The Bovine Leukemia Virus Encapsidation Signal Is Composed of RNA Secondary Structures

Abstract

The encapsidation signal of bovine leukemia virus (BLV) was previously shown by deletion analysis to be discontinuous and to extend into the 5′ end of the gag gene (L. Mansky et al., J. Virol. 69:3282–3289, 1995). The global minimum-energy optimal folding for the entire BLV RNA, including the previously mapped primary and secondary encapsidation signal regions, was analyzed. Two stable stem-loop structures (located just downstream of the gagstart codon) were predicted within the primary signal region, and one stable stem-loop structure (in the gag gene) was predicted in the secondary signal region. Based on these predicted structures, we introduced a series of mutations into the primary and secondary encapsidation signals in order to explore the sequence and structural information contained within these regions. The replication efficiency and levels of cytoplasmic and virion RNA were analyzed for these mutants. Mutations that disrupted either or both of the predicted stem-loop structures of the primary signal reduced the replication efficiency by factors of 7 and 40, respectively; similar reductions in RNA encapsidation efficiency were observed. The mutant with both stem-loop structures disrupted had a phenotype similar to that of a mutant containing a deletion of the entire primary signal region. Mutations that disrupted the predicted stem-loop structure of the secondary signal led to similar reductions (factors of 4 to 6) in both the replication and RNA encapsidation efficiencies. The introduction of compensatory mutations into mutants from both the primary and secondary signal regions, which restored the predicted stem-loop structures, led to levels of replication and RNA encapsidation comparable to those of virus containing the wild-type encapsidation signal. Replacement of the BLV RNA region containing the primary and secondary encapsidation signals with a similar region from human T-cell leukemia virus (HTLV) type 1 or type 2 led to virus replication at three-quarters or one-fifth of the level of the parental virus, respectively. The results from both the compensatory mutants and BLV-HTLV chimeras indicate that the encapsidation sequences are recognized largely by their secondary or tertiary structures.

Transmissible Gastroenteritis Coronavirus Genome Packaging Signal Is Located at the 5′ End of the Genome and Promotes Viral RNA Incorporation into Virions in a Replication-Independent Process

“…Packaging efficiency of L-R1-derived sequences. There is experimental evidence supporting the idea that genome packaging in some RNA viruses depends on the secondary structure of the PS (8,30,31). It has been shown that the L-R1 sequence was responsible for packaging in TGEV.…”

“…The continuity between the leader sequence (nt 1 to 99) and downstream sequences (nt 100 to 598) in the PS was required for packaging, since the insertion of ectopic viral sequences between the leader and R1 in sgmRNA L-3a-R1 led to a significant reduction in the packaging efficiency compared to that of sgmRNA L-R1. This continuity might be required to maintain the correct secondary structure of the PS, since structural requirements frequently determine packaging in RNA viruses (8,30,31). Further deletions along the TGEV PS prevented packaging of viral RNAs, indicating that the whole genomic region was necessary for its function.…”

Enhancing Protein Backbone Binding—A Fruitful Concept for Combating Drug‐Resistant HIV

“…Its relentless ability to mutate arises from a high mismatch error rate (10 À3 to 10 À5 nucleotide bases per cycle) of the viruss reverse transcriptase enzyme and the absence of exonuclease-based proofreading activity. [24,25] These factors, in conjunction with the viruss rapid replication cycle (10 10 virions per day) and genetic recombination ability, result in seemingly endless genetic diversification. [26,27] However, the number of variants within the quasi-species at a given time is limited by natural selection, deleterious mutations, and limited host cell availability, and by inactivation from the hosts immune system response.…”

Human T-cell leukemia virus type 1 Gag domains have distinct RNA-binding specificities with implications for RNA packaging and dimerization

“…To examine whether HTLV-1 MA and NC play similar roles as the analogous proteins in HTLV-2, fluorescence anisotropy (FA) binding assays were performed using fluorescently labeled RNAs derived from the previously proposed HTLV-1 ⌿ element (SL(450 -470), SL(474 -508), and SL(447-512) in Fig. 1A) (11,12). HTLV-1 SL(450 -470) and SL(474 -508) RNA sequences derived from a provirus cloned from the lymphocytic cell line CS-1 were previously used to replace BLV SL1 and SL2 individually and simultaneously.…”

“…downstream of the gag start codon and consists of two stemloop (SL) elements (10,11). Replacement of the BLV packaging signal with a similar region from either HTLV-1 or HTLV-2 led to only partial BLV replication defects, suggesting some level of conservation in the function of deltaretroviral packaging signals (11,12).…”