Biomedical subjects

Antonio Mas

Publications and source records attributed to Antonio Mas.

8 recordsLinked to original sources

PubMed2006

Host deadenylation-dependent mRNA decapping factors are required for a key step in brome mosaic virus RNA replication.

The genomes of positive-strand RNA [+RNA] viruses perform two mutually exclusive functions: they act as mRNAs for the translation of viral proteins and as templates for viral replication. A universal key step in the replication of +RNA viruses is the coordinated transition of the RNA genome from the cellular translation machinery to the viral replication complex. While host factors are involved in this step, their nature is largely unknown. By using the ability of the higher eukaryotic +RNA virus brome mosaic virus (BMV) to replicate in yeast, we previously showed that the host Lsm1p protein is required for efficient recruitment of BMV RNA from translation to replication. Here we show that in addition to Lsm1p, all tested components of the Lsm1p-7p/Pat1p/Dhh1p decapping activator complex, which functions in deadenylation-dependent decapping of cellular mRNAs, are required for BMV RNA recruitment for RNA replication. In contrast, other proteins of the decapping machinery, such as Edc1p and Edc2p from the deadenylation-dependent decapping pathway and Upf1p, Upf2p, and Upf3p from the deadenylation-independent decapping pathway, had no significant effects. The dependence of BMV RNA recruitment on the Lsm1p-7p/Pat1p/Dhh1p complex was linked exclusively to the 3' noncoding region of the BMV RNA. Collectively, our results suggest that the Lsm1p-7p/Pat1p/Dhh1p complex that transfers cellular mRNAs from translation to degradation might act as a key regulator in the switch from BMV RNA translation to replication.

Adenosine Monophosphate↗

PubMed2005

Information catastrophe in RNA viruses through replication thresholds.

RNA viruses are known to replicate at very high mutation rates. These rates are actually known to be close to their so-called error threshold. This threshold is in fact a critical point beyond which genetic information is lost through a so-called error catastrophe. However, the transition from a stable quasispecies to genetic drift and loss of information can also occur by crossing replication thresholds, below some replication rates, the viral population is suddenly unable to survive. Available data from hepatitis C virus population analysis [Mas, A., Ulloa, E., Bruguera, M., Furcić, I., Garriga, D., Fábregas, S., Andreu, D., Saiz, J.C., Díez, J., 2004. Hepatitis C virus population analysis of a single-source nosocomial outbreak reveals an inverse correlation between viral load and quasispecies complexity. J. Gen. Virol. 85, 3619-3626] can be interpreted through this theoretical view, providing evidence for such a replication threshold. Here a simple model is used in order to provide evidence for such a phenomenon, consistent with available data.

Cross Infection↗

PubMed2004

Molecular determinants of multi-nucleoside analogue resistance in HIV-1 reverse transcriptases containing a dipeptide insertion in the fingers subdomain: effect of mutations D67N and T215Y on removal of thymidine nucleotide analogues from blocked DNA primers.

Human immunodeficiency virus type 1 isolates having dipeptide insertions in the fingers subdomain of the reverse transcriptase (RT) show high level resistance to 3 '-azido-3 '-deoxythymidine (AZT) and other nucleoside analogues. Insertions are usually associated with thymidine analogue resistance mutations, such as T215Y. The resistance phenotype correlates with increased ATP-dependent phosphorolytic activity, which facilitates removal of thymidine analogues from inhibitor-terminated primers. In this report, we show that substituting Thr, Ser, or Asn for Tyr-215 in a multidrug-resistant RT, bearing a Ser-Ser insertion between codons 69 and 70, leads to AZT and stavudine resensitization through the loss of the ATP-mediated removal activity. The mutation D67N, which is rarely found in insertion-containing strains, had no effect on excision and a minor influence on resistance. Substituting Tyr-215 had a larger effect than deleting the dipeptide insertion. The presence of both the insertion and mutation T215Y in the wild-type BH10 RT conferred significant ATP-mediated removal activity and moderate resistance to AZT. However, resistance levels and unblocking activities were lower than those observed with the multidrug-resistant enzyme. Removal reactions can be inhibited by the next complementary dNTP. Both Tyr-215 and the dipeptide insertion affect RT-DNA.DNA-dNTP ternary complex formation, an effect that was not detected in the presence of foscarnet. Based on crystal structures of binary and ternary complexes of HIV-1 RT, we propose that Tyr-215 exerts its action by facilitating a proper orientation of the pyrophosphate donor molecule, whereas the effects on dNTP binding are indirect and could be related to significant conformational changes occurring during polymerization.

Adenosine Triphosphate↗

PubMed2004

Long-term suppression of plasma viremia with highly active antiretroviral therapy despite virus evolution and very limited selection of drug-resistant genotypes.

HIV-1 evolution and the possible emergence of mutations associated with resistance to antiretroviral inhibitors have been evaluated in a cohort of sixty-three patients successfully treated with highly active antiretroviral therapy (HAART). The patients under effective HAART were recruited in three different hospitals in Spain, and none of them had been treated (naïve) before entering this study. HIV-1 RNA levels, CD4+, and CD8+ T-cell counts were determined, and nucleotide sequences of proviral regions encoding protease and reverse transcriptase (RT) were obtained for longitudinal blood samples spanning a mean follow-up period of 88 weeks. Phylogenetic reconstructions and calculations of genetic distances among the different sequences of each patient were performed. All except one of the patients under study showed an early and sustained decrease in plasma HIV-1 RNA to levels that were below 200 copies/ml. The plasma viral decline paralleled a significant increase in the CD4+ T-lymphocyte counts. Amino acid sequence analyses revealed the occurrence of mutations associated with antiretroviral resistance in nine patients (14.3%) during HAART treatment, that in some cases could be attributed to excess G to A transitions. In six of the nine patients, the mutations conferred resistance to inhibitors administered in the treatment regime, although the mutations did not result in treatment failure. Sequence comparisons revealed viral evolution during the period of treatment in 47.5% of the patients. The results indicate successful suppression of HIV-1 under HAART for extended time periods, indistinguishable for patients in which evidence of virus evolution could or could not be documented.

Adult↗

PubMed2004

Similar HIV-1 evolution and immunological responses at 10 years despite several therapeutic strategies and host HLA Types.

Two sexual partners infected with related HIV-1 viruses and enrolled in different therapeutic strategies including structured treatment interruptions (STI) provided us an opportunity to compare long term (10 years) viral genetic evolution for closely related isolates in different hosts. HLA loci were molecularly typed and different genetic markers were studied. The viral genetic evolution was studied by sequencing pol and env genes. The HIV-specific CD4+ and CD8+ T cell responses were assessed by the lymphoproliferative response (LPR) and an ELISPOT assay, respectively. HLA class I loci of patients A and B were different and both of them were heterozygous for CCR5delta32 gene. During the two STI studies, viral load of both patients rebounded after treatment interruption and both developed a transitory strong helper and CTL responses. After definitive interruption of therapy, viral load remained below 5,000 copies/ml without therapy during the two years of follow-up. Two patients infected with related viruses showed a similar dynamics of viral evolution and CD4 T cells, despite hosts having a different HLA type and being treated with several therapeutic protocols, after 10 years of infection. These results suggest that, in this case, an effective immunological response to STI depended more on the virus than on the characteristics of the host.

Anti-HIV Agents↗

PubMed2004

Hepatitis C virus population analysis of a single-source nosocomial outbreak reveals an inverse correlation between viral load and quasispecies complexity.

The features of Hepatitis C virus (HCV) quasispecies within an envelope segment including the hypervariable region 1 were analysed at an early time point post-infection in seven patients that acquired HCV from a single common donor during a nosocomial outbreak. The grouping of patients according to viral load was reflected in the structure of the quasispecies. A higher viral load correlated with the presence of a predominant HCV genome and a corresponding lower quasispecies complexity. The quasispecies complexity itself was not correlated with HCV clearance or persistence. Thus, the relationship between an intrapatient HCV quasispecies and the clinical outcome of an HCV infection is more complex than previously anticipated.

Amino Acid Sequence↗

PubMed2002

Multidrug-resistant HIV-1 reverse transcriptase: involvement of ribonucleotide-dependent phosphorolysis in cross-resistance to nucleoside analogue inhibitors.

Human immunodeficiency virus type 1 (HIV-1) strains having a dipeptide insertion between codons 69 and 70 of the viral reverse transcriptase (RT) have been observed in isolates from patients treated with 3'-azido-3'-deoxythymidine (AZT) and other nucleoside analogues. These viruses contain additional mutations related to drug resistance and display reduced susceptibility to most nucleoside analogue inhibitors, including AZT. The mechanism of AZT resistance implies an increased ability of the multidrug-resistant (SS) RT to remove AZT-monophosphate (AZTMP) from blocked primers through a nucleotide-dependent reaction. We show that its higher ATP-dependent phosphorolytic activity is also detectable with primers terminated with 2',3'-didehydro-3'-deoxythymidine-5'-monophosphate (d4TMP) or 2',3'-dideoxythymidine-5'-monophosphate (ddTMP), but is significantly reduced when the dipeptide insertion is deleted. Removal of AZTMP, d4TMP and ddTMP can be inhibited by the next complementary deoxynucleoside triphosphate (dNTP). AZTMP removal reactions catalysed by SS RT were highly resistant to dNTP inhibition (IC(50)>0.25mM), while unblocking of d4TMP- and ddTMP-terminated primers was around tenfold more sensitive to inhibition by the next complementary dNTP. Both SS and mutant 2S0S RTs were able to unblock and extend primers terminated with 2',3'-dideoxycytidine-5'-monophosphate (ddCMP) in the presence of ATP, albeit very poorly. Under these conditions, none of the RTs was able to remove 2',3'-dideoxy-3'-thiacytidine-5'-monophosphate (3TCMP) from a terminated DNA primer. Resistance mediated by ATP-dependent phosphorolysis depends on the intracellular levels of dNTP. High levels as found in transformed cell lines (i.e. H-9, CEM lymphoblasts, SupT1 cells, etc.) may prevent repair of primers terminated with d4TMP. However, ATP-dependent phosphorolysis could be relevant for d4T resistance in cells having low levels of dNTPs. This proposal could explain why insertion-containing HIV-1 variants have been detected in the absence of AZT, during d4T treatment.

Amino Acid Sequence↗

PubMed2002

Insertions in the reverse transcriptase increase both drug resistance and viral fitness in a human immunodeficiency virus type 1 isolate harboring the multi-nucleoside reverse transcriptase inhibitor resistance 69 insertion complex mutation.

Recent studies have shown that the accumulation of multiple mutations associated with nucleoside reverse transcriptase inhibitor (NRTI) resistance may be grouped as multi-NRTI resistance (MNR) complexes. In this study, we have examined the viral fitness of recombinant viruses carrying the reverse transcriptase (RT) of a human immunodeficiency virus type 1 (HIV-1) primary isolate harboring mutations comprising the MNR 69 insertion complex. Different RT mutants were prepared in the sequence context of either the wild-type RT sequence of the HIV-1(BH10) isolate or the sequence found in a clinical HIV-1 isolate with the MNR 69 insertion mutation. As expected, in the presence of zidovudine, recombinant viruses harboring the MNR RT from the patient were more fit than wild-type viruses. However, in the absence of drug, the virus with the RT from the original clinical isolate (SS) was more fit than (i) the wild-type virus with an engineered serine insertion between residues 69 and 70 (T69SSS) and (ii) the recombinant virus with the MNR RT where the insertion was removed (2S0S). These results suggest that RT insertions, in the right sequence context (i.e., additional mutations contained in the MNR 69 insertion complex), enhance NRTI resistance and may improve viral fitness. Thus, comparing complex mutation patterns with viral fitness may help to elucidate the role of uncharacterized drug resistance mutations in antiretroviral treatment failure.

Adult↗