An RNA secondary structure juxtaposes two remote genetic signals for human T-cell leukemia virus type I RNA 3'-end processing.

Sequence analysis of the human T-cell leukemia virus type I (HTLV-I) long terminal repeat (LTR) does not reveal a polyadenylation consensus sequence, AAUAAA, close to the polyadenylation site at the 3' end of the viral RNA. Using site-directed mutagenesis, we demonstrated that two cis-acting signals are required for efficient RNA processing in HTLV-I LTR: (i) a remote AAUAAA hexamer at a distance of 276 nucleotides upstream of the polyadenylation site, and (ii) the 20-nucleotide GU-rich sequence immediately downstream from the poly(A) site. It has been postulated that the folding of RNA into a secondary structure juxtaposes the AAUAAA sequence, in a noncontiguous manner, to within 14 nucleotides of the polyadenylation site. To test this hypothesis, we introduced deletions and point mutations within the U3 and R regions of the LTR. RNA 3'-end processing occurred efficiently at the authentic HTLV-I poly(A) site after deletion of the sequences predicted to form the secondary structure. Thus, the genetic analysis supports the hypothesis that folding of the HTLV-I RNA in the U3 and R regions juxtaposes the AAUAAA sequence and the poly(A) site to the correct functional distance. This unique arrangement of RNA-processing signals is also found in the related retroviruses HTLV-II and bovine leukemia virus.     

Avian retroviral long terminal repeats bind CCAAT/enhancer-binding protein.

DNA-protein interactions involving enhancer and promoter sequences within the U3 regions of several avian retroviral long terminal repeats (LTRs) were studied by DNase I footprinting. The rat CCAAT/enhancer-binding protein, C/EBP, bound to all four viral LTRs examined. The Rous sarcoma virus binding site corresponded closely to the 5' limit of the LTR enhancer; nucleotides -225 to -188 were protected as a pair of adjacent binding domains. The Fujinami sarcoma virus LTR bound C/EBP at a single site at nucleotides -213 to -195. C/EBP also bound to the promoter region of the enhancerless Rous-associated virus-0 LTR at nucleotides -77 to -57. The avian myeloblastosis virus LTR bound C/EBP at three sites: nucleotides -262 to -246, -154 to -134, and -55 to -39. We have previously observed binding of C/EBP to an enhancer in the gag gene of avian retroviruses. A heat-treated nuclear extract from chicken liver bound to all of the same retroviral sequences as did C/EBP. Alignment of the avian retroviral binding sequences with the published binding sites for C/EBP in two CCAAT boxes and in the simian virus 40, polyoma, and murine sarcoma virus enhancers suggested TTGNNGCTAATG as a consensus sequence for binding of C/EBP. When two bases of this consensus sequence were altered by site-specific mutagenesis of the Rous sarcoma virus LTR, binding of the heat-stable chicken protein was eliminated.     

Human T-lymphotropic virus types I and II.

Human T-lymphotropic virus types I (HTLV-I) and II (HTLV-II) are members of a family of four known retroviruses that are oncogenic as opposed to cytopathic. This family includes HTLV-I and -II, bovine leukemia virus, and simian T-cell leukemia virus. The two types of HTLV are closely related, and for more than a decade we have been aware of the presence of these viruses in humans. In the first part of this article I summarize recent epidemiologic and clinical findings related to the presence of HTLV-I and -II in the Americas. In the second part, I discuss how these viruses may regulate themselves and how in turn they might cause leukemia and neurologic disease in humans.     

Avian Retrovirus pp32 DNA-Binding Protein I. Recognition of Specific Sequences on Retrovirus DNA Terminal Repeats

The avian retrovirus pp32 protein possesses a DNA-nicking activity which prefers supercoiled DNA as substrate. We have investigated the binding of pp32 to avian retrovirus long terminal repeat (LTR) DNA present in both supercoiled and linear forms. The cloned viral DNA was derived from unintegrated Schmidt-Ruppin A (SRA) DNA. A subclone of the viral DNA in pBR322 (termed pPvuII-DG) contains some src sequences, tandem copies of LTR sequences, and partial gag sequences in the order src-U(3) U(5):U(3) U(5)-gag. Binding of pp32 to supercoiled pPvuII-DG DNA followed by digestion of this complex with a multicut restriction enzyme (28 fragments total) permitted pp32 to preferentially retain on nitrocellulose filters two viral DNA fragments containing only LTR DNA sequences. In addition, pp32 also preferentially retained four plasmid DNA fragments containing either potential promoters or Tn3 "left-end" inverted repeat sequences. Mapping of the pp32 binding sites on viral LTR DNA was accomplished by using the DNase I footprinting technique. The pp32 protein, but not the avian retrovirus alphabeta DNA polymerase, is able to form a unique protein-DNA complex with selected regions of either SRA or Prague A LTR DNAs. Partial DNase I digestion of a 275-base pair SRA DNA fragment complexed with pp32 gives upon electrophoresis in denaturing gels a unique ladder pattern, with regions of diminished DNase I susceptibility from 6 to 10 nucleotides in length, in comparison with control digests in the absence of protein. The binding of pp32 to this fragment also yields enhanced DNase I-susceptible sites that are spaced between the areas protected from DNase I digestion. The protected region of this unique complex was a stretch of 170 +/- 10 nucleotides that encompasses the presumed viral promoter site in U(3), which is adjacent to the src region, extends through U(5), and proceeds past the joint into U(3) for about 34 base pairs. No specific protection or DNase I enhancement by pp32 was observed in experiments with a 435-base pair SRA DNA fragment derived from a part of U(3) and the adjacent src region or a 55-base pair DNA fragment derived from another part of U(3). The DNA sequence of Prague A DNA at the fused LTRs differs from that of SRA DNA. The alteration in the sequence at the juncture of the LTRs prevented pp32 from forming a stable complex in this region of the LTR. Our results are relevant to two aspects of the interaction between pp32 and LTR DNA. First, the pp32 protein in the presence of selected viral DNA restriction fragments possibly forms a higher order oligomer analogous to Escherichia coli DNA gyrase-DNA complexes or eucaryotic nucleosome structures. Second, the specificity of the binding suggests a role for pp32 and the protected DNA sequences in the retrovirus life cycle. The preferred sequences to which pp32 binds include two adjacent 15-base pair inverted terminal repeats at the joint between U(5) and U(3) in SRA DNA. This region is involved in circularization of linear DNA and is perhaps the site that directs integration into cellular DNA.     

Recent advances in detection of human T-cell leukemia viruses type I and type II infection

The human T-cell leukemia viruses type I (HTLV-I) and type II (HTLV-II) have been implicated in the pathogenesis of a variety of neoplastic and neurological disorders. Classical techniques for detection involve assay of serum for antibodies by Western blotting or ELISA, which do not discriminate between infection with HTLV-I and HTLV-II. In order to provide appropriate prognostic information to infected individuals and to obtain an accurate assessment of the prevalence of both retroviruses in the United States, we and others have applied the technique of enzymatic DNA amplification to detect HTLV-I and HTLV-II. These techniques allow rapid detection of viral nucleic acids in freshly isolated peripheral blood samples. Recent studies indicate an unusually high rate of HTLV-II infection among seropositive individuals in a sampling of New Orleans intravenous drug users, indicating a need for combined serological and molecular genetic screening of high-risk populations.