Nucleotide sequence analysis and enhancer function of long terminal repeats associated with an endogenous African green monkey retroviral DNA.

Abstract

The nucleotide sequence and enhancer activity of the long terminal repeats (LTRs) associated with a cloned endogenous African green monkey (AGM) retroviral DNA designated as X-AGM-1 was studied. A unique feature of the endogenous AGM proviral LTRs was the presence of multiple copies of two types of directty repeating units in the U3 region: 16 8-base-pair (bp) repeats were present in the 5' LTR and 12 were present in the 3' LTR which were bound by a 6-bp perfect direct repeat; tandem duplication of a 32-bp sequence resulted in 3.5 copies in the 5' LTR and 2.5 copies in the 3' LTR. Nucleotide sequence homology was seen between the 8-bp direct repeats located in the AGM proviral LTRs and a 10-bp repeat unit of the deca-satellite present in AGM cellular DNA. The 32-bp repeats of the AGM proviral LTFIs contained sequences which were related to the SV40 21-bp repeats and to the "core" of the SV40 72-bp enhancer element. Furthermore, the AGM provirus was distinct from known infectious retroviruses due to the presence of a primer-binding sequence complementary to the 3' terminus of mammalian tRNAGY. Functional analysis of the 3' LTR present in X-AGM-1 DNA by chloramphenicol acetyltransferase assay demonstrated enhancer activity associated with the 32-bp direct repeats. Sequences outside the 32-bp unit were necessary for full activator function, suggesting the presence of multiple enhapcer domains in the AGM provirus.

Chemical Induction of Endogenous Retrovirus Particles from the Vero Cell Line of African Green Monkeys

“…Endogenous retroviruses have been isolated from Old World monkeys, such as the baboon, langur, colobus monkey, and macaques, and from New World monkeys, such as the woolly monkey, squirrel monkey, and owl monkey, as well as from the gibbon ape and a prosimian tree shrew (9,21,33,62,67,(76)(77)(78)(79). However, there has been no report of endogenous retrovirus particles of AGM origin, even though endogenous retroviral sequences related to type C gammaretrovirus murine leukemia virus (MLV) and type D betaretrovirus SRV are present in the genome of African green monkeys and in cell lines derived from this species (56,83,85), and MLV-related viral DNAs cloned from AGM tissue were shown to contain functional long terminal repeats (LTRs) (36). Furthermore, African green monkey cell lines, such as Vero and CV-1 (30), have been widely used in research and diagnostics without any report of spontaneous release of virus particles.…”

“…We have recently developed a stepwise strategy for using chemical inducers to optimize induction conditions for investigating the presence of latent viruses (37). We have used this strategy to evaluate activation of endogenous retroviral sequences in Vero cells, which, although known to contain numerous copies of endogenous retroviral sequences due to their AGM species of origin (5,8,10,26,31,36,42,57,68,81), were not expected to contain an inducible virus, based upon the extensive testing history and broad use of the cell line (29,74). Here, we report that treatment of Vero cells with 5-azacytidine (AzaC) and with 5Ј-iodo-2Ј-deoxyuridine (IUdR) induced endogenous retroviral particles related to ancient sim-ian endogenous type D betaretrovirus (SERV) sequences that are present in all Old World monkeys (83) and distinct from pathogenic, type D simian retroviruses (SRVs) (46).…”