Yeast mutations in multiple complementation groups inhibit brome mosaic virus RNA replication and transcription and perturb regulated expression of the viral polymerase-like gene

Brome mosaic virus (BMV), a member of the alphavirus-like superfamily of positive-strand RNA viruses, encodes two proteins, 1a and 2a, that interact with each other, with unidentified host proteins, and with host membranes to form the viral RNA replication complex. Yeast expressing 1a and 2a support replication and subgenomic mRNA synthesis by BMV RNA3 derivatives. Using a multistep selection and screening process, we have isolated yeast mutants in multiple complementation groups that inhibit BMV-directed gene expression. Three complementation groups, represented by mutants mab1–1, mab2–1, and mab3–1 (for maintenance of BMV functions), were selected for initial study. Each of these mutants has a single, recessive, chromosomal mutation that inhibits accumulation of positive- and negative-strand RNA3 and subgenomic mRNA. BMV-directed gene expression was inhibited when the RNA replication template was introduced by in vivo transcription from DNA or by transfection of yeast with in vitro transcripts, confirming that cytoplasmic RNA replication steps were defective. mab1–1, mab2–1, and mab3–1 slowed yeast growth to varying degrees and were temperature-sensitive, showing that the affected genes contribute to normal cell growth. In wild-type yeast, expression of the helicase-like 1a protein increased the accumulation of 2a mRNA and the polymerase-like 2a protein, revealing a new level of viral regulation. In association with their other effects, mab1–1 and mab2–1 blocked the ability of 1a to stimulate 2a mRNA and protein accumulation, whereas mab3–1 had elevated 2a protein accumulation. Together, these results show that BMV RNA replication in yeast depends on multiple host genes, some of which directly or indirectly affect the regulated expression and accumulation of 2a.

Adeno-associated viral vector-mediated gene transfer results in long-term enzymatic and functional correction in multiple organs of Fabry mice

Fabry disease is a lysosomal storage disorder caused by a deficiency of the lysosomal enzyme α-galactosidase A (α-gal A). This enzyme deficiency leads to impaired catabolism of α-galactosyl-terminal lipids such as globotriaosylceramide (Gb3). Patients develop painful neuropathy and vascular occlusions that progressively lead to cardiovascular, cerebrovascular, and renal dysfunction and early death. Although enzyme replacement therapy and bone marrow transplantation have shown promise in the murine analog of Fabry disease, gene therapy holds a strong potential for treating this disease in humans. Delivery of the normal α-gal A gene (cDNA) into a depot organ such as liver may be sufficient to elicit corrective circulating levels of the deficient enzyme. To investigate this possibility, a recombinant adeno-associated viral vector encoding human α-gal A (rAAV-AGA) was constructed and injected into the hepatic portal vein of Fabry mice. Two weeks postinjection, α-gal A activity in the livers of rAAV-AGA-injected Fabry mice was 20–35% of that of the normal mice. The transduced animals continued to show higher α-gal A levels in liver and other tissues compared with the untouched Fabry controls as long as 6 months after treatment. In parallel to the elevated enzyme levels, we see significant reductions in Gb3 levels to near normal at 2 and 5 weeks posttreatment. The lower Gb3 levels continued in liver, spleen, and heart, up to 25 weeks with no significant immune response to the virus or α-gal A. Also, no signs of liver toxicity occurred after the rAAV-AGA administration. These findings suggest that an AAV-mediated gene transfer may be useful for the treatment of Fabry disease and possibly other metabolic disorders.

Sense- and antisense-mediated gene silencing in tobacco is inhibited by the same viral suppressors and is associated with accumulation of small RNAs

Antisense-mediated gene silencing (ASGS) and posttranscriptional gene silencing (PTGS) with sense transgenes markedly reduce the steady-state mRNA levels of endogenous genes similar in transcribed sequence. RNase protection assays established that silencing in tobacco plants transformed with plant-defense-related class I sense and antisense chitinase (CHN) transgenes is at the posttranscriptional level. Infection of tobacco plants with cucumber mosaic virus strain FN and a necrotizing strain of potato virus Y, but not with potato virus X, effectively suppressed PTGS and ASGS of both the transgenes and homologous endogenes. This suggests that ASGS and PTGS share components associated with initiation and maintenance of the silent state. Small, ca. 25-nt RNAs (smRNA) of both polarities were associated with PTGS and ASGS in CHN transformants as reported for PTGS in other transgenic plants and for RNA interference in Drosophila. Similar results were obtained with an antisense class I β-1,3-glucanase transformant showing that viral suppression and smRNAs are a more general feature of ASGS. Several current models hold that diverse signals lead to production of double-stranded RNAs, which are processed to smRNAs that then trigger PTGS. Our results provide direct evidence for mechanistic links between ASGS and PTGS and suggest that ASGS could join a common PTGS pathway at the double-stranded RNA step.

Provirus integration into a gene encoding a ubiquitin-conjugating enzyme results in a placental defect and embryonic lethality.

Ubiquitin-conjugating enzymes (E2 or Ubc) constitute a family of conserved proteins that play a key role in ubiquitin-dependent degradation of proteins in eukaryotes. We describe here a transgenic mouse strain where retrovirus integration into an Ubc gene, designated UbcM4, results in a recessive-lethal mutation. UbcM4 is the mouse homologue of the previously described human UbcH7 that is involved in the in vitro ubiquitination of several proteins including the tumor suppressor protein p53. The provirus is located in the first intron of the gene. When both alleles are mutated the level of steady-state mRNA is reduced by about 70%. About a third of homozygous mutant embryos die around day 11.5 of gestation. Embryos that survive that stage are growth retarded and die perinatally. The lethal phenotype is most likely caused by impairment of placenta development as this is the only organ that consistently showed pathological defects. The placental labyrinth is drastically reduced in size and vascularization is disturbed. The UbcM4 mouse mutant represents the first example in mammals of a mutation in a gene involved in ubiquitin conjugation. Its recessive-lethal phenotype demonstrates that the ubiquitin system plays an essential role during mouse development.

Site-specific integration by adeno-associated virus.

Adeno-associated virus (AAV) has attracted considerable interest as a potential vector for gene delivery. Wild-type virus is notable for the lack of association with any human disease and the ability to stably integrate its genome in a site-specific manner in a locus on human chromosome 19 (AAVS1). Use of a functional model system for AAV DNA integration into AAVS1 has allowed us to conclude that the recombination event is directed by cellular DNA sequences. Recombinant junctions isolated from our integration assay were analyzed and showed characteristics similar to those found in latently infected cell lines. The minimal DNA signals within AAVS1 required for targeted integration were identified and shown to contain functional motifs of the viral origin of replication. A replication mediated model of AAV DNA integration is proposed.

Fusigenic viral liposome for gene therapy in cardiovascular diseases.

To improve the efficiency of liposome-mediated DNA transfer as a tool for gene therapy, we have developed a fusigenic liposome vector based on principles of viral cell fusion. The fusion proteins of hemagglutinating virus of Japan (HVJ; also Sendai virus) are complexed with liposomes that encapsulate oligodeoxynucleotide or plasmid DNA. Subsequent fusion of HVJ-liposomes with plasma membranes introduces the DNA directly into the cytoplasm. In addition, a DNA-binding nuclear protein is incorporated into the HVJ-liposome particle to enhance plasmid transgene expression. The fusigenic viral liposome vector has proven to be efficient for the intracellular introduction of oligodeoxynucleotide, as well as intact genes up to 100 kbp, both in vitro and in vivo. Many animal tissues have been found to be suitable targets for fusigenic viral liposome DNA transfer. In the cardiovascular system, we have documented successful cytostatic gene therapy in models of vascular proliferative disease using antisense oligodeoxynucleotides against cell cycle genes, double-stranded oligodeoxynucleotides as "decoys" to trap the transcription factor E2F, and expression of a transgene encoding the constitutive endothelial cell form of nitric oxide synthase. Similar strategies are also effective for the genetic engineering of vein grafts and for the treatment of a mouse model of immune-mediated glomerular disease.

Plasmoviruses: nonviral/viral vectors for gene therapy.

We have generated a chimeric gene transfer vector that combines the simplicity of plasmids with the infectivity and long-term expression of retroviruses. We replaced the env gene of a Moloney murine leukemia virus-derived provirus by a foreign gene, generating a plasmid that upon transfer to tumor cells generates noninfectious retroviral particles carrying the transgene. We added to this plasmid an independent expression cassette comprising a cytomegalovirus promoter, an amphotropic retroviral envelope, and a polyadenylylation signal from simian virus 40. These constructs were designed to minimize the risk of recombination generating replication-competent retroviruses. Their only region of homology is a 157-bp sequence with 53% identity. We show that the sole transfection of this plasmid in various cell lines generates infectious but defective retroviral particles capable of efficiently infecting and expressing the transgene. The formation of infectious particles allows the transgene propagation in vitro. Eight days after transfection in vitro, the proportion of cells expressing the transgene is increased by 10-60 times. There was no evidence of replication-competent retrovirus generation in these experiments. The intratumoral injection of this plasmid, but not of the control vector lacking the env gene, led to foci of transgene-expressing cells, suggesting that the transgene had propagated in situ. Altogether, these "plasmoviruses" combine advantages of viral and non-viral vectors. They should be easy to produce in large quantity as clinical grade materials and should allow efficient and safe in situ targeting of tumor cells.

Treatment of a human breast cancer xenograft with an adenovirus vector containing an interferon gene results in rapid regression due to viral oncolysis and gene therapy.

Treatment of a human breast cancer cell line (MDA-MB-435) in nude mice with a recombinant adenovirus containing the human interferon (IFN) consensus gene, IFN-con1 (ad5/IFN), resulted in tumor regression in 100% of the animals. Tumor regression occurred when virus was injected either within 24 hr of tumor cell implantation or with established tumors. However, regression of the tumor was also observed in controls in which either the wild-type virus or a recombinant virus containing the luciferase gene was used, although tumor growth was not completely suppressed. Tumor regression was accompanied by a decrease in p53 expression. Two other tumors, the human myelogenous leukemic cell line K562 and the hamster melanoma tumor RPMI 1846, also responded to treatment but only with ad5/IFN. In the case of K562 tumors, there was complete regression of the tumor, and tumors derived from RPMI 1846 showed partial regression. We propose that the complete regression of the breast cancer with the recombinant virus ad5/IFN was the result of two events: viral oncolysis in which tumor cells are being selectively lysed by the replication-competent virus and the enhanced effect of expression of the IFN-con1 gene. K562 and RPMI 1846 tumors regressed only as a result of IFN gene therapy. This was confirmed by in vitro analysis. Our results indicate that a combination of viral oncolysis with a virus of low pathogenicity, itself resistant to the effects of IFN and IFN gene therapy, might be a fruitful approach to the treatment of a variety of different tumors, in particular breast cancers.

Integration of multiple repeats of geminiviral DNA into the nuclear genome of tobacco during evolution.

Integration of viral DNA into the host nuclear genome, although not unusual in bacterial and animal systems, has surprisingly not been reported for plants. We have discovered geminvirus-related DNA (GRD) sequences, in the form of distinct sets of multiple direct repeats comprising three related repeat classes, situated in a unique locus in the Nicotiana tabacum (tobacco) nuclear genome. The organization of these sequences is similar or identical in eight different tobacco cultivars we have examined. DNA sequence analysis reveals that each repeat has sequences most resembling those of the New World geminiviral DNA replication origin plus the adjacent AL1 gene, encoding the viral replication protein. We believe these GRD sequences originated quite recently in Nicotiana evolution through integration of geminiviral DNA by some combination of the processes of illegitimate recombination, amplification, deletions, and rearrangements. These events must have occurred in plant tissue that was subsequently able to contribute to meristematic tissue yielding gametes. GRD may have been retained in tobacco by selection or by random fixation in a small evolving population. Although we cannot detect transcription of these sequences, this does not exclude the possibility that they may originally have been expressed.

Gene codon composition determines differentiation-dependent expression of a viral capsid gene in keratinocytes in vitro and in vivo

By establishing mouse primary keratinocytes (KCs) in culture, we were able, for the first time, to express papillomavirus major capsid (L1) proteins by transient transfection of authentic or codon-modified L1 gene expression plasmids. We demonstrate in vitro and in vivo that gene codon composition is in part responsible for differentiation-dependent expression of L1 protein in KCs. L1 mRNA was present in similar amounts in differentiated and undifferentiated KCs transfected with authentic or codon-modified L1 genes and had a similar half-life, demonstrating that L1 protein production is posttranscriptionally regulated. We demonstrate further that KCs substantially change their tRNA profiles upon differentiation. Aminoacyl-tRNAs from differentiated KCs but not undifferentiated KCs enhanced the translation of authentic L1 mRNA, suggesting that differentiation-associated change to tRNA profiles enhances L1 expression in differentiated KCs. Thus, our data reveal a novel mechanism for regulation of gene expression utilized by a virus to direct viral capsid protein expression to the site of virion assembly in mature KCs. Analysis of two structural proteins of KCs, involucrin and keratin 14, suggests that translation of their mRNAs is also regulated, in association with KC differentiation in vitro, by a similar mechanism

Viral Delivery of the Fat-1 Gene to Treat Post-Traumatic Arthritis with Diet-Induced Obesity

Post-traumatic arthritis (PTA) is arthritis that develops following joint injury, including meniscus and ligament tears. Current treatments for PTA range from over-the-counter medication to knee replacement; however, in the presence of obesity, the levels of pro-inflammatory cytokines, such as interleukin-1 (IL-1) and tumor necrosis factor alpha (TNF-α,) are more elevated than in non-obese individuals. The role of fatty acids, obesity, and PTA has been examined, with omega-3 fatty acids showing promise as an anti-inflammatory after injury due to its ability to suppress IL-1 and TNF-α. Due to the difficulty in switching patients’ diets, an alternative solution to increasing omega-3 levels needs to be developed. The Fat-1 enzyme, an omega-3 desaturase that has the ability to convert omega-6 to omega-3 fatty acids, may be a good target for increasing the omega-3 levels in the body.

In the first study, we examined whether Fat-1 transgenic mice on a high-fat diet would exhibit lower levels of PTA degeneration following the destabilization of the medial meniscus (DMM). Both male and female Fat-1 and wild-type (WT) littermates were put on either a control diet (10% fat) or an omega-6 rich high-fat diet (60% fat) and underwent DMM surgery. Arthritic changes were examined 12 weeks post-surgery. Fat-1 mice on both the control and high-fat diet showed protection from PTA-related degeneration, while WT mice showed severe arthritic changes. These findings suggest that the omega-6/omega-3 ratio plays an important role in reducing PTA following injury, and demonstrates the potential therapeutic benefit of the Fat-1 enzyme in preventing PTA in both normal and obese patients following acute injury.

Following this, we needed to establish a translatable delivery mechanism for getting the Fat-1 enzyme, which is not present in mammalian cells, into patients. In the second study, we examined whether anti-inflammatory gene delivery of the Fat-1 enzyme would prevent PTA following DMM surgery. In vitro testing of both lentivirus (LV) and adeno-associated virus (AAV) was completed to confirm functionality and conformation of the Fat-1 enzyme after transduction. Male WT mice were placed on an omega-6 rich high-fat diet (60% fat) and underwent DMM surgery; either local or systemic AAV injections of the Fat-1 enzyme or Luciferase, a vector control, were given immediately following surgery. 12 weeks post-surgery, arthritic changes were assessed. The systemic administration of the Fat-1 enzyme showed protection from synovial inflammation and osteophyte formation, while administration of Luciferase did not confer protection. These findings suggest the utility of gene therapy to deliver the Fat-1 enzyme, which has potential as a therapeutic for injured obese patients for the prevention of PTA.

von Hippel Lindau binding protein 1-mediated degradation of integrase affects HIV-1 gene expression at a postintegration step

HIV-1 integrase, the viral enzyme responsible for provirus integration into the host genome, can be actively degraded by the ubiquitin-proteasome pathway. Here, we identify von Hippel-Lindau binding protein 1(VBP1), a subunit of the prefoldin chaperone, as an integrase cellular binding protein that bridges interaction between integrase and the cullin2 (Cul2)-based von Hippel-Lindau (VHL) ubiquitin ligase. We demonstrate that VBP1 and Cul2/VHL are required for proper HIV-1 expression at a step between integrase-dependent proviral integration into the host genome and transcription of viral genes. Using both an siRNA approach and Cul2/VHL mutant cells, we show that VBP1 and the Cul2/VHL ligase cooperate in the efficient polyubiquitylation of integrase and its subsequent proteasome-mediated degradation. Results presented here support a role for integrase degradation by the prefoldin-VHL-proteasome pathway in the integration-transcription transition of the viral replication cycle.

CHARACTERIZATION OF TOBACCO MOSAIC VIRUS DIRECTED REPROGRAMMING OF PHLOEM GENE EXPRESSION TO PROMOTE VIRAL PHLOEM LOADING AND SYSTMEIC MOVEMENT

Vascular phloem loading has long been recognized as an essential step in the establishment of a systemic virus infection. Yet little is known about this process and the mechanisms that control it. In this study, an interaction between the replication protein of Tobacco mosaic virus (TMV) and phloem specific auxin/indole acetic acid (Aux/IAA) transcriptional regulators was found to modulate virus phloem loading. Promoter expression studies show TMV 126/183 kDa interacting Aux/IAAs predominantly express and accumulate within the nuclei of phloem companion cells (CC). Furthermore, CC Aux/IAA nuclear localization is disrupted upon infection with an interacting virus but not during infection with a non-interacting virus. In situ analysis of virus spread shows the inability of TMV variants to disrupt Aux/IAA CC nuclear localization correlates with a reduced ability to load into the vascular tissue. Subsequent systemic movement assays also demonstrate that a virus capable of disrupting Aux/IAA localization is significantly more competitive at systemic movement than a non-interacting virus. Similarly, CC expression and over-accumulation of a degradation-resistant-interacting Aux/IAA protein was found to selectively inhibit TMV accumulation and phloem loading. Transcriptional expression studies demonstrate a role for interacting Aux/IAA proteins in the regulation of salicylic acid and jasmonic acid dependent host defense responses as well as virus specific movement factors including pectin methylesterase that are involved in regulating plasmodesmata size exclusion limits and promoting virus cell-to-cell movement. Further characterization of the phloem environment was done using two phloem specific promoters (pSUC2 and pSULTR2;2) to generate epitope-tagged polysomal-RNA complexes. Immuno-purification using the epitope tag allowed us to obtain mRNAs bound to polysomes (the translatome) specifically in phloem tissue. We found the phloem translatome is uniquely altered during TMV infection with 90% and 88% of genes down regulated in the pSUC2 and pSULTR2;2 phloem translatomes, compared to 31% of genes down regulated in the whole plant p35S translatome. Transcripts down regulated in phloem include genes involved in callose deposition at plasmodesmata, host defense responses, and RNA silencing. Combined, these findings indicate TMV reprograms gene expression within the vascular phloem as a means to enhance phloem loading and systemic spread.