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In the most extensive examination to date of the relationship between the pausing of reverse transcrip-tase (RT) and RNA secondary structures, pause events were found to be correlated to inverted repeats both ahead of, and behind the catalytic site in vitro. In addition pausing events were strongly associated with polyadenosine sequences and to a lesser degree diadenosines and monoadenosine residues. Pausing was also inversely proportional to the potential bond strength between the nascent strand and the template at the point of termination, for both mono and dinucleotides. A run of five adenosine and four uridine residues caused most pausing on the HIV-1 template, a region which is the site of much sequence heterogeneity in HIV-1. We propose that homopolyadenosine tracts can act as termination signals for RT in the context of inverted repeats as they do for certain RNA polymerases.

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Packaging of type C retrovirus genomic RNAs into budding virions requires a highly specific interaction between the viral Gag precursor and unique cis-acting packaging signals on the full-length RNA genome, allowing the selection of this RNA species from among a pool of spliced viral RNAs and similar cellular RNAs. This process is thought to involve RNA secondary and tertiary structural motifs since there is little conservation of the primary sequence of this region between retroviruses. To confirm RNA secondary structures, which we and others have predicted for this region, disruptive, compensatory, and deletion mutations were introduced into proviral constructs, which were then assayed in a permissive cell line. Disruption of either of two predicted stem-loops was found to greatly reduce RNA encapsidation and replication, whereas compensatory mutations restoring base pairing to these stem-loops had a wild-type phenotype. A GGNGR motif was identified in the loops of three hairpins in this region. Results were consistent with the hypothesis that the process of efficient RNA encapsidation is linked to dimerization. Replication and encapsidation were shown to occur at a reduced rate in the absence of the previously described kissing hairpin motif.

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The role of the RNA secondary structure in the 5' packaging signal region of human immunodeficiency virus type 1 (HIV-1) in initiating translation of gag mRNA has been investigated both in vitro and in the presence of cellular cofactors in vivo. Heat denaturation of the structure and mutagenic deletion both lead to an increase in levels of translated products, indicating that the structure is a significant inhibitor of translation. The proximity of the gag AUG to the packaging signal structure suggested that it might function as an internal ribosome entry site. However, in both a cell-free system and eukaryotic cells, translation will initiate at a novel upstream initiation codon introduced within the 5' noncoding region. This codon is utilized exclusively, resulting in gag protein products with an extra 11 amino acids at the amino terminus, which, when expressed in T lymphocytes, are confined intracellularly, probably because of the lack of an N-terminal glycine myristoylation signal. Deletion of the secondary structure abolishes gag production even in the presence of tat and rev in trans. Using dicistronic constructs containing the HIV-1 5' leader cloned between two heterologous open reading frames, we were unable to detect any significant expression of the second open reading frame that would have been supportive of an internal ribosome entry site mechanism. Using mutant proviruses either lacking the entire packaging signal structure region or containing the introduced upstream initiation codon in long-term replication studies, we were unable to detect reverse transcriptase activity in culture supernatants. The 5' packaging signal structure of HIV-1 does not serve as an internal ribosome entry site. The translation of gag is consistent with ribosomal scanning. However, the packaging signal structure causes significant translational inhibition.

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A stable secondary structure model is presented for the region 3' of the primer-binding site to 130 bases into the gag sequence of the prototype type D retrovirus Mason-Pfizer monkey virus. Using biochemical probing of RNA from this region in association with free energy minimization, we have identified a stem-loop structure in the region, which from other studies has been shown to be important for genomic RNA encapsidation. The structure involves a highly stable stem of five G-C pairs terminating in a heptaloop. Comparison of the Mason-Pfizer monkey virus structure with one predicted for squirrel monkey retrovirus demonstrates an identical stem and a common ACC motif in the loop. Free energy studies of the secondary structure of the 5' regions of eight other retroviruses predict stem loops which have similar GAYC motifs. We believe this may represent a common structural and sequence motif which among other functions may be involved in genomic RNA packaging in these viruses.

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AIM: To estimate measles vaccine efficacy in the field in New Zealand, during the 1991 measles epidemic. METHODS: Notifications in the Wellington Area Health Board region from the first 21 weeks of the 1991 measles epidemic (1444 cases) were analysed to estimate vaccine efficacy. Estimates of 70% and 80% immunisation coverage were used in this analysis for the age groups of 1-4 years, 5-9 years, 10-14 years, and 15-19 years. RESULTS: A trend was demonstrated, suggesting a reduction in vaccine efficacy (VE) for the two older age groups at 70% coverage (1-4 years 69% VE, 5-9y 68%, 10-14y 46%, 15-19y 47%) and 80% coverage (1-4y 82%, 5-9y 81%, 10-14y 69%, 15-19y 69%). Potential sources of bias are highlighted and their impact on the vaccine efficacy estimates discussed. CONCLUSIONS: There are multiple sources of bias that exist. If New Zealand is to improve and monitor the quality of its vaccination programmes, good quality information is required, which may include the need for immunisation registers, or ongoing case control studies in certain circumstances.

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Interaction of cis-acting RNA sequences with nucleocapsid proteins is one of the critical events leading to retroviral genomic RNA packaging. We have derived a potentially stable secondary structure for the packaging signal region of human immunodeficiency virus strain IIIB, using a combination of biochemical analysis and computer modelling. This region encompasses the major splice donor (SD), which is found in a highly structured conserved stem-loop. Comparison with other published human immunodeficiency virus type 1 sequences shows almost absolute nucleotide conservation in base-paired regions required to maintain this structure. Presently and previously described packaging-defective mutants would disrupt the structure. The structure depends on base pairing between nucleotide sequences 5' of the major SD which are common to both genomic and subgenomic RNAs and sequences 3' of SD which are unique to the unspliced RNA. This may explain how in retroviruses such as Rous sarcoma virus, mutations in regions common to genomic and subgenomic RNA might prevent packaging of the unspliced mRNA by disrupting a signal structure which can exist only in the genomic RNA species.

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