MicroRNAs (miRNAs) are small (approximately 22-nucleotide) RNAs that in lower organisms serve important regulatory roles in development and gene expression, typically by forming imperfect duplexes with target messenger RNAs. miRNAs have also been described in mammalian cells and in infections with Epstein-Barr virus (EBV), but the function of most of them is unknown. Although one EBV miRNA probably altered the processing of a viral mRNA, the regulatory significance of this event is uncertain, because other transcripts exist that can supply the targeted function. Here we report the identification of miRNAs encoded by simian virus 40 (SV40) and define their functional significance for viral infection. SVmiRNAs accumulate at late times in infection, are perfectly complementary to early viral mRNAs, and target those mRNAs for cleavage. This reduces the expression of viral T antigens but does not reduce the yield of infectious virus relative to that generated by a mutant lacking SVmiRNAs. However, wild-type SV40-infected cells are less sensitive than the mutant to lysis by cytotoxic T cells, and trigger less cytokine production by such cells. Thus, viral evolution has taken advantage of the miRNA pathway to generate effectors that enhance the probability of successful infection.

DNA tumor viruses such as simian virus 40 (SV40) express dominant acting oncoproteins that exert their effects by associating with key cellular targets and altering the signaling pathways they govern. Thus, tumor viruses have proved to be invaluable aids in identifying proteins that participate in tumorigenesis, and in understanding the molecular basis for the transformed phenotype. The roles played by the SV40-encoded 708 aminoacid large T antigen (T antigen), and 174 amino acid small T antigen (t antigen), in transformation have been examined extensively. These studies have firmly established that large T antigen's inhibition of the p53 and Rbfamily of tumor suppressors and small T antigen's action on the pp2A phosphatase, are important for SV40-induced transformation. It is not yet clear if the Rb, p53 and pp2A proteins are the only targets through which SV40 transforms cells, or whether additional targets await discovery. Finally, expression of SV40 oncoproteins in transgenic mice results in effects ranging from hyperplasia to invasive carcinoma accompanied by metastasis, depending on the tissue in which they are expressed. Thus, the consequences of SV40 action on these targets depend on the cell type being studied. The identification of additional cellular targets important for transformation, and understanding the molecular basis for the cell type-specific action of the viral T antigens are two important areas through which SV40 will continue to contribute to our understanding of cancer.

Simian virus 40 (SV40) encodes two proteins, large T antigen and small t antigen that contribute to virus-induced tumorigenesis. Both proteins act by targeting key cellular regulatory proteins and altering their function. Known targets of the 708-amino-acid large T antigen include the three members of the retinoblastoma protein family (pRb, p107, and p130), members of the CBP family of transcriptional adapter proteins (cap-binding protein [CBP], p300, and p400), and the tumor suppressor p53. Small t antigen alters the activity of phosphatase pp2A and transactivates the cyclin A promoter. The first 82 amino acids of large T antigen and small t antigen are identical, and genetic experiments suggest that an additional target(s) important for transformation interacts with these sequences. This region contains a motif similar to the J domain, a conserved sequence found in the DnaJ family of molecular chaperones. We show here that mutations within the J domain abrogate the ability of large T antigen to transform mammalian cells. To examine whether a purified 136-amino-acid fragment from the T antigen amino terminus acts as a DnaJ-like chaperone, we investigated whether this fragment stimulates the ATPase activity of two hsc70s and discovered that ATP hydrolysis is stimulated four-to ninefold. In addition, ATPase-defective mutants of full-length T antigen, as well as wild-type small t antigen, stimulated the ATPase activity of hsc70. T antigen derivatives were also able to release an unfolded polypeptide substrate from an hsc70, an activity common to DnaJ chaperones. Because the J domain of T antigen plays essential roles in viral DNA replication, transcriptional control, virion assembly, and tumorigenesis, we conclude that this region may chaperone the rearrangement of multiprotein complexes.Simian virus 40 (SV40) encodes two proteins involved in tumorigenesis, the large and small tumor antigens. Large tumor antigen (T antigen) orchestrates many aspects of productive viral infection and is necessary and in many cases sufficient for tumorigenesis. T antigen is a 708-amino-acid multifunctional protein that elicits cellular transformation by acting on multiple targets, including members of the retinoblastoma tumor suppressor family (pRb, p107, and p130), members of the CBP family of transcriptional coactivators (CREB-binding protein [CBP], p300, and p400), and the tumor suppressor, p53. It is likely that additional T antigen targets important for transformation await discovery. T antigen sequences important for transformation map to two different regions of the molecule: the amino-terminal domain, which encompasses the first 125 amino acids, and a region located within the carboxyterminal half of the molecule (Fig. 1). Major questions that remain to be answered are the following. How does T antigen act on each of the cellular targets? How does the concerted action of T antigen on these multiple targets lead to tumorigenesis?Evidence that one or more independent transforming functions reside in the carboxy-terminal half o...

At this time, about 3,000 different viruses are recognized, but metagenomic studies suggest that these viruses are a small fraction of the viruses that exist in nature. We have explored viral diversity by deep sequencing nucleic acids obtained from virion populations enriched from raw sewage. We identified 234 known viruses, including 17 that infect humans. Plant, insect, and algal viruses as well as bacteriophages were also present. These viruses represented 26 taxonomic families and included viruses with single-stranded DNA (ssDNA), double-stranded DNA (dsDNA), positive-sense ssRNA [ssRNA(+)], and dsRNA genomes. Novel viruses that could be placed in specific taxa represented 51 different families, making untreated wastewater the most diverse viral metagenome (genetic material recovered directly from environmental samples) examined thus far. However, the vast majority of sequence reads bore little or no sequence relation to known viruses and thus could not be placed into specific taxa. These results show that the vast majority of the viruses on Earth have not yet been characterized. Untreated wastewater provides a rich matrix for identifying novel viruses and for studying virus diversity.Importance At this time, virology is focused on the study of a relatively small number of viral species. Specific viruses are studied either because they are easily propagated in the laboratory or because they are associated with disease. The lack of knowledge of the size and characteristics of the viral universe and the diversity of viral genomes is a roadblock to understanding important issues, such as the origin of emerging pathogens and the extent of gene exchange among viruses. Untreated wastewater is an ideal system for assessing viral diversity because virion populations from large numbers of individuals are deposited and because raw sewage itself provides a rich environment for the growth of diverse host species and thus their viruses. These studies suggest that the viral universe is far more vast and diverse than previously suspected.

The p53 protein is apparently central to the development of human cancers because both alleles are often found to be mutated in different tumour types. In addition, wild-type p53 can inhibit transformation by viral and cellular oncogenes in vitro, so p53 has been classified as a tumour suppressor. Investigations of the normal function of p53 have indicated that at least one of its functions could involve the activation of gene expression through the binding of specific DNA-regulatory sequences. Also, overexpression of p53 can mediate growth arrest and repress transcription from a variety of promoters. We demonstrate here both in vivo and in vitro that expression of wild-type p53 specifically represses the activity of promoters whose initiation is dependent on the presence of a TATA box. Promoters whose accurate transcription is directed by a pyrimidine-rich initiator element, however, are immune to the effects of p53. Furthermore, we observe that repression is mediated by an interaction of p53 with basal transcription factor(s). Thus, p53 appears to repress the activity of certain promoters through direct communication with TATA box-dependent basal transcription machinery.

In the classic paradigm of mammalian cell cycle control, Rb functions to restrict cells from entering S phase by sequestering E2F activators (E2f1, E2f2 and E2f3), which are invariably portrayed as the ultimate effectors of a transcriptional program that commit cells to enter and progress through S phase1, 2. Using a panel of tissue-specific cre-transgenic mice and conditional E2f alleles we examine the effects of E2f1, E2f2 and E2f3 triple deficiency in murine ES cells, embryos and small intestines. We show that in normal dividing progenitor cells E2F1-3 function as transcriptional activators, but contrary to current dogma, are dispensable for cell division and instead are necessary for cell survival. In differentiating cells they function in complex with Rb as repressors to silence E2F targets and facilitate exit from the cell cycle. The inactivation of Rb in differentiating cells resulted in a switch of E2F1-3 from repressors to activators, leading to the superactivation of E2F responsive targets and ectopic cell divisions, and loss of E2f1-3 completely suppressed these phenotypes. This work contextualizes the activator versus repressor functions of E2F1-3 in vivo, revealing distinct roles in dividing versus differentiating cells and in normal versus cancer-like cell cycles in vivo.

Simian virus 40 (SV40) is a small DNA tumor virus that has been extensively characterized due to its relatively simple genetic organization and the ease with which its genome is manipulated. The large and small tumor antigens (T antigens) are the major regulatory proteins encoded by SV40. Large T antigen is responsible for both viral and cellular transcriptional regulation, virion assembly, viral DNA replication, and alteration of the cell cycle. Deciphering how a single protein can perform such numerous and diverse functions has remained elusive. Recently it was established that the SV40 T antigens, including large T antigen, are molecular chaperones, each with a functioning DnaJ domain. The molecular chaperones were originally identified as bacterial genes essential for bacteriophage growth and have since been shown to be conserved in eukaryotes, participating in an array of both viral and cellular processes. This review discusses the mechanisms of DnaJ/Hsc70 interactions and how they are used by T antigen to control viral replication and tumorigenesis. The use of the DnaJ/Hsc70 system by SV40 and other viruses suggests an important role for these molecular chaperones in the regulation of the mammalian cell cycle and sheds light on the enigmatic SV40 T antigen—a most amazing molecule

Mutants of animal viruses can be isolated in bacteria by recombinant DNA methods. Since no viral functions are required for propagation of recombinants in bacteria, viral mutants with lethal changes in cis- or trans-acting elements can be isolated, as well as partially or conditionally defective mutants. In the cases of viruses with small DNA genomes, such as the tumorigenic simian virus 40 (SV40), the entire viral DNA can be inserted into the bacterial plasmid pBR322 and cloned in Escherichia coli. Recombinant plasmids with a single copy of SV40 DNA cause morphological transformation of mouse cells in culture with the same efficiency as SV40 DNA isolated from virus-infected monkey cells, but the recombinant DNA is noninfectious and replicates poorly in permissive cells. However, SV40 DNA excised from the plasmid replicates as well as authentic viral DNA and is fully infectious. SV40 mutants with small deletions or base substitutions have been isolated by in vitro site-specific or random local mutagenesis of recombinant DNA followed by cloning in E. coli. Many of the mutants thus isolated are defective in specific viral functions.