Complex effect of adenovirus early region proteins on innate immune system.

Adenoviruses (Ads) cause acute and persistent infections. The genome of Ads has five early transcription units that are the first viral genes expressed during an active infection. The Early Region 1A (E1A) gene of the adenovirus genome is crucial for adenovirus transformation of the host cell. Ads E1A block some aspects of the innate immune system to enable viruses to invade the host cell. E1A suppresses nitric oxide (NO) production through transcriptional control of the inducible NO synthase (iNOS) gene. This inhibition of NO production may enable the virus to persist in human tissue because NO is an antiviral effector of the innate immune system. E1A also blocks secretory leukoprotease inhibitor (SLPI) and elafin/skin-derived antileukoproteinase (SKALP) secretion by alveolar epithelial cells. Recent scientific evidence suggests that SLPI and elafin/SKALP have broad-spectrum antibiotic activities that include bactericidal and antifungal properties. The inhibition of inflammation by Ad early region proteins is complex, as certain early region proteins can promote as well as inhibit inflammation depending on the genetic context of the virus. E1A DNA and protein are frequently detected in the lungs of chronic obstructive pulmonary disease (COPD) patients and it is associated with an increased inflammatory response. E1A enhances intercellular adhesion molecule-1 and interleukin-8 mRNA expression with lipopolysaccharide stimulation. Understanding the roles of the Ad gene products in the induction and inhibition of innate inflammatory functions will help us to clarify the pathogenesis of the chronic respiratory illness including COPD.

Adenovirus E1A orchestrates the urokinase-plasminogen activator system and upregulates PAI-2 expression, supporting a tumor suppressor effect.

Invasiveness and metastatic potential are the two most important properties defining malignancy. The adeno-virus E1A (Ad-E1A) gene has a dual effect as a proliferative gene and as a tumor-suppressor gene, decreasing tumor growth and the metastatic potential of malignant cells. In order to study genes related with the antimetastatic effect of Ad-E1A in human cells, we performed a microarray analysis using OncoChiptrade mark. In three independent experiments, NIH3T3, IMR90 and MDA MB 435 cells were infected with pLPC retroviruses carrying the adenovirus 12S E1A gene or the GFP gene. We analyzed cDNA expression by using the CNIO OncoChipTM, a cDNA microarray containing a total of 6386 genes represented by 7237 clones. uPA, uPAr, tPA, PAI-1 and PAI-2 were also studied at RNA and protein levels. Microarrays of cDNA expression, RT-PCR and Western blot performed in IMR90 E1A-expressing cells showed downregulation of uPA, uPAr, tPA, PAI-1 and upregulation of PAI-2. These results were confirmed in NIH3T3 and MDA MB 435 breast carcinoma cells, with PAI-2 upregulation by RT-PCR and Western blot. In addition, zymographic analysis demonstrated that E1A expression greatly reduced the gelatinase activity of the pro-MMP2 and -MMP9 proteins. We propose that adenovirus E1A may orchestrate the expression of most members of the urokinase-plasminogen activation system, downregulating potentially invasive genes and upregulating PAI-2, which is associated with a better prognosis in human tumors.

Induction and inhibition of innate inflammatory responses by adenovirus early region proteins.

First-generation adenovirus (Ad) gene therapy vectors deleted for the E1A, E1B, and E3 regions and carrying foreign genes under the control of strong foreign promoters induce high-level innate inflammatory responses within the first 24 hrs after transduction. Both uptake of the capsid and expression of gene products encoded by the vector contribute to the innate inflammatory response. Natural infections by Ad are frequently asymptomatic, suggesting that Ad has potent methods of inhibiting inflammation. The inability of Ad vectors to counter inflammatory responses suggests that the products of the Ad genes deleted in vector construction play critical roles in inhibiting these responses. Genetic analysis of the roles of Ad early region gene functions in vivo demonstrated that a virus made replication-incompetent by deletion of the preterminal protein gene and deleted for the transcriptional activation function of E1A effectively inhibits the innate inflammatory processes induced by Ad vectors. The mechanism(s) by which the Ad early region proteins inhibit inflammation is complex, as certain early region proteins can promote as well as inhibit inflammation, depending on the genetic context of the virus. Understanding of the roles of the Ad gene products in the induction and inhibition of innate inflammatory functions offers potential for the development of non-inflammatory vectors as well as for understanding of the mechanisms by which inflammation is regulated.

Molecular regulation and biological function of adenovirus early genes: the E4 ORFs.

Over the past few years there have been a number of interesting advances in our understanding of the functions encoded by the adenovirus early transcription unit 4 (Ad E4). A large body of recent data demonstrates that E4 proteins encompass an unexpectedly diverse collection of functions required for efficient viral replication. E4 gene products operate through a complex network of protein interactions with key viral and cellular regulatory components involved in transcription, apoptosis, cell cycle control and DNA repair, as well as host cell factors that regulate cell signaling, posttranslational modifications and the integrity of nuclear multiprotein complexes known as nuclear bodies (NBs) or PML oncogenic domains (PODs). As understood at present, some of the lytic functions overlap with roles in oncogenic transformation of primary mammalian cells. These observations, together with findings that E4 proteins substantially affect cell toxicity and the immune response of the host have profound implications for the development of Ad vectors for gene therapy. In this article we will summarize recent findings regarding the diverse functions of E4 gene products in the context of earlier work. We will emphasize the interaction of E4 proteins with cellular and viral interaction partners, the role of these interactions for lytic virus growth and how these interactions may contribute to viral oncogenesis. Finally, we will discuss their role in Ad vector and adeno-associated virus infections.

Induction of cell death by adenoviruses.

Adenoviruses have proved to be excellent tools for gaining insight into the regulation, and deregulation, of the mammalian cell cycle. With the widespread clinical use of gene therapy fast approaching, there comes a need for a better understanding of how the cell death process is regulated. A greater understanding will allow the development of therapeutic approaches that both maximise transgene expression while minimising cytotoxicity to the target cell. Consequently, much adenovirus research has centered on understanding the mechanisms governing adenovirus induced cell death or apoptosis. This review discusses recent advances in the field of adenovirus cell death regulation and evaluates the roles of implicated gene products and their respective data. The data suggest the existence of multiple virus gene products involved in cell death regulation and point towards several distinct, yet related, cell death pathways. A discussion of the shortcomings of current adenoviral research, along with a proposed model based upon the data is also given.

The adenovirus type 5 E1b 55K and E4 Orf3 proteins associate in infected cells and affect ND10 components.

Three early proteins expressed by adenovirus type 5, E1b 55K, E4 Orf3 and E4 Orf6, are involved in regulating late viral gene expression. It has previously been shown that 55K associates with Orf6. Here we show that 55K also associates with Orf3 and that this interaction is necessary for 55K to localize to the nuclear matrix fraction of the cell. From our data, we infer that the Orf3 and Orf6 interactions with 55K may be mutually exclusive. The Orf3 protein is also known to associate with and cause the reorganization of cell nucleus structures known as ND10 or PODs. Consistent with the observed increase in the biochemical interaction between 55K and Orf3 in the absence of Orf6, the 55K association with Orf3 in ND10 was also found to increase in the absence of Orf6. The most studied cellular component of ND10 is PML, a complex protein present in a range of isoforms, some of which are modified by conjugation to the small ubiquitin-like protein PIC-1. The pattern of PML isoforms was altered in adenovirus-infected cells, in that a number of additional isoform bands appeared in an Orf3-dependent manner, one of which became predominant later in infection. As for ND10 reorganization, neither Orf6 nor 55K was required for this effect. Therefore it is likely that these changes in PML are related to the changes in ND10 structure that occur during infection.