Genetic and molecular characterization of the Rev protein of equine infectious anemia virus

Members of the lentivirus subfamily of retroviruses are characterized as causing slow, chronic disease. Atypical of other lentiviruses, such as human immunodeficiency virus type 1, equine infectious anemia virus (EIAV) may produce a rapid, variable disease course in horses. Infected horses may undergo an, acute episode of disease involving viremia, fever, and thrombocytopenia. Following this acute period, horses may resolve to an inapparent infection or suffer a chronic period of additional cycles of viremia, fever, and thrombocytopenia. Horses which survive clinical episodes usually become inapparent carriers of the virus for life. Numerous virus and host factors contribute to the phenotypic manifestations of disease. These include, but are not limited to, the rate of virus replication, the host immune response, and genetic variation of the virus. Genetic variation in EIAV has been identified in a region overlapped by the genes encoding the transmembrane protein and the trans-regulatory protein Rev. Rev is a nucleocytoplasmic transport protein which regulates the expression of viral structural proteins and progeny RNA molecules during the late phase of virus replication. Therefore, factors which modulate Rev activity may result in changes in virus replication and ultimately contribute to virus pathogenesis in vivo. The long term goal of this research project is to determine the contribution of Rev variation to the manifestation of clinical disease during an EIAV infection. To accomplish this goal, we first determined that genetic variation in Rev can modulate the rate of virus replication in vitro;We then characterized variation within Rev in an experimentally-infected pony. Functional analysis of the dominant Rev variants at various time points during infection indicated that the variants dominant during the clinical periods possessed more nuclear export activity than those dominant in the acute or aclinical periods. To further understand the significance of Rev variation, we also mapped the cis- and trans-acting elements required for the nuclear export and alternative splicing activities of Rev. Specifically, we mapped the alternative splicing domain of Rev and identified the RRE of EIAV. These studies provide strong evidence that variation in Rev contributes to clinical disease during the course of an EIAV infection

Shelia Moore and Bob Belshan in a Joint Senior Recital

This is the program for the senior recital of pianist Shelia Moore and tenor Bob Belshan. Lois Kirkpatrick assisted. The recital took place on November 5, 1974, in Mitchell Hall

NF45 and NF90 Bind HIV-1 RNA and Modulate HIV Gene Expression

A previous proteomic screen in our laboratory identified nuclear factor 45 (NF45) and nuclear factor 90 (NF90) as potential cellular factors involved in human immunodeficiency virus type 1 (HIV-1) replication. Both are RNA binding proteins that regulate gene expression; and NF90 has been shown to regulate the expression of cyclin T1 which is required for Tat-dependent trans-activation of viral gene expression. In this study the roles of NF45 and NF90 in HIV replication were investigated through overexpression studies. Ectopic expression of either factor potentiated HIV infection, gene expression, and virus production. Deletion of the RNA binding domains of NF45 and NF90 diminished the enhancement of HIV infection and gene expression. Both proteins were found to interact with the HIV RNA. RNA decay assays demonstrated that NF90, but not NF45, increased the half-life of the HIV RNA. Overall, these studies indicate that both NF45 and NF90 potentiate HIV infection through their RNA binding domains

Binding of Equine Infectious Anemia Virus Rev to an Exon Splicing Enhancer Mediates Alternative Splicing and Nuclear Export of Viral mRNAs

In addition to facilitating the nuclear export of incompletely spliced viral mRNAs, equine infectious anemia virus (EIAV) Rev regulates alternative splicing of the third exon of the tat/rev mRNA. In the presence of Rev, this exon of the bicistronic RNA is skipped in a fraction of the spliced mRNAs. In this report, the cis-acting requirements for exon 3 usage were correlated with sequences necessary for Rev binding and transport of incompletely spliced RNA. The presence of a purine-rich exon splicing enhancer (ESE) was required for exon 3 recognition, and the addition of Rev inhibited exon 3 splicing. Glutathione-S-transferase (GST)-Rev bound to probes containing the ESE, and mutation of GAA repeats to GCA within the ESE inhibited both exon 3 recognition in RNA splicing experiments and GST-Rev binding in vitro. These results suggest that Rev regulates alternative splicing by binding at or near the ESE to block SR protein-ESE interactions. A 57-nucleotide sequence containing the ESE was sufficient to mediate Rev-dependent nuclear export of incompletely spliced RNAs. Rev export activity was significantly inhibited by mutation of the ESE or by trans-complementation with SF2/ASF. These results indicate that the ESE functions as a Rev-responsive element and demonstrate that EIAV Rev mediates exon 3 exclusion through protein-RNA interactions required for efficient export of incompletely spliced viral RNAs

Thermosensitive Gel Containing Cellulose Acetate Phthalate-Efavirenz Combination Nanoparticles for Prevention of HIV-1 Infection

The objective of this investigation was to develop and evaluate a nano-microbicide containing a combination of cellulose acetate phthalate (HIV-1 entry inhibitor) and efavirenz (anti-HIV agent) for HIV prophylaxis. Cellulose acetate phthalate-efavirenz combination nanoparticles (CAP-EFV-NPs) were fabricated by the nanoprecipitation method and were characterized for particle size, zeta potential and encapsulation efficiency of efavirenz. CAP-EFV-NPs were incorporated into a thermosensitive gel (CAP-EFV-NP-Gel). CAP-EFV-NPs, CAP-EFV-NP-Gel and efavirenz solution were evaluated for cytotoxicity to HeLa cells and for in vitro short-term (1-day) and long-term (3-day) prophylaxis against HIV-1 infection in TZM-bl cells. CAP-EFV-NPs had size \u3c 100 nm, negative surface charge and encapsulation efficiency of efavirenz was \u3e 98%. CAP-EFV-NPs and CAP-EFV-NP-Gel were significantly less toxic (P \u3c 0 01) to HeLa cells as compared to efavirenz solution. CAP-EFV-NPs showed significantly higher prophylactic activity (P \u3c 0 01) against HIV-1 infection to TZM-bl cells as compared to efavirenz solution and blank CAP nanoparticles. CAP-EFV-NP-Gel can be a promising nano-microbicide for long-term HIV prophylaxis

In Vitro Data Suggest a Role for PMS2 Kozak Sequence Mutations in Lynch Syndrome Risk

This study investigates the role of 5′ UTR PMS2 Kozak sequence genetic variation in cancer risk. It accomplishes this through the development of a mid-throughput reporter assay where variants can be tested for protein translation efficiency. The results highlight the importance of continued study of the Kozak sequence related to human disease

Vpx is Critical for SIVmne infection of pigtail macaques

<p>Abstract</p> <p>Background</p> <p>Viral protein X (Vpx) of SIV has been reported to be important for establishing infection <it>in vivo</it>. Vpx has several different activities <it>in vitro</it>, promoting preintegration complex import into the nucleus in quiescent lymphocytes and overcoming a block in reverse transcription in macrophages. Vpx interacts with the DDB1-CUL4-DCAF1 E3 ligase complex, which may or may not be required for the ascribed functions. The goal of the current study was to determine whether these activities of Vpx are important <it>in vivo</it>.</p> <p>Results</p> <p>An infectious, pathogenic clone of SIVmne was used to examine correlations between Vpx functions <it>in vitro </it>and <it>in vivo</it>. Three previously described HIV-2 Vpx mutants that were shown to be important for nuclear import of the preintegration complex in quiescent lymphocytes were constructed in SIVmne: A <it>vpx</it>-deleted virus, a truncation of Vpx at amino acid 102 that deletes the C-terminal proline-rich domain (X(102)), and a mutant with tyrosines 66, 69, and 71 changed to alanine (X(y-a)). All mutant viruses replicated similarly to wild type SIVmne027 in primary pigtail macaque PBMCs, and were only slightly retarded in CEMx174 cells. However, all the <it>vpx </it>mutant viruses were defective for replication in both human and pigtail monocyte-derived macrophages. PCR assays demonstrated that the efficiency of reverse transcription and the levels of viral integration in macrophages were substantially reduced for the <it>vpx </it>mutant viruses. <it>In vitro</it>, the X(y-a) mutant, but not the X(102) mutant lost interaction with DCAF1. The wild type SIVmne027 and the three <it>vpx </it>mutant SIVs were inoculated by the intra-rectal route into pigtail macaques. Peak levels of plasma viremia of the <it>vpx </it>mutant SIVs were variable, but consistently lower than that observed in macaques infected with wild type SIVmne. <it>In situ </it>hybridization for SIV demonstrated that compared to wild type SIVmne infected macaques five of the six animals inoculated with the <it>vpx </it>mutant SIVs had only low levels of SIV-expressing cells in the rectum, most intestinal epithelial tissues, spleen, and mesenteric and peripheral nodes.</p> <p>Conclusions</p> <p>This work demonstrates that the activities of Vpx to overcome restrictions in culture <it>in vitro </it>are also likely to be important for establishment of infection <it>in vivo </it>and suggest that both the nuclear localization and DCAF1-interaction functions of Vpx are critical <it>in vivo</it>.</p

Structural Model of the Rev Regulatory Protein from Equine Infectious Anemia Virus

Rev is an essential regulatory protein in the equine infectious anemia virus (EIAV) and other lentiviruses, including HIV-1. It binds incompletely spliced viral mRNAs and shuttles them from the nucleus to the cytoplasm, a critical prerequisite for the production of viral structural proteins and genomic RNA. Despite its important role in production of infectious virus, the development of antiviral therapies directed against Rev has been hampered by the lack of an experimentally-determined structure of the full length protein. We have used a combined computational and biochemical approach to generate and evaluate a structural model of the Rev protein. The modeled EIAV Rev (ERev) structure includes a total of 6 helices, four of which form an anti-parallel four-helix bundle. The first helix contains the leucine-rich nuclear export signal (NES). An arginine-rich RNA binding motif, RRDRW, is located in a solvent-exposed loop region. An ERLE motif required for Rev activity is predicted to be buried in the core of modeled structure where it plays an essential role in stabilization of the Rev fold. This structural model is supported by existing genetic and functional data as well as by targeted mutagenesis of residues predicted to be essential for overall structural integrity. Our predicted structure should increase understanding of structure-function relationships in Rev and may provide a basis for the design of new therapies for lentiviral diseases

Targeted Sequencing for Hereditary Breast and Ovarian Cancer in BRCA1/2-Negative Families Reveals Complex Genetic Architecture and Phenocopies

Approximately 20% of breast cancer cases are attributed to increased family risk, yet variation in BRCA1/2 can only explain 20%-25% of cases. Historically, only single gene or single variant testing were common in at-risk family members, and further sequencing studies were rarely offered after negative results. In this study, we applied an efficient and inexpensive targeted sequencing approach to provide molecular diagnoses in 245 human samples representing 134 BRCA mutation-negative (BRCAX) hereditary breast and ovarian cancer (HBOC) families recruited from 1973 to 2019 by Dr. Henry Lynch. Sequencing identified 391 variants, which were functionally annotated and ranked based on their predicted clinical impact. Known pathogenic CHEK2 breast cancer variants were identified in five BRCAX families in this study. While BRCAX was an inclusion criterion for this study, we still identified a pathogenic BRCA2 variant (p.Met192ValfsTer13) in one family. A portion of BRCAX families could be explained by other hereditary cancer syndromes that increase HBOC risk: Li-Fraumeni syndrome (gene: TP53) and Lynch syndrome (gene: MSH6). Interestingly, many families carried additional variants of undetermined significance (VOUSs) that may further modify phenotypes of syndromic family members. Ten families carried more than one potential VOUS, suggesting the presence of complex multi-variant families. Overall, nine BRCAX HBOC families in our study may be explained by known likely pathogenic/pathogenic variants, and six families carried potential VOUSs, which require further functional testing. To address this, we developed a functional assay where we successfully re-classified one family\u27s PMS2 VOUS as benign

Analysis of the EIAV Rev-Responsive Element (RRE) Reveals a Conserved RNA Motif Required for High Affinity Rev Binding in Both HIV-1 and EIAV

A cis-acting RNA regulatory element, the Rev-responsive element (RRE), has essential roles in replication of lentiviruses, including human immunodeficiency virus (HIV-1) and equine infection anemia virus (EIAV). The RRE binds the viral trans-acting regulatory protein, Rev, to mediate nucleocytoplasmic transport of incompletely spliced mRNAs encoding viral structural genes and genomic RNA. Because of its potential as a clinical target, RRE-Rev interactions have been well studied in HIV-1; however, detailed molecular structures of Rev-RRE complexes in other lentiviruses are still lacking. In this study, we investigate the secondary structure of the EIAV RRE and interrogate regulatory protein-RNA interactions in EIAV Rev-RRE complexes. Computational prediction and detailed chemical probing and footprinting experiments were used to determine the RNA secondary structure of EIAV RRE-1, a 555 nt region that provides RRE function in vivo. Chemical probing experiments confirmed the presence of several predicted loop and stem-loop structures, which are conserved among 140 EIAV sequence variants. Footprinting experiments revealed that Rev binding induces significant structural rearrangement in two conserved domains characterized by stable stem-loop structures. Rev binding region-1 (RBR-1) corresponds to a genetically-defined Rev binding region that overlaps exon 1 of the EIAV rev gene and contains an exonic splicing enhancer (ESE). RBR-2, characterized for the first time in this study, is required for high affinity binding of EIAV Rev to the RRE. RBR-2 contains an RNA structural motif that is also found within the high affinity Rev binding site in HIV-1 (stem-loop IIB), and within or near mapped RRE regions of four additional lentiviruses. The powerful integration of computational and experimental approaches in this study has generated a validated RNA secondary structure for the EIAV RRE and provided provocative evidence that high affinity Rev binding sites of HIV-1 and EIAV share a conserved RNA structural motif. The presence of this motif in phylogenetically divergent lentiviruses suggests that it may play a role in highly conserved interactions that could be targeted in novel anti-lentiviral therapies