIV RNA to facilitate the export of intron-containing HIV mRNAs from the nucleus to the cytoplasm, and, as such, HIV cannot replicate in the absence of Rev. We have developed cell-intact and cell-free assays based upon a robust firefly split-luciferase complementation system, both of which quantify Rev-Rev interaction. Using the cell-based system we show that additional Crm1 did not impact the interaction, whereas excess Rev reduced it. Furthermore, when a series of mutant Revs were tested, there was a strong correlation between the results of the cell-based assay and the results of a functional Rev trans-complementation infectivity assay. Of interest, a camelid nanobody (NB) that was known to inhibit Rev function enhanced Rev-Rev interaction in the cell-based system. We observed a similar increase in Rev-Rev interaction in a cell-free system, when cell lysates expressing Rev-NLUC or CLUC-Rev were simply mixed. In the cell-free system Rev-Rev interaction occurred within minutes and was inhibited by excess Rev. The levels of interaction between the mutant Revs tested varied by mutant type. Treatment of Rev lysates with RNAse minimally reduced the degree of interaction whereas addition of HIV RRE RNA enhanced the interaction. Purified GST-Rev protein inhibited the interaction. The Z-factor (Z') for the cell-free system was ∼0.85 when tested in 96-well format, and the anti-Rev NB enhanced the interaction in the cell-free system. Thus, we have developed both cell-intact and cell-free systems that can reliably, rapidly, and reproducibly quantify Rev-Rev interaction. These assays, particularly the cell-free one, may be useful in screening and identifying compounds that inhibit Rev function on a high throughput basis.

A Novel, Fully Spliced, Accessory Gene in Equine Lentivirus with Distinct Rev-Responsive Element.

All lentiviruses encode the accessory protein Rev, whose main biological function is to mediate the nuclear export of unspliced and incompletely spliced viral transcripts by binding to a viral cis-acting element (termed the Rev-responsive element, RRE) within the env-encoding region. Equine infectious anemia virus (EIAV) is a member of the lentivirus genus in the Retroviridae family and is considered an important model for the study of lentivirus pathogenesis. Here, we identified a novel transcript from the EIAV genome that encoded a viral protein, named Mat, with an unknown function. The transcript mat was fully spliced and comprised parts of the coding regions of MA and TM. Interestingly, the expression of Mat depended on Rev and the chromosome region maintenance 1 (CRM1) pathway. Rev could specifically bind to Mat mRNA to promote its nuclear export. We further identified that the first exon of Mat mRNA, which was located within the Gag-encoding region, acted as an unreported RRE. Altogether, we identified a novel fully spliced transcript mat with an unusual RRE, which interacted with Rev for nuclear export through the CRM1 pathway. These findings updated the EIAV genome structure, highlighted the diversification of posttranscriptional regulation patterns in EIAV, and may help to expand the understanding of gene transcription and expression of lentivirus. IMPORTANCE In lentiviruses, the nuclear export of viral transcripts is an important step in controlling viral gene expression. Generally, the unspliced and incompletely spliced transcripts are exported via the CRM1-dependent export pathway in a process mediated by the viral Rev protein by binding to the Rev-responsive element (RRE) located within the Env-coding region. However, the completely spliced transcripts are exported via an endogenous cellular pathway, which was Rev independent. Here, we identified a novel fully spliced transcript from EIAV and demonstrated that it encoded a viral protein, termed Mat. Interestingly, we determined that the expression of Mat depended on Rev and identified that the first exon of Mat mRNA could specifically bind to Rev and be exported to the cytoplasm, which suggested that the first exon of Mat mRNA was a second RRE of EIAV. These findings provided important insights into the Rev-dependent nuclear export of completely spliced transcripts in lentiviruses.

Keap1 recognizes EIAV early accessory protein Rev to promote antiviral defense.

The Nrf2/Keap1 axis plays a complex role in viral susceptibility, virus-associated inflammation and immune regulation in host cells. However, whether or how the Nrf2/Keap1 axis is involved in the interactions between equine lentiviruses and their hosts remains unclear. Here, we demonstrate that the Nrf2/Keap1 axis was activated during EIAV infection. Mechanistically, EIAV-Rev competitively binds to Keap1 and releases Nrf2 from Keap1-mediated repression, leading to the accumulation of Nrf2 in the nucleus and promoting Nrf2 responsive genes transcription. Subsequently, we demonstrated that the Nrf2/Keap1 axis represses EIAV replication via two independent molecular mechanisms: directly increasing antioxidant enzymes to promote effective cellular resistance against EIAV infection, and repression of Rev-mediated RNA transport through direct interaction between Keap1 and Rev. Together, these data suggest that activation of the Nrf2/Keap1 axis mediates a passive defensive response to combat EIAV infection. The Nrf2/Keap1 axis could be a potential target for developing strategies for combating EIAV infection.

HIV Rev-isited.

The human immunodeficiency virus type 1 (HIV-1) proteome is expressed from alternatively spliced and unspliced genomic RNAs. However, HIV-1 RNAs that are not fully spliced are perceived by the host machinery as defective and are retained in the nucleus. During late infection, HIV-1 bypasses this regulatory mechanism by expression of the Rev protein from a fully spliced mRNA. Once imported into the nucleus, Rev mediates the export of unprocessed HIV-1 RNAs to the cytoplasm, leading to the production of the viral progeny. While regarded as a canonical RNA export factor, Rev has also been linked to HIV-1 RNA translation, stabilization, splicing and packaging. However, Rev's functions beyond RNA export have remained poorly understood. Here, we revisit this paradigmatic protein, reviewing recent data investigating its structure and function. We conclude by asking: what remains unknown about this enigmatic viral protein?

Impact of Nuclear Export Pathway on Cytoplasmic HIV-1 RNA Transport Mechanism and Distribution.

HIV-1 full-length RNA (referred to as HIV-1 RNA here) serves as the viral genome in virions and as a template for Gag/Gag-Pol translation. We previously showed that HIV-1 RNA, which is exported via the CRM1 pathway, travels in the cytoplasm mainly through diffusion. A recent report suggested that the export pathway used by retroviral RNA could affect its cytoplasmic transport mechanism and localization. HIV-1 RNA export is directed by the viral protein Rev and the cis-acting element, Rev response element (RRE). When Rev/RRE is replaced with the constitutive transport element (CTE) from Mason-Pfizer monkey virus (MPMV), HIV-1 RNA is exported through the NXF1 pathway. To determine the effects of the export pathway on HIV-1 RNA, we tracked individual RNAs and found that the vast majority of cytoplasmic HIV-1 RNAs travel by diffusion regardless of the export pathway. However, CTE-containing HIV-1 RNA diffuses at a rate slower than that of RRE-containing HIV-1 RNA. Using in situ hybridization, we analyzed the subcellular localizations of HIV-1 RNAs in cells expressing a CTE-containing and an RRE-containing provirus. We found that these two types of HIV-1 RNAs have similar subcellular distributions. HIV-1 RNA exported through the NXF1 pathway was suggested to cluster near centrosomes. To investigate this possibility, we measured the distances between individual RNAs to the centrosomes and found that HIV-1 RNAs exported through different pathways do not exhibit significantly different distances to centrosomes. Therefore, HIV-1 RNAs exported through CRM1 and NXF1 pathways use the same RNA transport mechanism and exhibit similar cytoplasmic distributions.IMPORTANCE The unspliced HIV-1 full-length RNA (HIV-1 RNA) is packaged into virions as the genome and is translated to generate viral structural proteins and enzymes. To serve these functions, HIV-1 RNA must be exported from the nucleus to the cytoplasm. It was recently suggested that export pathways used by HIV-1 RNA could affect its cytoplasmic transport mechanisms and distribution. In the current report, we examined the HIV-1 RNA transport mechanism by following the movement of individual RNAs and identifying the distribution of RNA using in situ hybridization. Our results showed that whether exported by the CRM1 or NXF1 pathway, HIV-1 RNAs mainly use diffusion for cytoplasmic travel. Furthermore, HIV-1 RNAs exported using the CRM1 or NXF1 pathway are well mixed in the cytoplasm and do not display export pathway-specific clustering near centrosomes. Thus, the export pathways used by HIV-1 RNAs do not alter the cytoplasmic transport mechanisms or distribution.

Identification of the nuclear and nucleolar localization signals of the Feline immunodeficiency virus Rev protein.

Lentivirus genomes code for a regulatory protein essential for virus replication termed Rev. The Rev protein binds to partially spliced and unspliced viral RNAs and mediates their nuclear export. Therefore, Rev possesses functional domains that enable its shuttling between the cytoplasm and the nucleus. The Feline immunodeficiency virus (FIV), a lentivirus, can lead to an immunodeficiency syndrome after a long incubation period, similar to that associated with the human immunodeficiency virus type 1 (HIV-1). The FIV Rev functional domains have been predicted only by homology with those of HIV-1 Rev. In the present study, the nuclear and nucleolar localization signals (NLS and NoLS, respectively) of the FIV Rev were examined. A series of FIV Rev deletion mutants fused to the enhanced green fluorescent protein (EGFP) were used to localize the NLS in a region spanning amino acids (aa) 81-100. By using alanine substitution mutants, basic residues present between the amino acids (aa) 84-99 of the FIV Rev protein sequence were identified to form the NLS, whereas those between aa 82-95 were associated with the NoLS function. These results further enhance our understanding of how Rev exerts its role in the replication cycle of lentiviruses.

Characterization of Signal Sequences Determining the Nuclear/Nucleolar Import and Nuclear Export of the Caprine Arthritis-Encephalitis Virus Rev Protein.

Caprine arthritis-encephalitis virus (CAEV), a lentivirus, relies on the action of the Rev protein for its replication. The CAEV Rev fulfills its function by allowing the nuclear exportation of partially spliced or unspliced viral mRNAs. In this study, we characterized the nuclear and nucleolar localization signals (NLS and NoLS, respectively) and the nuclear export signal (NES) of the CAEV Rev protein. These signals are key actors in the nucleocytoplasmic shuttling of a lentiviral Rev protein. Several deletion and alanine substitution mutants were generated from a plasmid encoding the CAEV Rev wild-type protein that was fused to the enhanced green fluorescent protein (EGFP). Following cell transfection, images were captured by confocal microscopy and the fluorescence was quantified in the different cell compartments. The results showed that the NLS region is localized between amino acids (aa) 59 to 75, has a monopartite-like structure and is exclusively composed of arginine residues. The NoLS was found to be partially associated with the NLS. Finally, the CAEV Rev protein's NES mapped between aa 89 to 101, with an aa spacing between the hydrophobic residues that was found to be unconventional as compared to that of other retroviral Rev/Rev-like proteins.

BioID screening of biotinylation sites using the avidin-like protein Tamavidin 2-REV identifies global interactors of stimulator of interferon genes (STING).

Stimulator of interferon genes (STING) mediates cytosolic DNA-induced innate immune signaling via membrane trafficking. The global identification of proteins that spatiotemporally interact with STING will provide a better understanding of its trafficking mechanisms and of STING signaling pathways. Proximity-dependent biotin identification (BioID) is a powerful technology to identify physiologically relevant protein-protein interactions in living cells. However, biotinylated peptides are rarely detected in the conventional BioID method, which uses streptavidin beads to pull down biotinylated proteins, because the biotin-streptavidin interaction is too strong. As a result, only nonbiotinylated peptides are identified, which cannot be distinguished from peptides of nonspecifically pull-downed proteins. Here, we developed a simple method to efficiently and specifically enrich biotinylated peptides using Tamavidin 2-REV, an engineered avidin-like protein with reversible biotin-binding capability. Using RAW264.7 macrophages stably expressing TurboID-fused STING, we identified and quantified >4,000 biotinylated peptides of STING-proximal proteins. Various endoplasmic reticulum-associated proteins were biotinylated in unstimulated cells, and STING activation caused biotinylation of many proteins located in the Golgi and endosomes. These proteins included those known to interact with activated STING, such as TANK-binding kinase 1 (TBK1), several palmitoyl transferases, and p62/sequestosome 1 (SQSTM1). Furthermore, interferon-induced transmembrane protein 3 (IFITM3), an endolysosome-localized antiviral protein, bound to STING at the late activation stage. These dynamic interaction profiles will provide detailed insights into STING signaling; we propose that our approach using Tamavidin 2-REV would be useful for BioID-based and other biotinylation-based peptide identification methods.

The Jembrana disease virus Rev protein: Identification of nuclear and novel lentiviral nucleolar localization and nuclear export signals.

The lentiviral Rev protein, which is a regulatory protein essential for virus replication, has been first studied in the human immunodeficiency virus type 1 (HIV-1). The main function of Rev is to mediate the nuclear exportation of viral RNAs. To fulfill its function, Rev shuttles between the cytoplasm and the nucleus. The Jembrana disease virus (JDV), a lentivirus, is the etiologic agent of the Jembrana disease which was first described in Bali cattle in Indonesia in 1964. Despite the high mortality rate associated with JDV, this virus remains poorly studied. Herein the subcellular distribution of JDV Rev, the nuclear and nucleolar localization signals (NLS and NoLS, respectively) and the nuclear export signal (NES) of the protein were examined. JDV Rev fused to the enhanced green fluorescent protein (EGFP) predominantly localized to the cytoplasm and nucleolus of transfected cells, as determined by fluorescence microscopy analyses. Through transfection of a series of deletion mutants of JDV Rev, it was possible to localize the NLS/NoLS region between amino acids (aa) 74 to 105. By substituting basic residues with alanine within this sequence, we demonstrated that the JDV Rev NLS encompasses aa 76 to 86, and is exclusively composed of arginine residues, whereas a bipartite NoLS was observed for the first time in any retroviral Rev/Rev-like proteins. Finally, a NES was identified downstream of the NLS/NoLS and encompasses aa 116 to 128 of the JDV Rev protein. The JDV Rev NES was found to be of the protein kinase A inhibitor (PKI) class instead of the HIV-1 Rev class. It also corresponds to the most optimal consensus sequence of PKI NES and, as such, is novel among lentiviral Rev NES.

Evolution of the HIV-1 Rev Response Element during Natural Infection Reveals Nucleotide Changes That Correlate with Altered Structure and Increased Activity over Time.

The HIV-1 Rev response element (RRE) is a cis-acting RNA element characterized by multiple stem-loops. Binding and multimerization of the HIV Rev protein on the RRE promote the nucleocytoplasmic export of incompletely spliced mRNAs, an essential step in HIV replication. Most of our understanding of the Rev-RRE regulatory axis comes from studies of lab-adapted HIV clones. However, in human infection, HIV evolves rapidly, and mechanistic studies of naturally occurring Rev and RRE sequences are essential to understanding this system. We previously described the functional activity of two RREs found in circulating viruses in a patient followed during the course of HIV infection. The early RRE was less functionally active than the late RRE, despite differing in sequence by only 4 nucleotides. In this study, we describe the sequence, function, and structural evolution of circulating RREs in this patient using plasma samples collected over 6 years of untreated infection. RRE sequence diversity varied over the course of infection, with evidence of selection pressure that led to sequence convergence as disease progressed being found. An increase in RRE functional activity was observed over time, and a key mutation was identified that correlates with a major conformational change in the RRE and increased functional activity. Additional mutations were found that may have contributed to increased activity as a result of greater Shannon entropy in RRE stem-loop II, which is key to primary Rev binding.IMPORTANCE HIV-1 replication requires interaction of the viral Rev protein with a cis-acting regulatory RNA, the Rev response element (RRE), whose sequence changes over time during infection within a single host. In this study, we show that the RRE is subject to selection pressure and that RREs from later time points in infection tend to have higher functional activity. Differences in RRE functional activity are attributable to specific changes in RNA structure. Our results suggest that RRE evolution during infection may be important for HIV pathogenesis and that efforts to develop therapies acting on this viral pathway should take this into account.

[Construction and application of PGRN and Rev-erbß double genes knockout HEK293 cell lines].

In order to study the molecular mechanism and physiological significance of the interaction between PGRN and Rev-erbß, the PGRN gene in HEK293 (Rev-erbß-/-) marked as C3-6 cell lines was knocked out by CRISPR/Cas9 system to generate the Rev-erbß and PGRN double genes knockout HEK293 cell lines. First, four sgRNAs were designed for PGRN gene, and PGRN sgRNA2 and sgRNA3 with the higher activity were used to construct the Lentiviral vector, pLenti/CMV-Loxp-Cas9-sgRNA2-U6-sgRNA3-U6-Loxp-EF1α-Puro. Then, the lentivirus vector carrying Cas9 and double PGRN sgRNA were used to infect HEK293 C3-6 cells. Through drug screening, cloning and sequencing, we obtained the monoclonal HEK293 (Rev-erbß-/-; PGRN-/-) marked as C3-6/23 cell lines. Using qRT-PCR and Western blotting, we detected PGRN mRNA and protein expression in C3-6/23 cell lines. Finally, genetic complementation was used to study the effect of PGRN-mediated Rev-erbß on the regulation of the target gene promoter transcriptional activity in the C3-6/23 cell lines. In HEK293 C3-6/23 cell lines, the two DNA chains of PGRN gene were both deletion mutagenesis, and the expression mRNA and protein of PGRN did not reach the detection level. At the same time, the interaction between PGRN and Rev-erbß enhanced the regulation of Rev-erbß on the transcription of target gene promoter in the cell lines. Using CRISPR/Cas9 system, we successfully constructed the double knockout HEK293 (Rev-erbß-/-; PGRN-/-) monoclonal cell lines. The study found that PGRN could affect Rev-erbß on the regulation of target gene promoter transcription in the C3-6/23 cell lines; however, the mechanism of PGRN involvement in mediating Rev-erbß in transcriptional regulation remains to be further studied.

Multifunctional Enzyme Packaging and Catalysis in the Qß Protein Nanoparticle.

The simultaneous expression in Escherichia coli cells of the Qß virus-like particle (VLP) capsid protein and protein "cargo" tagged with a positively charged Rev peptide sequence leads to the spontaneous self-assembly of VLPs with multiple copies of the cargo inside. We report the packaging of four new enzymes with potential applications in medicine and chemical manufacturing. The captured enzymes are active while inside the nanoparticle shell and are protected from environmental conditions that lead to free-enzyme destruction. We also describe genetic modifications to the packaging scheme that shed light on the self-assembly mechanism of this system and allow indirect control over the internal packaging density of cargo. The technology was extended to create, via self-assembly, VLPs that simultaneously display protein ligands on the exterior and contain enzymes within. Inverse relationships were observed between the size of both the packaged and externally displayed protein or domains and nanoparticle yield. These results provide a general method for the rapid creation of robust protein nanoparticles with desired catalytic and targeting functionalities.

Modeling circadian variability of core-clock and clock-controlled genes in four tissues of the rat.

Circadian clocks, present in almost all cells of the body, are entrained to rhythmic changes in the environment (e.g. light/dark cycles). Genes responsible for this timekeeping are named core-clock genes, which through transcriptional feedback interactions mediated by transcription factor binding to Ebox/RRE/Dbox elements can generate oscillatory activity of their expression. By regulating the transcription of other clock-controlled genes (CCGs) circadian information is transmitted to tissue and organ levels. Recent studies have indicated that there is a considerable variability of clock-controlled gene expression between tissues both with respect to the circadian genes that are regulated and to their phase lags. In this work, a mathematical model was adapted to explore the dynamics of core-clock and clock-controlled genes measured in four tissues of the rat namely liver, muscle, adipose, and lung. The model efficiently described the synchronous rhythmicity of core-clock genes and further predicted that their phases are mainly regulated by Per2 and Cry1 transcriptional delays and Rev-Erba and Cry1 degradation rates. Similarly, after mining databases for potential Ebox/RRE/Dbox elements in the promoter region of clock-controlled genes, the phase variabilities of the same genes between different tissues were described. The analysis suggests that inter-tissue circadian variability of the same clock-controlled genes is an inherent component of homeostatic function and may arise due to different transcription factor activities on Ebox/RRE/Dbox elements.

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.

Differential interaction between human and murine Crm1 and lentiviral Rev proteins.

Mice have multiple obstacles to HIV replication, including a block of unspliced and partially spliced viral mRNA nuclear export. In human, Rev binds to the Rev-response element and human (h) Crm1, facilitating nuclear export of RRE-containing viral RNAs. Murine (m) Crm1 is less functional than hCrm1 in this regard. Here we demonstrated that in biochemical experiments mCrm1 failed to interact with HIV Rev whereas hCrm1 did. In genetic experiments in human cells, we observed a modest but significant differential effect between mCrm1 and hCrm1, which was also true of other lentiviral Revs tested. Triple mutant hCrm1 P411T-M412V-F414S behaved similarly to mCrm1, whereas mCrm1 with T411P-V412M-S414F regained some activity, although contribution of additional residues to its function can not be excluded. Similar results were observed in murine cells. This suggests a differential interaction between hCrm1 and mCrm1 and many lentiviral Revs, which may partially explain the HIV replicative defect in mice.

Identification of a homogenous structural basis for oligomerization by retroviral Rev-like proteins.

BACKGROUND: Rev-like proteins are post-transcriptional regulatory proteins found in several retrovirus genera, including lentiviruses, betaretroviruses, and deltaretroviruses. These essential proteins mediate the nuclear export of incompletely spliced viral RNA, and act by tethering viral pre-mRNA to the host CRM1 nuclear export machinery. Although all Rev-like proteins are functionally homologous, they share less than 30% sequence identity. In the present study, we computationally assessed the extent of structural homology among retroviral Rev-like proteins within a phylogenetic framework. RESULTS: We undertook a comprehensive analysis of overall protein domain architecture and predicted secondary structural features for representative members of the Rev-like family of proteins. Similar patterns of α-helical domains were identified for Rev-like proteins within each genus, with the exception of deltaretroviruses, which were devoid of α-helices. Coiled-coil oligomerization motifs were also identified for most Rev-like proteins, with the notable exceptions of HIV-1, the deltaretroviruses, and some small ruminant lentiviruses. In Rev proteins of primate lentiviruses, the presence of predicted coiled-coil motifs segregated within specific primate lineages: HIV-1 descended from SIVs that lacked predicted coiled-coils in Rev whereas HIV-2 descended from SIVs that contained predicted coiled-coils in Rev. Phylogenetic ancestral reconstruction of coiled-coils for all Rev-like proteins predicted a single origin for the coiled-coil motif, followed by three losses of the predicted signal. The absence of a coiled-coil signal in HIV-1 was associated with replacement of canonical polar residues with non-canonical hydrophobic residues. However, hydrophobic residues were retained in the key 'a' and 'd' positions, and the α-helical region of HIV-1 Rev oligomerization domain could be modeled as a helical wheel with two predicted interaction interfaces. Moreover, the predicted interfaces mapped to the dimerization and oligomerization interfaces in HIV-1 Rev crystal structures. Helical wheel projections of other retroviral Rev-like proteins, including endogenous sequences, revealed similar interaction interfaces that could mediate oligomerization. CONCLUSIONS: Sequence-based computational analyses of Rev-like proteins, together with helical wheel projections of oligomerization domains, reveal a conserved homogeneous structural basis for oligomerization by retroviral Rev-like proteins.