E4 engages uPAR and enolase-1 and activates urokinase to exert antifibrotic effects.

Fibroproliferative disorders such as systemic sclerosis (SSc) have no effective therapies and result in significant morbidity and mortality. We recently demonstrated that the C-terminal domain of endostatin, known as E4, prevented and reversed both dermal and pulmonary fibrosis. Our goal was to identify the mechanism by which E4 abrogates fibrosis and its cell surface binding partner(s). Our findings show that E4 activated the urokinase pathway and increased the urokinase plasminogen activator (uPA) to type 1 plasminogen activator inhibitor (PAI-1) ratio. In addition, E4 substantially increased MMP-1 and MMP-3 expression and activity. In vivo, E4 reversed bleomycin induction of PAI-1 and increased uPA activity. In patients with SSc, the uPA/PAI-1 ratio was decreased in both lung tissues and pulmonary fibroblasts compared with normal donors. Proteins bound to biotinylated-E4 were identified as enolase-1 (ENO) and uPA receptor (uPAR). The antifibrotic effects of E4 required uPAR. Further, ENO mediated the fibrotic effects of TGF-ß1 and exerted TGF-ß1-independent fibrotic effects. Our findings suggest that the antifibrotic effect of E4 is mediated, in part, by regulation of the urokinase pathway and induction of MMP-1 and MMP-3 levels and activity in a uPAR-dependent manner, thus promoting extracellular matrix degradation. Further, our findings identify a moonlighting function for the glycolytic enzyme ENO in fibrosis.

Effects of the deletion of early region 4 (E4) open reading frame 1 (orf1), orf1-2, orf1-3 and orf1-4 on virus-host cell interaction, transgene expression, and immunogenicity of replicating adenovirus HIV vaccine vectors.

The global health burden engendered by human immunodeficiency virus (HIV)-induced acquired immunodeficiency syndrome (AIDS) is a sobering reminder of the pressing need for a preventative vaccine. In non-human primate models replicating adenovirus (Ad)-HIV/SIV recombinant vaccine vectors have been shown to stimulate potent immune responses culminating in protection against challenge exposures. Nonetheless, an increase in the transgene carrying capacity of these Ad vectors, currently limited to approximately 3000 base pairs, would greatly enhance their utility. Using a replicating, E3-deleted Ad type 5 host range mutant (Ad5 hr) encoding full-length single-chain HIVBaLgp120 linked to the D1 and D2 domains of rhesus macaque CD4 (rhFLSC) we systematically deleted the genes encoding early region 4 open reading frame 1 (E4orf1) through E4orf4. All the Ad-rhFLSC vectors produced similar levels of viral progeny. Cell cycle analysis of infected human and monkey cells revealed no differences in virus-host interaction. The parental and E4-deleted viruses expressed comparable levels of the transgene with kinetics similar to Ad late proteins. Similar levels of cellular immune responses and transgene-specific antibodies were elicited in vaccinated mice. However, differences in recognition of Ad proteins and induced antibody subtypes were observed, suggesting that the E4 gene products might modulate antibody responses by as yet unknown mechanisms. In short, we have improved the transgene carrying capacity by one thousand base pairs while preserving the replicability, levels of transgene expression, and immunogenicity critical to these vaccine vectors. This additional space allows for flexibility in vaccine design that could not be obtained with the current vector and as such should facilitate the goal of improving vaccine efficacy. To the best of our knowledge, this is the first report describing the effects of these E4 deletions on transgene expression and immunogenicity in a replicating Ad vector.

[Development of a novel adenovirus vector exhibiting microRNA-mediated suppression of the leaky expression of adenovirus genes].

Replication-incompetent adenovirus (Ad) vectors are widely used in gene therapy studies because they are beneficial as a gene delivery vehicle enabling high-titer production and highly efficient gene transfer into a wide spectrum of dividing and non-dividing cells in vitro and in vivo. Theoretically, Ad genes should not be expressed following transduction with a replication-incompetent Ad vector. However, leaky expression of viral genes is known to occur following transduction with a conventional Ad vector, which leads to a cellular immunity against Ad proteins as well as Ad protein-induced toxicity. Such Ad protein-induced cellular immunity and toxicity frequently cause both an elimination of Ad vector-transduced cells and tissue damage, leading to short-lived transgene expression. To date, no detailed analysis of the leaky expression profile of Ad genes has been performed. First, we systematically examined the expression profiles of Ad genes in cells using real-time RT-PCR following transduction with a conventional Ad vector. The results revealed that significant expression was found for E2A, E4, and pIX genes. Next, in order to suppress the leaky expression of Ad genes, complementary sequences for microRNA (miRNA) were inserted into the 3'-untranslated region of the E2A, E4, or pIX genes. miRNAs are an approximately 22-nt length non-coding RNA, and bind to imperfectly complementary sequences in the 3'-untranslated region of target mRNA, leading to suppression of gene expression via post-transcriptional regulation. Incorporation of the miRNA-targeted sequences significantly suppressed the leaky expression of Ad genes in an miRNA-dependent manner.

[Identification of a non-essential region for replication of fowl adenovirus QU strain].

The avirulent QU strain of fowl adenovirus, a member of duck adenovirus type 1, could be a potential vector in recombinant vaccine development. To identify a non-essential region for replication of QU virus, a 3.4 kb fragment near the E4 region of QU virus genome was amplified by PCR to construct a plasmid pADGFP, in which ORF1, ORF8 and ORF9 was replaced with a system expressing enhanced green fluorescence protein. Further, a recombinant virus rQUGFP was constructed by homologous recombination after pADGFP and QU virus were co-transfected into chick embryo fibroblast. The one step growth curve of the rQUGFP was found to be identical with that of parent QU virus and the TCID50 titers of different generation recombinants maintained stable. These findings suggest that the region including ORF1, ORF8 and ORF9 of QU virus genome is dispensable for virus replication, and the foreign gene inserted into virus genome can be efficiently and stably expressed. The work lays the foundation for further studies of developing this virus as a vector of recombinant vaccine.

Rescue of chimeric adenoviral vectors to expand the serotype repertoire.

The successful use of any adenoviral vectors is predicated upon the use of a serotype that is not neutralized by circulating antibodies. However, efforts to develop a diverse repertoire of serologically distinct adenovirus vectors may be hindered by the necessity to generate cell lines to allow for the successful propagation of vectors deleted of essential genes. A strategy to construct chimeric adenoviruses whereby the rescue and propagation of an E1-deleted HAdV-B-derived adenoviral vector can be achieved using existing cell lines such as HEK 293 is reported. It is further shown that this strategy may be more widely applicable.

Analysis of early region 4 of porcine adenovirus type 3.

The early region 4 (E4) of porcine adenovirus (PAdV)-3, located at the right-hand end of the genome is transcribed in a leftward direction and has the potential to encode seven (p1-p7) open reading frames (ORFs). To determine the role of each protein in viral replication, we constructed full-length PAdV-3 genomic clones containing deletions of individual E4 ORF or combined deletions of the neighboring ORFs. Transfection of swine testicular (ST) cells with individual E4 mutant plasmid DNAs generated PAdV-3 E4 mutant viruses except with plasmids containing a deletion of ORF p3, ORF p2+ p3 or ORF p3+ p4. Each of the mutants was further analyzed for growth kinetics, and early/late protein synthesis. Mutant viruses carrying deletions in ORF p1, ORF p2 or ORF p4 showed growth characteristics similar to that of wild-type PAdV-3. Early/late protein synthesis was also indistinguishable from that of wild-type PAdV-3. However, mutant viruses carrying deletions in ORF p5, ORF p6 or ORF p7 showed a modest effect in their ability to grow in porcine cells and express early proteins. These results suggest that the E4 ORF p3 (showing low homology with non-essential human adenovirus (HAdV)-9-E4 ORF1 encoded proteins) is essential for the replication of PAdV-3 in vitro. In contrast, the E4 ORF p7 (showing homology to essential HAdV-2 34 kDa protein) is not essential for replication of PAdV-3 in vitro. Moreover, successful deletion of 1.957 kb fragment in E4 region increased the available capacity of replication-competent PAdV-3 (E3 + E4 deleted) to approximately 4.3 kb and that of replication-defective PAdV-3 (E1 + E3 + E4 deleted) to approximately 7 kb. This is extremely useful for the construction of PAdV-3 vectors that express multiple genes and/or regulatory elements for gene therapy and vaccination.

Mutational analysis of early region 4 of bovine adenovirus type 3.

The primary objective of characterizing bovine adenovirus type 3 (BAV3) in greater detail is to develop it as a vector for gene therapy and vaccination of humans and animals. A series of BAV3 early region 4 (E4) deletion-mutant viruses, containing deletions in individual E4 open reading frames (Orf) or combinations of Orfs, were generated by transfecting primary fetal bovine retinal cells with E4-modified genomic DNA. Each of these mutants was further analyzed for growth kinetics, viral DNA accumulation, and early-late protein synthesis. Mutant viruses carrying deletions in Orf1, Orf2, Orf3, or Orf4 showed growth characteristics similar to those of the E3-deleted BAV3 (BAV302). DNA accumulation and early/late protein synthesis were also indistinguishable from those of BAV302. However, mutant viruses carrying a deletion in Orf5, Orfs 1-3 (BAV429), or Orfs 3-5 (BAV430) were modestly compromised in their ability to grow in bovine cells and express early/late proteins. E4 mutants containing larger deletions, Orfs 1-3 (BAV429) and Orfs 3-5 (BAV430), were further tested in a cotton rat model. Both mutants replicated as efficiently as BAV3 or BAV302 in the lungs of cotton rats. BAV3-specific IgA and IgG responses were detected in serum and at the mucosal surfaces in cotton rats inoculated with mutant viruses. In vitro and in vivo characterization of these E4 mutants suggests that none of the individual E4 Orfs are essential for viral replication. Moreover, successful deletion of a 1.5-kb fragment in the BAV3 E4 region increased the available insertion capacity of replication-competent BAV3 vector (E3-E4 deleted) to approximately 4.5 kb and that of replication-defective BAV3 vector (E1a-E3-E4 deleted) to approximately 5.0 kb. This is extremely useful for the construction of BAV3 vectors that express multiple genes and/or regulatory elements for gene therapy and vaccination.

Novel role for E4 region genes in protection of adenovirus vectors from lysis by cytotoxic T lymphocytes.

Target cells infected with adenovirus (Ad) vectors containing intact E3 and E4 regions were found to be relatively resistant to lysis by Ad-specific cytotoxic T lymphocytes. Elements from both the E3 and the E4 regions were required for this effect, leading to the identification of a previously undescribed role for E4 gene products in resistance to cytolysis.

In vitro and in vivo biology of recombinant adenovirus vectors with E1, E1/E2A, or E1/E4 deleted.

Isogenic, E3-deleted adenovirus vectors defective in E1, E1 and E2A, or E1 and E4 were generated in complementation cell lines expressing E1, E1 and E2A, or E1 and E4 and characterized in vitro and in vivo. In the absence of complementation, deletion of both E1 and E2A completely abolished expression of early and late viral genes, while deletion of E1 and E4 impaired expression of viral genes, although at a lower level than the E1/E2A deletion. The in vivo persistence of these three types of vectors was monitored in selected strains of mice with viral genomes devoid of transgenes to exclude any interference by immunogenic transgene-encoded products. Our studies showed no significant differences among the vectors in the short-term maintenance and long-term (4-month) persistence of viral DNA in liver and lung cells of immunocompetent and immunodeficient mice. Furthermore, all vectors induced similar antibody responses and comparable levels of adenovirus-specific cytotoxic T lymphocytes. These results suggest that in the absence of transgenes, the progressive deletion of the adenovirus genome does not extend the in vivo persistence of the transduced cells and does not reduce the antivirus immune response. In addition, our data confirm that, in the absence of transgene expression, mouse cellular immunity to viral antigens plays a minor role in the progressive elimination of the virus genome.

Blockage by adenovirus E4orf6 of transcriptional activation by the p53 tumor suppressor.

The adenovirus E4orf6 protein is shown here to interact with the cellular tumor suppressor protein p53 and to block p53-mediated transcriptional activation. The adenovirus protein inhibited the ability of p53 to bind to human TAFII31, a component of transcription factor IID (TFIID). Earlier work demonstrated that the interaction of p53 with TAFII31 involves a sequence near the NH2-terminus of p53, whereas the E4orf6-p53 interaction occurs within amino acids 318 to 360 of p53. Thus, the E4orf6 protein interacts at a site on p53 distinct from the domain that binds to TAFII31 but nevertheless inhibits the p53-TAFII31 interaction.

Antibodies to the E4, E6, and E7 proteins of human papillomavirus (HPV) type 16 in patients with HPV-associated diseases and in the normal population.

In a cross-sectional study, titers of antibodies to the E4 and E7 proteins of human papillomavirus (HPV) type 16 were measured by peptide-based enzyme-linked immunosorbent assay in 1707 sera. Sera were obtained from healthy individuals (ages 1 to 95 years), from patients with HPV-associated infection (cervical intraepithelial neoplasia and cervical cancer), and from patients who were at high risk for HPV infection (attending a sexually transmitted disease clinic or referred to a colposcopist because of an abnormal Papanicolaou smear). The prevalence of anti-E7 antibodies increased with age, although the overall prevalence in the adult population was low (10.36%) compared to the frequent detection of HPV 16 DNA in the population. This suggests that only a fraction of patients infected with HPV 16 develop an anti-E7 response. The age distribution of anti-E4 antibodies showed a different pattern, i.e., the prevalence was low in the adult population (1.14%) but exceeded 20% in children and teenagers. As the specificity of the anti-E4 reaction was supported by a highly significant association with anti-E6 positivity in children's sera (p = 0.002), it was assumed that infection with HPV 16 can occur frequently early in life. As compared to healthy controls, patients at high risk for HPV infection had a significantly higher frequency (p < 0.001) of antibodies to the HPV 16 E4 protein (but not to the E6 or the E7 protein) in their sera. Therefore, we conclude that in adults E4-specific antibodies may be a marker for virus replication.