Messa a punto di sistemi per il controllo e la terapia delle malattie virali

A livello globale una delle problematiche più urgenti della sanità pubblica umana e veterinaria è rappresentata dal controllo delle infezioni virali. L’emergenza di nuove malattie, la veloce diffusione di patologie finora confinate ad alcune aree geografiche, lo sviluppo di resistenza dei patogeni alle terapie utilizzate e la mancanza di nuove molecole attive, sono gli aspetti che influiscono più negativamente livello socio-economico in tutto il mondo. Misure per limitare la diffusione delle infezioni virali prevedono strategie per prevenire e controllare le infezioni in soggetti a rischio . Lo scopo di questa tesi è stato quello di indagare il possibile utilizzo di prototipi virali utilizzati come modello di virus umani per valutare l’efficacia di due diversi metodi di controllo delle malattie virali: la rimozione mediante filtrazione di substrati liquidi e gli antivirali di sintesi e di origine naturale. Per quanto riguarda la rimozione di agenti virali da substrati liquidi, questa è considerata come requisito essenziale per garantire la sicurezza microbiologica non solo di acqua ad uso alimentare , ma anche dei prodotti utilizzati a scopo farmaceutico e medico. Le Autorità competenti quali WHO ed EMEA hanno redatto delle linee guida molto restrittive su qualità e sicurezza microbiologica dei prodotti biologici per garantire la rimozione di agenti virali che possono essere trasmessi con prodotti utilizzati a scopo terapeutico. Nell'industria biomedicale e farmaceutica c'è l'esigenza di una tecnologia che permetta la rimozione dei virus velocemente, in grande quantità, a costi contenuti, senza alterare le caratteristiche del prodotto finale . La collaborazione con l’azienda GVS (Zola Predosa, Italia) ha avuto come obiettivo lo studio di una tecnologia di filtrazione che permette la rimozione dei virus tramite membrane innovative e/o tessuti-non-tessuti funzionalizzati che sfruttano l’attrazione elettrostatica per ritenere ed asportare i virus contenuti in matrici liquide. Anche gli antivirali possono essere considerati validi mezzi per il controllo delle malattie infettive degli animali e nell’uomo quando la vaccinazione non è realizzabile come ad esempio in caso di scoppio improvviso di un focolaio o di un attacco bioterroristico. La scoperta degli antivirali è relativamente recente ed il loro utilizzo è attualmente limitato alla patologia umana, ma è in costante aumento l’interesse per questo gruppo di farmaci. Negli ultimi decenni si è evidenziata una crescente necessità di mettere a punto farmaci ad azione antivirale in grado di curare malattie ad alta letalità con elevato impatto socio-economico, per le quali non esiste ancora un’efficace profilassi vaccinale. Un interesse sempre maggiore viene rivolto agli animali e alle loro patologie spontanee, come modello di studio di analoghe malattie dell’uomo. L’utilizzo di farmaci ad azione antivirale in medicina veterinaria potrebbe contribuire a ridurre l’impatto economico delle malattie limitando, nel contempo, la disseminazione dei patogeni nell’ambiente e, di conseguenza, il rischio sanitario per altri animali e per l’uomo in caso di zoonosi. Le piante sono sempre state utilizzate dall’industria farmaceutica per l’isolamento dei composti attivi e circa il 40% dei farmaci moderni contengono principi d’origine naturale. Alla luce delle recenti emergenze sanitarie, i fitofarmaci sono stati considerati come una valida per migliorare la salute degli animali e la qualità dei prodotti da essi derivati. L’obiettivo del nostro studio è stato indagare l’attività antivirale in vitro di estratti naturali e di molecole di sintesi nei confronti di virus a RNA usando come prototipo il Canine Distemper Virus, modello di studio per virus a RNA a polarità negativa, filogeneticamente correlato al virus del morbillo umano. La scelta di questo virus è dipesa dal fatto che rispetto ai virus a DNA e ai retrovirus attualmente l’offerta di farmaci capaci di contrastare le infezioni da virus a RNA è molto limitata e legata a molecole datate con alti livelli di tossicità. Tra le infezioni emergenti causate da virus a RNA sono sicuramente da menzionare quelle provocate da arbovirus. Le encefaliti virali da arbovirus rappresentano una emergenza a livello globale ed attualmente non esiste una terapia specifica. Una delle molecole più promettenti in vitro per la terapia delle infezioni da arbovirus è la ribavirina (RBV) che, con il suo meccanismo d’azione pleiotropico, si presta ad essere ulteriormente studiata in vivo per la sua attività antivirale nei confronti delle infezioni da arbovirus. Uno dei fattori limitanti l’utilizzo in vivo di questa molecola è l’incapacità della molecola di oltrepassare la barriera emato-encefalica. Nel nostro studio abbiamo messo a punto una formulazione per la somministrazione endonasale di RBV e ne abbiamo indagato la diffusione dalla cavità nasale all’encefalo attraverso l’identificazione e quantificazione della molecola antivirale nei diversi comparti cerebrali . Infine è stato condotto un esperimento in vivo per valutare l’efficacia di un composto a base di semi di Neem, di cui sono già note le proprietà antimicrobiche, nei confronti dell’infezione da orf virus, una zoonosi a diffusione mondiale, che ha un elevato impatto economico in aree ad alta densità ovi-caprina e può provocare lesioni invalidanti anche nell’uomo.

Strukturelle und funktionelle Untersuchungen zur Bedeutung der endogenen retroviralen Insertion HERV-K(C4) im C4-Gen des Menschen

Der Längenpolymorphismus des C4-Gens beruht auf der An- oder Abwesenheit einer 6.4 kb langen Insertion im Intron 9. Es handelt sich dabei um einen eigenständigen bisher noch nicht beschriebenen Virus-Typ, der alle Sequenzmerkmale der Familie der humanen endogenen Retroviren (HERV) trägt und zu den HERV-K Viren gehört. Der Provirus wurde als HERV-K(C4) bezeichnet. Die Orientierung dieses retroviralen Elements ist entgegengesetzt zu der Transkriptionsrichtung des C4-Gens. Mittels RT-PCR, RNase Protection Assays und Northern-Blot Analysen konnte der Nachweis von HERV-K(C4)-Antisense mRNA-Transkripten in verschiedenen humanen Zellinien und Geweben erbracht werden. Die retroviralen Transkripte schlossen am 5'- und 3'-Ende Sequenzen des C4-Exon 9 und Exon 10 ein, so daß diese wahrscheinlich "readthrough" Transkripte darstellen, die durch einen 5' des LTR2 gelegenen Promotor initiiert oder im Zusammenhang mit der C4-Expression transkribiert und reguliert werden. Weiterhin konnten insgesamt 4 HERV-K(C4)-mRNA Spezies, einschließlich einer Vollängen-RNA detektiert werden. Die drei subgenomischen mRNAs werden vermutlich durch einfaches und mehrfaches Spleißen generiert. Die quantitative Analyse in verschiedenen humanen Zellinien ergab, daß HERV-K(C4) durchschnittlich mit einer Kopienanzahl zwischen ca.1 bis 100 Transkripten in einer Zelle vorkommt, so daß es sich um low abundance mRNAs handelt. Mittels eines Reportergen-System konnte eine Aktivität des LTR2-Promotors in der Sense-Orientierung des Retrovirus nachgewiesen werden, die nach Stimulation mit IFN- signifikant abnahm. Ein humanes Modell-Systems wurde etabliert, um die Theorie einer Antisense-Abwehr gegen exogene Retroviren in HepG2-Zellen zu überprüfen. Die Theorie basiert auf dem Nachweis von HERV-K(C4)-Antisense-Transkripten, die über eine Heteroduplexbildung mit der Sense-mRNA von verwandten, infektiösen Retroviren eine mögliche Blockierung deren Translation erwirken könnten. Es konnte eine signifikante Abnahme der retroviralen Expression von bis zu 45% nach steigenden Dosen an IFN- in HepG2-Zellen nachgewiesen werden. Der funktionell aktive 3'-LTR-Sense Promotor sowie der Nachweis von HERV-K(C4)-Antisense Transkripten sprechen für die bedeutende Rolle von HERV-K(C4) bei der Genregulation und Schutz gegen exogene Retroviren, wodurch eine Selektion stattgefunden hat.

Establishment of murine cell lines by constitutive and conditional immortalization

Mouse cell lines were immortalized by introduction of specific immortalizing genes. Embryonic and adult animals and an embryonal stem cell line were used as a source of primary cells. The immortalizing genes were either introduced by DNA transfection or by ecotropic retrovirus transduction. Fibroblasts were obtained by expression of SV40 virus large T antigen (TAg). The properties of the resulting fibroblast cell lines were reproducible, independent of the donor mouse strains employed and the cells showed no transformed properties in vitro and did not form tumors in vivo. Endothelial cell lines were generated by Polyoma virus middle T antigen expression in primary embryonal cells. These cell lines consistently expressed relevant endothelial cell surface markers. Since the expression of the immortalizing genes was expected to strongly influence the cellular characteristics fibroblastoid cells were reversibly immortalized by using a vector that allows conditional expression of the TAg. Under inducing conditions, these cells exhibited properties that were highly similar to the properties of constitutively immortalized cells. In the absence of TAg expression, cell proliferation stops. Cell growth is resumed when TAg expression is restored. Gene expression profiling indicates that TAg influences the expression levels of more than 1000 genes that are involved in diverse cellular processes. The data show that conditionally immortalized cell lines have several advantageous properties over constitutively immortalized cells.

Evidence for a retroviral insertion in TRPM1 as the cause of congenital stationary night blindness and leopard complex spotting in the horse.

Leopard complex spotting is a group of white spotting patterns in horses caused by an incompletely dominant gene (LP) where homozygotes (LP/LP) are also affected with congenital stationary night blindness. Previous studies implicated Transient Receptor Potential Cation Channel, Subfamily M, Member 1 (TRPM1) as the best candidate gene for both CSNB and LP. RNA-Seq data pinpointed a 1378 bp insertion in intron 1 of TRPM1 as the potential cause. This insertion, a long terminal repeat (LTR) of an endogenous retrovirus, was completely associated with LP, testing 511 horses (χ(2)=1022.00, p<0.0005), and CSNB, testing 43 horses (χ(2)=43, p<0.0005). The LTR was shown to disrupt TRPM1 transcription by premature poly-adenylation. Furthermore, while deleterious transposable element insertions should be quickly selected against the identification of this insertion in three ancient DNA samples suggests it has been maintained in the horse gene pool for at least 17,000 years. This study represents the first description of an LTR insertion being associated with both a pigmentation phenotype and an eye disorder.

EXPRESSION AND IMMUNOLOGICAL CHARACTERIZATION OF HERV-K TRANSMEMBRANE ENVELOPE PROTEIN IN HUMAN BREAST CANCER

The human endogenous retrovirus K (HERV-K) env gene encodes envelope protein comprising surface (SU) and transmembrane (TM) domains. Having shown the exclusive expression of SU in human breast cancer and the stimulation of SU-specific immune responses in patients with breast cancer, our research here confirmed and extended the data by investigating the expression of HERV-K TM envelope domain and the induction of specific immune responses against TM in breast cancer patients. We found HERV-K TM mRNA and protein expression only in human breast cancer cells but not in normal controls. The specific immune responses against TM domain were induced in mice determined by enzyme-linked immunosorbent assay (ELISA) and IFN-γ enzyme-linked immunosorbent spot (ELISPOT) assay. Furthermore, ELISA detected higher titers of anti-HERV-K TM Env IgG antibodies in sera of breast cancer patients. In addition, the magnitude of the anti-HERV TM B cell response was correlated with the disease stage. Peripheral blood mononuclear cells (PBMCs) before and after in vitro stimulation (IVS) with HERV-K TM from patients with breast cancer as well as healthy controls were tested for T cell responses against HERV-K TM domain by ELISPOT assay. Breast cancer patients (n=21) had stronger HERV-K TM-specific cellular responses than healthy controls (n=12) (P < 0.05). These findings suggest, for the first time, that HERV-K TM expression was enhanced in human breast cancer cells and was able to induce specific B cell and T cell immune responses in breast cancer patients. This study provides support for HERV-K TM as a promising source of antigen for anti-tumor immunotherapy, prevention, diagnosis, and prognosis.

Detection and sequence analysis of a nickel-induced mutation in a retroviral model system

We have developed a novel way to assess the mutagenicity of environmentally important metal carcinogens, such as nickel, by creating a positive selection system based upon the conditional expression of a retroviral transforming gene. The target gene is the v-mos gene in MuSVts110, a murine retrovirus possessing a growth temperature dependent defect in expression of the transforming gene due to viral RNA splicing. In normal rat kidney cells infected with MuSVts110 (6m2 cells), splicing of the MuSVts110 RNA to form the mRNA from which the transforming protein, p85$\sp{\rm gag-mos}$, is translated is growth-temperature dependent, occurring at 33 C and below but not at 39 C and above. This splicing "defect" is mediated by cis-acting viral sequences. Nickel chloride treatment of 6m2 cells followed by growth at 39 C, allowed the selection of "revertant" cells which constitutively express p85$\sp{\rm gag-mos}$ due to stable changes in the viral RNA splicing phenotype, suggesting that nickel, a carcinogen whose mutagenicity has not been well established, could induce mutations in mammalian genes. We also show by direct sequencing of PCR-amplified integrated MuSVts110 DNA from a 6m2 nickel-revertant cell line that the nickel-induced mutation affecting the splicing phenotype is a cis-acting 70-base duplication of a region of the viral DNA surrounding the 3$\sp\prime$ splice site. These findings provide the first example of the molecular basis for a nickel-induced DNA lesion and establish the mutagenicity of this potent carcinogen. ^

Identification of cadmium-induced mutations in a mammalian cell line

Epidemiological studies have shown cadmium to induce cancer in humans, while experimental studies have proven this metal to be a potent tumor inducer in animals. However, cadmium appears nonmutagenic in most prokaryotic and eukaryotic mutagenesis assays. In this study, we present the identification of mutations in normal rat kidney cells infected with the mutant MuSVts110 retrovirus (6m2 cells) as a result of treatment with cadmium chloride. The detection of these mutations was facilitated by the use of a novel mutagenesis assay established in this laboratory. The 6m2 reversion assay is a positive selection system based on the conditional expression of the MuSVts110 v-mos gene. In MuSVts110 the gag and mos genes are fused out of frame, thus the translation of the v-mos sequence requires a frameshift in the genomic RNA. In 6m2 cells this frameshift is accomplished by the temperature-dependent splicing of the primary MuSVts110 transcript. Splicing of MuSVts110, which is mediated by cis-acting sequences, occurs when 6m2 cells are grown at 33$\sp\circ$C and below, but not at 39$\sp\circ$C. Therefore, 6m2 cells appear transformed at low growth temperatures, but take on a morphologically normal appearance when grown at high temperatures. The treatment of 6m2 cells with cadmium chloride resulted in the outgrowth of a number of cells that reverted to the transformed state at high growth temperatures. Analysis of the viral proteins expressed in these cadmium-induced 6m2 revertants suggested that they contained mutations in their MuSVts110 DNA. Sequencing of the viral DNA from three revertants that constitutively expressed the P85$\sp{gag{-}mos}$ transforming protein revealed five different mutations. The Cd-B2 revertant contained three of those mutations: an A-to-G transition 48 bases downstream of the MuSVts110 3$\sp\prime$ splice site, plus a G-to-T and an A-to-T transversion 84 and 100 bases downstream of the 5$\sp\prime$ splice site, respectively. The Cd-15-5 revertant also contained a point mutation, a T-to-C transition 46 bases downstream of the 5$\sp\prime$ splice site, while Cd-10-5 contained a three base deletion of MuSVts110 11 bases upstream of the 3$\sp\prime$ splice site. A fourth revertant, Cd-10, expressed a P100$\sp{gag{-}mos}$ transforming protein, and was found to have a two base deletion. This deletion accomplished the frameshift necessary for v-mos expression, but did not alter MuSVts110 RNA splicing and the expression of p85$\sp{gag{-}mos}.$ Lastly, sequencing of the MuSVts110 DNA from three spontaneous revertants revealed the same G to T transversion in each one. This was the same mutation that was found in the Cd-B2 revertant. These findings provide the first example of mutations resulting from exposure to cadmium and suggest, by the difference in each mutation, the complexity of the mechanism utilized by cadmium to induce DNA damage. ^

Pre-therapy inflammation and coagulation activation and long-term CD4 count responses to the initiation of antiretroviral therapy

OBJECTIVES Pre-antiretroviral therapy (ART) inflammation and coagulation activation predict clinical outcomes in HIV-positive individuals. We assessed whether pre-ART inflammatory marker levels predicted the CD4 count response to ART. METHODS Analyses were based on data from the Strategic Management of Antiretroviral Therapy (SMART) trial, an international trial evaluating continuous vs. interrupted ART, and the Flexible Initial Retrovirus Suppressive Therapies (FIRST) trial, evaluating three first-line ART regimens with at least two drug classes. For this analysis, participants had to be ART-naïve or off ART at randomization and (re)starting ART and have C-reactive protein (CRP), interleukin-6 (IL-6) and D-dimer measured pre-ART. Using random effects linear models, we assessed the association between each of the biomarker levels, categorized as quartiles, and change in CD4 count from ART initiation to 24 months post-ART. Analyses adjusted for CD4 count at ART initiation (baseline), study arm, follow-up time and other known confounders. RESULTS Overall, 1084 individuals [659 from SMART (26% ART naïve) and 425 from FIRST] met the eligibility criteria, providing 8264 CD4 count measurements. Seventy-five per cent of individuals were male with the mean age of 42 years. The median (interquartile range) baseline CD4 counts were 416 (350-530) and 100 (22-300) cells/μL in SMART and FIRST, respectively. All of the biomarkers were inversely associated with baseline CD4 count in FIRST but not in SMART. In adjusted models, there was no clear relationship between changing biomarker levels and mean change in CD4 count post-ART (P for trend: CRP, P = 0.97; IL-6, P = 0.25; and D-dimer, P = 0.29). CONCLUSIONS Pre-ART inflammation and coagulation activation do not predict CD4 count response to ART and appear to influence the risk of clinical outcomes through other mechanisms than blunting long-term CD4 count gain.

HETEROGENEITY IN VIRUS EXPRESSION, TUMORIGENICITY, AND IN VITRO GROWTH PROPERTIES OF DRUG-RESISTANT CLONES OF AN RIII MOUSE MAMMARY TUMOR CELL LINE

Four 8-azaguanine (AG)-resistant and 5-bromodeoxyuridine (BUdR)-resistant clones of a mouse mammary adenocarcinoma cell line, RIII 7387, were developed and analyzed for their tumorigenic properties, in vitro characteristics, and virus expression. These characteristics were analyzed for relationships of any of the cellular parameters and the ability of these lines to produce tumors in syngeneic animals.^ The results of this study demonstrated that the parental line consists of a heterogeneous population of cells. Doubling times, saturation densities, and 2-deoxy-D-glucose uptake varied between sublines. In addition, while all sublines were found to express both B-type and C-type viral antigenic markers, levels of the major B-type and C-type viral proteins varied in the subclones. The sublines also differed markedly in their response to the presence of dexamethasone, glutathione, and insulin in the tissue culture medium.^ Variations in retrovirus expression were convirmed by electron microscopy. Budding and extracellular virus particles were seen in the majority of the cell lines. Virus particles in one of the BUdR-resistant lines, BUD9, were found however, only in inclusions and vacuoles. The AG-resistant subline AGE11 was observed to be rich in intracytoplasmic A particles. The examination of these cell lines for the presence of retroviral RNA-dependent DNA polymerase (RT) activity revealed that some B-type RT activity could be found in the culture fluid of most of the cell lines but that little C-type RT activity could be found suggesting that the C-type virus particles expressed by these RIII clones contain a defective RT.^ Tumor clones also varied in their ability to form tumors in syngeneic RIII mice. Tumor incidence ranged from 50% to 100%. The majority of the tumors regressed within 30 days post infection.^ Statistical analysis indicated that while these clones varied in their characteristics, there was no correlation between the ability of these cell lines to form tumors in syngeneic mice and any of the other characteristics examined.^ These studies have confirmed and extended the growing evidence that tumors, regardless of their natural origin, consist of heterogeneous subpopulations of cells which may vary widely in their in vitro growth behavior, their antigenic expression, and their malignant properties. ^

CHARACTERIZATION AND COMPARISON OF SUBHUMAN PRIMATE AND HUMAN TYPE C RETROVIRAL GENE PRODUCTS

The viral specific precursor polyproteins of simian sarcoma/simian associated virus (SiSV/SiAV), baboon endogenous viruses (BaEV), and three human isolate retroviruses, have been analyzed by radioimmunoprecipitation and tryptic peptide mapping. Cells infected with the BaEV isolates are characterized by identical precursor polyproteins: gPr80-85('env), Pr70-71('gag), and Pr65-67('gag). By tryptic digest mapping, m7-BaEV and 455K-BaEV were shown to be highly related. By comparison, mapping studies showed that BILN-BaEV was less highly related to m7-BaEV than was 455K-BaEV. Chase-incubated cells infected with BaEV also contained a stable, p28-related polyprotein termed P72('gag). This polyprotein appeared to arise by posttranslational modification of Pr70-71('gag). Tryptic digest mapping of BaEV and HL23V precursor polyproteins suggested that the BaEV-like component of HL23V was more closely related to m7-BaEV than to 455K-BaEV or BILN-BaEV.^ The intracellular precursor polyproteins of SiSV(SiAV) and gibbon ape leukemia virus (GaLV) were compared to the intracellular proteins of the human retrovirus isolates, HL23V, HEL12V, and A1476V. Cells infected with SiSV(SiAV) were characterized by polyproteins Pr200('gag-pol), gPr80('env), Pr80('gag), pr60('gag), and Pr40('gag). We have found that the human isolates are identical to true SiAV with regard to the size and structure of their precursor polyproteins. Both gPr80('env) and Pr60('gag) of SiAV were identical by tryptic peptide mapping to the respective proteins from the three human retroviral isolates examined. We have also shown that these viruses differ significantly from each of the GaLV isolates studied. Since SiAV differs substantially from any known GaLV isolate, we feel that it is unlikely that SiAV is a subtype of GaLV which exists today in the gibbon gene pool. The experimental evidence suggests that SiAV may be an exogenous human retrovirus that was transmitted originally into the human gene pool in the distant past by cross-species infection with GaLV(,SF) or with the GaLV(,SF) progenitor virus. It is, therefore, quite possible that SiAV expression in the pet woolly monkey arose from a recent infection of that monkey with SiAV from humans.^

STUDIES ON THE MECHANISM OF ASSEMBLY OF MURINE LEUKEMIA VIRUS

The purpose of this research was to elucidate the mechanism of assembly of retroviruses, specifically of murine leukemia virus, as studied through the treatment of virus-infected cells with interferon and through the use of temperature sensitive (ts) mutants. Our studies have shown a rapid and specific association of Rauscher murine leukemia virus (R-MuLV) precursor polyprotein Pr65('gag) with cytoskeletal elements in infected mouse fibroblasts. The Pr65('gag) associated with Nonidet P-40 (NP40)-insoluble cytoskeletal structures appeared to be subphosphorylated in comparison to NP40-soluble Pr65('gag). The association of Pr65('gag) with skeletal elements could be disrupted by extraction of the cytoskeleton with sodium deoxycholate, an ionic detergent. Both the skeleton-associated Pr65('gag) and its NP40-soluble counterpart were labeled with {('3)H}-palmitate, indicating their probable association with lipids presumably in the plasma membrane. Pr65('gag) molecules bound to skeletal elements in the infected cell appeared to be more stable to proteolytic processing than NP40-soluble Pr65('gag). Our studies with certain ts mutants of murine leukemia virus, defective in virus assembly, including Mo-MuLV ts3 and R-MuLV ts17, ts24, ts25 and ts26, have shown that virions released at 39(DEGREES)C (nonpermissive temperature) had high levels of uncleaved Pr65('gag) relative to that seen in virions released at 33(DEGREES)C (permissive temperature). Examination of cell extracts revealed that Pr54('gag) was more stable to processing at 39(DEGREES)C than at 33(DEGREES)C, whereas the 'env' and glycosylated 'gag' proteins were processed to the same extent at both temperatures. Detergent extraction of pulse-labeled cells to generate an NP40-insoluble cytoskeleton-enriched fraction showed that in ts3-, ts17- and ts24-infected cells, Pr65('gag) accumulated in the cytoskeleton-enriched fraction. In contrast, cells infected with ts25 or ts26 showed no preferential localization of Pr65('gag) in the cytoskeleton in a short pulse, but instead, Pr65('gag) accumulated in both the NP40-soluble and -insoluble fractions during a chase-incubation. The association of Pr65('gag) with cytoskeletal elements in the cell was neither increased nor decreased by blocking virus assembly and release with interferon. Based on these and other results, we have proposed a model for the active role of cytoskeleton-associated Pr65('gag) in retrovirus assembly.^

Molecular mechanisms of NF-kappaB activation in metastatic pancreatic cancer cells

Pancreatic adenocarcinoma is the fourth leading cause of adult cancer death in the United States. At the time of diagnosis, most patients with pancreatic cancer have advanced and metastatic disease, which makes most of the traditional therapeutic strategies are ineffective for pancreatic cancer. A better understanding of the molecular basis of pancreatic cancer will provide the approach to identify the new strategies for early diagnosis and treatment. NF-κB is a family of transcription factor that play important roles in immune response, cell growth, apoptosis, and tumor development. We have shown that NF-κB is constitutively activated in most human pancreatic tumor tissues and cell lines, but not in the normal tissues and HPV E6E7 gene-immortalized human pancreatic ductal epithelial cells (HPDE/E6E7). By infecting the pancreatic cancer cell line Aspc-1 with a replication defective retrovirus expressing phosphorylation-defective IκBα (IκBαM), the constitutive NF-κB activation is blocked. Subsequent injection of this Aspc-1/IκBαM cells into the pancreas of athymic nude mice showed that liver metastasis is suppressed by the blockade of NF-κB activation. Current studies showed that an autocrine mechanism accounts for the constitutive activation of NF-κB in metastatic human pancreatic cancer cell lines, but not in nonmetastatic human pancreatic cancer cell lines. Further investigation showed that interleukin-1α (IL-1α) was the primary cytokine secreted by these cells that activates NF-κB. Inhibition of IL-1α activity suppressed the constitutive activation of NF-κB and the expression of its downstream target gene, uPA, in metastatic pancreatic cancer cell lines. Even though IL-1α is one of the previously identified NF-κB downstream target genes, our results demonstrate that regulation of IL-1α expression is independent of NF-κB and primarily dependent on AP-1 activity, which is in part induced by overexpression of EGF receptors and activation of MAP kinases. In conclusion, our findings suggest a possible mechanism by which NF-κB is constitutively activated in metastatic human pancreatic cancer cells and a possible missing mechanistic links between inflammation and cancer. ^

Cross -packing among retroviruses and characterization of the feline immunodeficiency virus (FIV) packaging signal: Implications for the development of FIV-based gene transfer systems

Feline immunodeficiency virus (FIV)-based gene transfer systems are being seriously considered for human gene therapy as an alternative to vectors based on primate lentiviruses, a genetically complex group of retroviruses capable of infecting non-dividing cells. The greater phylogenetic distance between the feline and primate lentiviruses is thought to reduce chances of the generation of recombinant viruses. However, safety of FIV-based vector systems has not been tested experimentally. Since primate lentiviruses such as human and simian immunodeficiency viruses (HIV/SIV) can cross-package each other's genomes, we tested this trait with respect to FIV. Unexpectedly, both feline and primate lentiviruses were reciprocally able to both cross-package and propagate each other's RNA genomes. This was largely due to the recognition of viral packaging signals by the heterologous proteins. However, a simple retrovirus such as Mason-Pfizer monkey virus (MPMV) was unable to package FIV RNA. Interestingly, FIV could package MPMV RNA, but not propagate it for further steps of replication. These findings suggest that upon co-infection of the same host, cross-packaging may allow distinct retroviruses to generate chimeric variants with unknown pathogenic potential. ^ In order to understand the packaging determinants in FIV, we conducted a detailed mutational analysis of the region thought to contain FIV packaging signal. We show that the first 90–120 nt of the 5′ untranslated region (UTR) and the first 90 nt of gag were simultaneously required for efficient FIV RNA packaging. These results suggest that the primary FIV packaging signal is multipartite and discontinuous, composed of two core elements separated by 150 nt of the 5 ′UTR. ^ The above studies are being used towards the development of safer FIV-based self-inactivating (SIN) vectors. These vectors are being designed to eliminate the ability of FIV transfer vector RNAs to be mobilized by primate lentiviral proteins that may be present in the target cells. Preliminary test of the first generation of these vectors has revealed that they are incapable of being propagated by feline proteins. The inability of FIV transfer vectors to express packageable vector RNA after integration should greatly increase the safety of FIV vectors for human gene therapy. ^

SIRE-1, a copia/Ty1-like retroelement from soybean, encodes a retroviral envelope-like protein

The soybean genome hosts a family of several hundred, relatively homogeneous copies of a large, copia/Ty1-like retroelement designated SIRE-1. A copy of this element has been recovered from a Glycine max genomic library. DNA sequence analysis of two SIRE-1 subclones revealed that SIRE-1 contains a long, uninterrupted, ORF between the 3′ end of the pol ORF and the 3′ long terminal repeat (LTR), a region that harbors the env gene in retroviral genomes. Conceptual translation of this second ORF produces a 70-kDa protein. Computer analyses of the amino acid sequence predicted patterns of transmembrane domains, α-helices, and coiled coils strikingly similar to those found in mammalian retroviral envelope proteins. In addition, a 65-residue, proline-rich domain is characterized by a strong amino acid compositional bias virtually identical to that of the 60-amino acid, proline-rich neutralization domain of the feline leukemia virus surface protein. The assignment of SIRE-1 to the copia/Ty1 family was confirmed by comparison of the conceptual translation of its reverse transcriptase-like domain with those of other retroelements. This finding suggests the presence of a proretrovirus in a plant genome and is the strongest evidence to date for the existence of a retrovirus-like genome closely related to copia/Ty1 retrotransposons.

Gene therapy in allergic encephalomyelitis using myelin basic protein-specific T cells engineered to express latent transforming growth factor-β1

A myelin basic protein (MBP)-specific BALB/c T helper 1 (Th1) clone was transduced with cDNA for murine latent transforming growth factor-β1 (TGF-β1) by coculture with fibroblasts producing a genetically engineered retrovirus. When SJL x BALB/c F1 mice, immunized 12–15 days earlier with proteolipid protein in complete Freund’s adjuvant, were injected with 3 × 106 cells from MBP-activated untransduced cloned Th1 cells, the severity of experimental allergic encephalomyelitis (EAE) was slightly increased. In contrast, MBP-activated (but not resting) latent TGF-β1-transduced T cells significantly delayed and ameliorated EAE development. This protective effect was negated by simultaneously injected anti-TGF-β1. The transduced cells secreted 2–4 ng/ml of latent TGF-β1 into their culture medium, whereas control cells secreted barely detectable amounts. mRNA profiles for tumor necrosis factor, lymphotoxin, and interferon-γ were similar before and after transduction; interleukin-4 and -10 were absent. TGF-β1-transduced and antigen-activated BALB/c Th1 clones, specific for hemocyanin or ovalbumin, did not ameliorate EAE. Spinal cords from mice, taken 12 days after receiving TGF-β1-transduced, antigen-activated cells, contained detectable amounts of TGF-β1 cDNA. We conclude that latent TGF-β1-transduced, self-reactive T cell clones may be useful in the therapy of autoimmune diseases.