No abstract

We are studying the diversity of and relationships among papillomaviruses (PVs) to understand the modes and timescales of PV evolution and in the hope of finding animal PVs that may serve as model systems for disease caused by human PVs (HPVs). Toward this goal, we have examined 326 genital samples from rhesus monkeys and long-tailed macaques with a PCR protocol optimized for detecting genital HPV types. In 28 of the rhesus monkey samples, we found amplicons derived from 12 different and novel PV genomes, RhPV-a to RhPV-m, with the likely taxonomic status of "type." The frequency with which novel RhPVs were detected suggests that rhesus monkeys may play host to PVs with a diversity similar to that of humans. In phylogenetic trees, all 12 of the different RhPVs and the previously described type RhPV-1 were members of the genital HPV supergroup and formed three minor branches distinct from the 11 branches formed by genital HPVs. We also identified a novel PV amplicon, MfPV-a, from a long-tailed macaque, a species belonging to the same genus as rhesus monkeys. MfPV-a turned out to be a close relative of five RhPVs. It appears that the evolution of primate lineages leading to the genus Macaca and to humans created transmission barriers for PVs, resulting in viral evolution closely linked to the host. Additional support for the linked-evolution hypothesis comes from considering the phylogenetic association of two other ape and monkey PVs with the genital HPVs, the supergroup formed by at least seven ungulate PVs, and the isolated phylogenetic position of the only known bird PV.

Bovine leukemia virus (BLV) encodes at least two regulatory proteins, Rex and Tax. Tax, the transactivating protein, stimulates the long terminal repeat to promote viral transcription and may be involved in tumorigenesis. Rex is involved in the transition from early expression of regulatory proteins to later expression of viral structural proteins. We have targeted ribozymes against the mRNA encoding Rex and Tax. The ribozymes consist of the hammerhead catalytic motif flanked by antisense sequences that hybridize with the complementary rex/tax mRNA. To evaluate cleavage in a cell-free system, we transcribed portions of rex/tax mRNA and incubated them with synthetic RNA ribozymes. A ribozyme was identified that cleaves >80% of the target RNA. Synthetic DNA encoding this ribozyme was cloned into the expression vector pRc/RSV and transfected into BLV-infected bat lung cells. Intracellular cleavage of rex/tax mRNA was confirmed by reverse transcriptase PCR. In cells expressing the ribozyme, viral expression was markedly inhibited. Expression of the BLV core protein p24 was inhibited by 61%, and reverse transcriptase activity in supernatant was inhibited by 92%. Ribozyme inhibition of BLV expression suggests that cattle expressing these sequences may be able to control BLV replication.Bovine leukemia virus (BLV), a retrovirus structurally similar to human T-cell leukemia viruses I and II (HTLV-I and -II), causes persistent lymphocytosis and B-lymphocyte lymphoma in cattle and sheep (1). After initial infection, BLV expresses a doubly spliced transcript encoding the regulatory proteins Rex and Tax (2). Tax trans-activates the viral long terminal repeat and also cellular promoters, including c-fos and somatostatin (3). Cotransfection experiments have shown that Tax is necessary for viral expression in vitro (4). Rex regulates the transition from early expression of the doubly spliced transcript encoding regulatory proteins to the later expression of singly spliced or unspliced transcripts that express the env gene or the gag and pol genes (5). Recently, the 3' region of HTLV-I and BLV has been shown to encode RNA with alternative splice patterns that may express other regulatory proteins (6-8).Because of the critical role of regulatory proteins such as Tax and Rex in the HTLV/BLV group of viruses, we hypothesized that the rex/tax mRNA would be a rational target for ribozyme-mediated inhibition. The hammerhead motif, first identified in plant RNA pathogens (9, 10), cleaves the phosphodiester bond downstream of a GUC triplet (9,10). By flanking the hammerhead motif with antisense sequences, Haseloff and Gerlach (11) demonstrated cleavage of specific target RNAs. In this study, we demonstrate a ribozyme that cleaves rex/tax mRNA and, when transfected into BLV-infected cells, markedly inhibits viral expression.The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact...

The retinoic acid receptor beta2 (RARbeta2) protein is a putative tumor suppressor that inhibits proliferation and can induce apoptosis when introduced into breast, cervical, lung, and pancreatic cancer cell lines. To determine if RARbeta2 suppresses proliferation of mammary-derived cancer cells in vivo, we transduced MDA-MB-435 breast cancer cells with the LXSN retroviral vector containing RARbeta2 and implanted LXSN vector- or RARbeta2-transduced cells into the mammary fat pads of nude and severe combined immune deficiency (SCID) mice. We analyzed the xenografts for several tumor parameters, including tumor size, inflammation, vascularity, mitoses, tumor recurrence at the primary site following resection, and metastases. We found that 19 of 52 mice inoculated with vector-transduced cells developed metastases in multiple organs while only one of 55 mice receiving RARbeta2-transduced cells displayed evidence of metastases (p < 0.000001, combined experiments, two-tailed Fisher's exact test). Moreover, RARbeta2-tumor cell recipient mice had a lower incidence of post-resection tumor recurrence (8/55 vs. 25/52, p = 0.0004), 34% less necrosis (in three of four experiments, p = 0.001), and 39% fewer mitoses in tumor tissue (p < 0.000001). Our findings suggest that RARbeta2 may play a role in inhibiting the metastatic cascade in a mouse mammary gland xenograft tumor model and is a potential candidate for therapeutic intervention in human breast cancer.