Retinoblastoma family proteins as key targets of the small DNA virus oncoproteins

RB, the most investigated tumor suppressor gene, is the founder of the RB family of growth/tumor suppressors, which comprises also p107 (RBL1) and Rb2/p130 (RBL2). The protein products of these genes, pRb, p107 and pRb2/p130, respectively, are also known as 'pocket proteins', because they share a 'pocket' domain responsible for most of the functional interactions characterizing the activity of this family of cellular factors. The interest in these genes and proteins springs essentially from their ability to regulate negatively cell cycle processes and for their ability to slow down or abrogate neoplastic growth. The pocket domain of the RB family proteins is dramatically hampered in its functions by the interference of a number of proteins produced by the small DNA viruses. In the last two decades, the 'viral hypothesis' of cancer has received a considerable renewed impulse from the notion that small DNA viruses, such as Adenovirus, Human papillomavirus (HPV) and Polyomavirus, produce factors that can physically interact with major cellular regulators and alter their function. These viral proteins (oncoproteins) act as multifaceted molecular devices that have evolved to perform very specific tasks. Owing to these features, viral oncoproteins have been widely employed as invaluable experimental tools for the identification of several key families of regulators, particularly of the cell cycle homeostasis. Adenovirus early-region 1A (E1A) is the most widely investigated small DNA tumor virus oncoprotein, but relevant interest in human oncology is raised by the E1A-related E7 protein from transforming HPV strains and by Polyomavirus oncoproteins, particularly large and small T antigens from Simian virus 40, JC virus and BK virus.     

Mechanisms by which DNA tumor virus oncoproteins target the Rb family of pocket proteins

Small DNA tumor viruses have evolved different mechanisms to abrogate the function of the retinoblastoma tumor suppressor (pRb). Studies of these viruses have been invaluable in uncovering the central role of the Rb family of pocket proteins in cell cycle control. While the molecular mechanisms by which the viral oncoproteins inactivate the Rb family are still being elucidated, it is clear that targeting of this family is required both for viral replication and for virus-induced transformation of mammalian cells. This review compares and contrasts the approaches DNA tumor viruses have evolved to antagonize Rb family members--ranging from relatively simple equilibrium dissociation of pRb from cellular pRb-binding factors to chaperone-mediated alterations in pocket protein stability and phosphorylation levels. The review will focus on the viral oncoproteins adenovirus E1A, human papillomavirus E7 and the large T antigens of several polyomaviruses. An understanding of these mechanisms may provide further insight into the regulation and functions of Rb family members as well as uncover new targets for the development of novel anti-viral agents, particularly against human papillomavirus, which is a significant cause of human cancer.     

Viral carcinogenesis: revelation of molecular mechanisms and etiology of human disease

The RNA and DNA tumor viruses have made fundamental contributions to two major areas of cancer research. Viruses were vital, first, to the discovery and analysis of cellular growth control pathways and the synthesis of current concepts of cancer biology and, second, to the recognition of the etiology of some human cancers. Transforming retroviruses carry oncogenes derived from cellular genes that are involved in mitogenic signalling and growth control. DNA tumor viruses encode oncogenes of viral origin that are essential for viral replication and cell transformation; viral oncoproteins complex with cellular proteins to stimulate cell cycle progression and led to the discovery of tumor suppressors. Viral systems support the concept that cancer development occurs by the accumulation of multiple cooperating events. Viruses are now accepted as bona fide etiologic factors of human cancer; these include hepatitis B virus, Epstein-Barr virus, human papillomaviruses, human T-cell leukemia virus type I and hepatitis C virus, plus several candidate human cancer viruses. It is estimated that 15% of all human tumors worldwide are caused by viruses. The infectious nature of viruses distinguishes them from all other cancer-causing factors; tumor viruses establish long-term persistent infections in humans, with cancer an accidental side effect of viral replication strategies. Viruses are usually not complete carcinogens, and the known human cancer viruses display different roles in transformation. Many years may pass between initial infection and tumor appearance and most infected individuals do not develop cancer, although immunocompromised individuals are at elevated risk of viral-associated cancers. Variable factors that influence viral carcinogenesis are reviewed, including possible synergy between viruses and environmental cofactors. The difficulties in establishing an etiologic role for a virus in human cancer are discussed, as well as the different approaches that proved viral links to cancer. Future directions for tumor virus studies are considered.     

Control of Polyomavirus T-antigen and DNA synthesis in mouse embryo fibroblast cells by vitamin A

Vitamin A (retinoic acid, 10(-6) M) treatment of confluent mouse embryo cells for only 7 h resulted in optimal inhibition of Polyomavirus replication. Depending upon the input multiplicity of virus, one could wait until between 12 and 18 h postinfection to add vitamin A and still observe maximal inhibition of virus yields. Taken together, and assuming the same kinetics before and after virus infection, these results suggested that the inhibitory action of vitamin A occurred between 19 and 25 h into the Polyomavirus replication cycle. In this model system, such a time corresponded to the onset of T-antigen expression and virus-induced cellular DNA synthesis. Analysis of both viral and virus-induced cellular DNA synthesis by the method of Hirt (J. Mol. Biol., 26: 365-369, 1967) and by cesium chloride gradients suggested that vitamin A preferentially inhibited viral, more than virus-induced cellular, DNA synthesis in confluent cell monolayers. Vitamin A also concomitantly inhibited Polyomavirus T-antigen expression in such confluent cultures. In contrast, viral DNA synthesis and infectious virus yields were not significantly inhibited by vitamin A in subconfluent cell cultures. The antagonistic effect of vitamin A on Polyomavirus replication in confluent monolayers could be blocked with cycloheximide, a reversible protein synthesis inhibitor. This suggested that vitamin A inhibition of Polyomavirus replication was indirect and mediated by a newly synthesized protein. Taken together, these results suggest that vitamin A induced a protein in confluent, but not subconfluent, cells, which blocked the expression of Polyomavirus T-antigen. Decreased amounts of T-antigen most likely reduced Polyomavirus and cellular DNA synthesis and virus yield.     

Biological activities and molecular targets of the human papillomavirus E7 oncoprotein.

The human papillomavirus (HPV) E7 protein is one of only two viral proteins that remain expressed in HPV-associated human cancers. HPV E7 proteins share structural and functional similarities with oncoproteins encoded by other small DNA tumor viruses such as adenovirus E1A and SV40 large tumor antigen. The HPV E7 protein plays an important role in the viral life cycle by subverting the tight link between cellular differentiation and proliferation in normal epithelium, thus allowing the virus to replicate in differentiating epithelial cells that would have normally withdrawn from the cell division cycle. The transforming activities of E7 largely reflect this important function.     

PP2A fulfills its promises as tumor suppressor: which subunits are important?

Reversible phosphorylation of proteins, catalyzed by kinases and phosphatases, is a key regulatory mechanism in the control of multiple cellular signal transduction pathways. Uncontrolled regulation by the altered phosphorylation state of the components of these pathways often leads to increased cell proliferation and cell transformation. Many viruses encode oncogenic proteins, required for their efficient viral replication, which deregulate the activity of host cell proteins. This might program cells to a malignant state, underlying the molecular mechanism of tumor formation and cancer development. Recent studies reveal a role for a specific form of protein phosphatase 2A (PP2A) in viral-induced cell transformation by interaction with the small t antigen (ST) of the DNA tumor simian virus 40 (SV40).     

Polyomaviruses and cancer--interplay between viral proteins and signal transduction pathways

Polyomaviruses are highly suspected to be involved in the development of cancer. A strong correlation has been established between the activity of an early viral genome and the development of a transformed phenotype. Polyomavirus transforming antigens (T-antigens) are the major suspects in the process of deregulating cellular equilibrium. Multiple interactions between T-antigens and cellular regulatory proteins have been detected at different regulatory levels including signal transduction, gene expression, cell cycle progression, and possible DNA repair. In this context, we are reviewing the most recent experimental evidence which, in combination with more than thirty years of studies of polyomaviruses, could help us understand whether and how viral infection contributes to the development of malignant transformation.     

Phosphorylation of the retinoblastoma protein is modulated in mouse kidney cells infected with polyomavirus.

Lytic infection with polyomavirus, an oncogenic DNA-containing virus, leads in G0-arrested primary baby mouse kidney (BMK) cell cultures to a mitotic host reaction. In the present work, we examined the expression of the retinoblastoma gene (RB) and of its product (Rb) in virus-infected BMK with the aim of correlating its modulation with the sequential activation of cellular processes leading to the induction of S phase by virus. In contrast to cell cycle-regulated genes whose expression is induced by viral infection, expression of RB is not altered during the transition from G0/G1 to S phase. In BMK cell cultures irreversibly arrested in the G0 phase of the cell cycle, an unphosphorylated species is the only detectable form of the RB protein (Rb). Time course analysis showed that in polyoma-infected cells induced to re-enter the S phase of the cell cycle the appearance of the phosphorylated forms of Rb coincided in time with the accumulation of large T antigen and preceded DNA synthesis. During the late phase of infection, the majority of Rb was present as phosphorylated forms. Ongoing DNA synthesis was not required for the cells to phosphorylate Rb, indicating that this post-translational modification takes place during the activation of the cellular DNA-synthesizing apparatus. Using hamster anti-polyoma tumor serum, it was observed that the underphosphorylated form of Rb co-precipitated with polyoma large T antigen extracted from infected cells late during infection. Our data add more evidence to the proposal that interactions between viral early proteins encoded by DNA tumor viruses and the product of RB may play a pivotal role in the mitogenic effect induced by viral infection.     

Kaposi's Sarcoma-Associated Herpesvirus-Encoded Oncogenes and Oncogenesis

Molecular biologic studies of Kaposi's sarcoma-associated herpesvirus(KSHV) have identified a number of potential viral oncogenesthat may contribute to KSHV-related neoplasia including a D-typecyclin, an IL-6-like cytokine, and a novel member of the interferonregulatory factor family. KSHV is functionally related to otherDNA tumor viruses by encoding specific proteins to inhibit pRb,pro-apoptotic, and interferon-signaling tumor suppressor pathways.The virus appears to employ molecular piracy of cellular regulatorygenes as a mechanism to avoid cellular antiviral responses.The transparency of the KSHV genome allows ready identificationof the cellular regulatory pathways which may be involved intransformation by KSHV. This provides strong support to thenotion that some tumor suppressor pathways serve the dual functionof being antiviral pathways to induce cell cycle arrest, apoptosis,and enhanced cell-mediated immunity in response to virus infection.Neoplasia may result from specific viral strategies to overcomethese host defense pathways.     

Identification of a novel activity of human papillomavirus type 16 E6 protein in deregulating the G1/S transition

In this study we show that E6 of human papillomavirus has the ability to deregulate the cell cycle G1/S transition. In rodent immortalized fibroblasts (NIH3T3) serum deprivation or over-expression of the cyclin-dependent kinase inhibitors, p16(INK4a) or p27(KIP1), leads to G1 cell cycle arrest. HPV16 E6 overcomes the antiproliferative signals, gaining the ability to drive serum-deprived and p16(INK4a) or p27(KIP1) over-expressing cells into S phase. E6 protein from the benign HPV type 1 displays a similar activity to HPV16 E6 to deregulate the G1/S transition. Thus, this activity appears to be conserved between E6 proteins from non-oncogenic and oncogenic HPV types. Furthermore, we show that HPV16 E6 is not able to circumvent a G1 arrest imposed by pRb mutant in which all CDK phosphorylation sites have been mutated. These data indicate that the viral protein acts upstream of pRb and its mechanism in promoting cell cycle progression is dependent on pRb phosphorylation. In summary, this study describes a novel biological function of HPV E6 and shows that the S phase entry, required for viral DNA replication, is not exclusively controlled by E7, but that E6 also is involved in this event.     

Cell transformation by the middle T-antigen of polyoma virus.

The polyoma virus region expressed early in the lytic cycle encodes three proteins, or T-antigens, that together cause the infected cell to enter the cell cycle and so provide a suitable cellular environment for replication of the viral genome. Under some circumstances infection does not kill the cell, but the T-antigens are still produced, resulting in the cell becoming transformed and tumorigenic. Most of this transforming action is exerted by the middle T-antigen, which has the ability to convert established cell lines to an oncogenic state. Middle T is a membrane bound polypeptide that interacts with a number of the proteins used by tyrosine kinase associated receptors to stimulate mitogenesis, so MT can be considered as a permanently active analogue of a receptor. Through a defined series of interactions, MT assembles a large multi-protein complex at the cell membrane, consisting of MT, the core dimer of protein phosphatase 2A, an src-family tyrosine kinase, and via phosphotyrosines, ShcA, phosphatidylinositol (3') kinase, and phospholipase Cgamma-1. Tyrosine phosphorylation stimulates PI3K and PLCgamma-1 enzymatic activity, and on ShcA creates binding sites for Grb2 with its associated Sos1 and Gab1. This activates p21(ras), and hence, the MAP kinase cascade. Consequently, MT can be used as a model for studying cell transformation and growth factor receptor signalling pathways.     

Emerging roles of DNA tumor viruses in cell proliferation: new insights into genomic instability

The small DNA virus proteins E1A and E1B from human Adenovirus, E6 and E7 from human papillomavirus, and large T and small T antigens from SV40, are multifaceted molecular tools that can carry out an impressive number of tasks in the host cell. These viral factors, collectively termed 'oncoproteins' for their ability to induce cancer, can be viewed as paradigmatic oncogenic factors which can disrupt checkpoint controls at multiple levels--they interfere with both 'gatekeeper' cellular functions, including major control pathways of cell cycle and apoptosis, and with 'caretaker' functions, thereby inducing mitotic abnormalities and increasing genomic instability. Both E1A and E7 have been recently found to interact physically with the Ran GTPase. This interaction is key in uncoupling the centrosome cycle from the cell cycle, highlighting a direct link between viral infection and the induction of genomic instability. Further expanding our current knowledge in this field will be crucial to elucidate viral strategies leading to cellular transformation and cancer progression, as well as design novel preventive or therapeutic approaches to human cancer.     

Cell alterations induced by the large T-antigens of SV40 and polyoma virus

The large T-antigens of SV40 and polyoma virus are nuclear, multifunctional proteins that are essential for replication of the respective viruses. They can also 'transform' cells in culture to varying extents; both can immortalise primary cells, and SV40 large T can additionally induce full transformation. Recently, p105Rb, the protein product of the anti-oncogene RB-1, has been shown to interact with both large T-antigens. SV40 large T-antigen also binds to a p105Rb related protein, p107. SV40 large T differs from that of polyoma virus in its ability to associate with another anti-oncogene product, p53. The significance of these properties to the transforming potential of both viruses is considered.     

Interaction of SV40 large T antigen with components of the nucleo/cytoskeleton.

The SV40 large T antigen is a viral oncoprotein which performs multiple interactions with cellular factors to achieve a proliferative state required for viral replication as well as for transformation. The major targets in this scenario are members of the Rb family, pRb, p107, and p130, and tumor suppressor protein p53. These interactions of large T with Rb proteins and p53 are required but not sufficient for transformation. To search for unknown interaction partners of large T that might participate in its transforming activity we employed the yeast two-hybrid system. Screening a cDNA library from a large T-induced brain tumor cell line revealed a total of 86 positive clones representing 37 individual clones. Of these, four clones were selected for further analyses. Interestingly, the cDNA inserts of these clones coded for different components of the cytoskeleton, lamin C, laminin gamma1, thymosin beta4, and gelsolin. Complex formation between large T and these proteins was confirmed in vitro. Interaction of large T with these components might influence activities such as intracellular transport, signal transduction, adhesion, or migration.