Effect of mutations in the nucleocapsid protein (NCp7) upon Pr160(gag-pol) and tRNA(Lys) incorporation into human immunodeficiency virus type 1

COS-7 cells were transfected with DNAs containing mutations in the NCp7 sequences of human immunodeficiency virus. Selective incorporation into the virus of tRNA(Lys) was measured by two-dimensional polyacrylamide gel electrophoresis, and Pr160(gag-pol) incorporation into the virus was detected in Western blots of viral protein. Mutations tested included cysteine and histidine mutations in either of the Cys-His boxes, as well as mutations in the N- and C-terminal flanking regions and in the linker region between the two Cys-His boxes. Of 10 mutations tested, only 2 inhibited tRNA(Lys) incorporation: a P31L mutation in the linker region and a deletion which removed both Cys-His boxes and the linker region (deltaK14-T50). The P31L mutation prevents the incorporation of Pr160(gag-pol) into the virus. Cotransfection of COS cells with both P31L DNA and a plasmid coding only for unprocessed Pr160(gag-pol) resulted in the viral incorporation of Pr160(gag-pol) and the rescue of selective packaging of tRNA(Lys) into the virion. In the deltaK14-T50 mutant, Pr160(gag-pol) is incorporated into the virus. Selective tRNA(Lys) packaging is not rescued by cotransfection with a plasmid coding for Pr160(gag-pol) but is rescued by cotransfection with DNA coding for wild-type Pr55(gag). Since Pr55(gag) does not by itself selectively package tRNA(Lys), the deltaK14-T50 mutation may be affecting tRNA(Lys) binding to a cytoplasmic Pr55(gag)/Pr160(gag-pol) complex.     

Peptide therapy for treatment of allergic diseases

G.U wobble pairs are crucial to many examples of RNA-protein recognition. We previously concluded that the G.U wobble pair in the acceptor helix of Escherichia coli alanine tRNA (tRNA(Ala)) is recognized indirectly by alanyl-tRNA synthetase (AlaRS), although direct recognition may play some role. Our conclusion was based on the finding that amber suppressor tRNA Ala with G.U shifted to an adjacent helical site retained substantial but incomplete Ala acceptor function in vivo. Other researchers concluded that only direct recognition is operative. We report here a repeat of our original experiment using tRNA(Lys) instead of tRNA(Ala). We find, as in the original experiment, that a shifted G.U confers Ala acceptor activity. Moreover, the modified tRNA(Lys) was specific for Ala, corroborating our original conclusion and making it more compelling.     

Functional replacement of hamster lysyl-tRNA synthetase by the yeast enzyme requires cognate amino acid sequences for proper tRNA recognition

We cloned the cDNA encoding a 597-aa hamster lysyl-tRNA synthetase. This enzyme is a close homologue of the 591-aa Saccharomyces cerevisiae enzyme, with the noticeable exception of their 60-aa N-terminal regions, which differ significantly. Several particular features of this polypeptide fragment from the hamster lysyl-tRNA synthetase suggest that it is implicated in the assembly of that enzyme within the multisynthetase complex. However, we show that this protein domain is dispensable in vivo to sustain growth of CHO cells. The cross-species complementation was investigated in the lysine system. The mammalian enzyme functionally replaces a null-allele of the yeast KRS1 gene. Conversely, the yeast enzyme cannot rescue Lys-101 cells, a CHO cell line with a temperature-sensitive lysyl-tRNA synthetase. The yeast and mammalian enzymes, overexpressed in yeast, were purified to homogeneity. The hamster lysyl-tRNA synthetase efficiently aminoacylates both mammalian and yeast tRNA(Lys), whereas the yeast enzyme aminoacylates mammalian tRNA(Lys) with a catalytic efficiency 20-fold lower, as compared to its cognate tRNA. The 152-aa C-terminus extremity of the hamster enzyme provides the yeast enzyme with the capacity to complement Lys-101 cells. This hybrid protein is fairly stable and aminoacylates both yeast and mammalian tRNA(Lys) with similar catalytic efficiencies. Because this C-terminal polypeptide fragment is likely to make contacts with the acceptor stem of tRNA(Lys), we conclude that it should carry the protein determinants conferring specific recognition of the cognate tRNA acceptor stem and therefore contributes an essential role in the operational RNA code for amino acids.     

Cassava vein mosaic virus (CsVMV), type species for a new genus of plant double stranded DNA viruses?

The complete sequence of 8159 nucleotides of the double stranded DNA genome of cassava vein mosaic virus (CsVMV) was determined (# U59751) and revealed a significant difference in genome organization when compared with a previous report (# U20341). When transferred to cassava plants by microbombardment, the full length CsVMV clone was infectious, confirming the genome organization here described. Sequence comparisons between CsVMV and members of the genera Caulimovirus and Badnavirus revealed high homologies between consensus sequences of several proteins that are indispensable for virus replication, including a potential transactivator factor not reported previously. The presence of a sequence complementary to a plant Met tRNA confirms that CsVMV is a plant pararetrovirus and is most closely related to members of the genus Caulimovirus as previously assessed. However, differences in genome organization, number and size of the ORFs, in addition to sequence comparisons with other plant pararetroviruses, shows that either the genetic variability of caulimoviruses is much greater than previously thought, or that CsVMV is the unique representative of a new genus within the Caulimoviridae family. On the basis of this study, it is proposed to upgrade the floating genus Caulimovirus to the family level and to divide the Caulimoviridae family into at least three genera with CsVMV being the type member of a new genus.