RESUMO
Simian immunodeficiency virus (SIV) and human immunodeficiency virus (HIV-1) preferentially select and use tRNA(Lys,3) as the primer for initiation of reverse transcription. Previous studies have shown that HIV-1 can be forced to use tRNA(Glu) if mutations are made within the primer-binding site (PBS) and a region upstream, A-loop, to be complementary to the 3'-terminal 18 nucleotides and anticodon loop of tRNA(Glu). To examine the primer preference of SIV, mutations were made within the PBS of SIV(smmPBj) to be complementary to tRNA(Glu). Analysis of the production of infectious virus revealed that SIV(smmPBj) with the PBS complementary to tRNA(Glu) retained approximately 80% infectivity of the wild type. However, modification of the U5 of SIV(smmPBj) to alter nucleotides to be complementary to the anticodon of tRNA(Glu), in combination with the PBS complementary to tRNA(Glu), drastically reduced the production of infectious SIV(smmPBj) to less than 1% that of wild type. The replication of SIV(smmPBj) with the PBS complementary to tRNA(Glu) was similar to that of the wild type virus, while the replication of SIV(smmPBj) with PBS and A-loop complementary to tRNA(Glu) was delayed compared to that of wild type virus. Analysis of the PBS regions revealed that the virus with the PBS complementary to tRNA(Glu) reverted quickly, within 4 days, to be complementary to tRNA(Lys,3), while the virus with PBS and A-loop complementary to tRNA(Glu) retained the PBS for a longer time during in vitro culture although following extended replication both the A-loop and PBS of SIV(smmPBj) reverted to be complementary to tRNA(Lys,3). RNA modeling of SIV(smmPBj) U5-PBS by m-fold revealed two potential A-loop regions. Mutations in either A-loop drastically effected replication in human PBMC. Analysis of the A-loops following in vitro replication revealed that both reverted to the wild type sequence. The results of these studies demonstrate that SIV(smmPBj), like HIV-1, preferentially utilizes tRNA(Lys,3) as a primer for reverse transcription for high level replication, but unlike HIV-1 selection may involve the use of two adenosine-rich loops.
Assuntos
RNA de Transferência de Ácido Glutâmico/metabolismo , Transcrição Reversa , Vírus da Imunodeficiência Símia/crescimento & desenvolvimento , Vírus da Imunodeficiência Símia/genética , Replicação Viral , Sequência de Bases , Sítios de Ligação/genética , Linhagem Celular , Células Cultivadas , Humanos , Leucócitos Mononucleares , Modelos Moleculares , Dados de Sequência Molecular , Mutação , Conformação de Ácido Nucleico , RNA de Transferência de Ácido Glutâmico/química , RNA Viral/química , RNA Viral/genéticaRESUMO
OBJECTIVE: Bacterial vaginosis (BV) is characterized by a shift in vaginal tract ecology, which includes a decrease in the concentration and/or prevalence of facultative lactobacilli. Currently, mechanisms which could account for the disappearance of lactobacilli are not well understood. The objective of this study was to determine whether vaginal streptococci/enterococci can produce bacteriocin-like inhibitors antagonistic to vaginal lactobacilli. METHODS: Seventy strains of vaginal streptococci or enterococci were tested for antagonistic activities against vaginal lactobacilli using the deferred antagonism technique. RESULTS: One strain, Enterococcus faecium 62-6, which strongly inhibited growth of lactobacilli was selected for further characterization. The spectrum of inhibitory activity of strain 62-6 included Gram-positive organisms from the vaginal environment, although native lactobacilli from the same host were resistant to inhibitor action. Following growth in MRS broth the strain 62-6 inhibitor was shown to be heat- (100 degrees C, 30 minutes), cold- (4 degrees C, less than 114 days) and pH- (4-7) stable. The sensitivity of inhibitor-containing supernatants to pepsin and alpha-chymotrypsin suggested an essential proteinaceous component. The inhibitor was sensitive to lipase but resistant to lysozyme. Dialysis of inhibitor-containing culture supernatants suggested a molecular mass greater than 12,000 Da. All physicochemical properties were consistent with its classification as a bacteriocin-like inhibitor. Kinetic assays demonstrated a sharp onset of inhibitor production coinciding with a concentration of 62-6 of 10(7) cfu/ml, suggesting that production may be regulated by quorum sensing. CONCLUSIONS: These results may have clinical significance as a novel mechanism to account for the decline of vaginal Lactobacillus populations and contribute to both the establishment and recurrence of BV.