The 3¢-azido-3¢-deoxythymidine (AZT)-resistant phenotype of a heavily mutated human immunode®ciency virus type 1 (HIV-1) reverse transcriptase (RT) carrying a dipeptide (Ser-Ser) insertion between codons 69 and 70 as well as other mutations related to resistance to RT inhibitors has been studied. Recombinant virus carrying this variant RT (termed SS RT) showed reduced susceptibility to all nucleoside RT inhibitors in clinical use, particularly to AZT. In the presence of ATP, recombinant SS RT had an increased ability to remove the 3¢-terminal nucleotide from AZTterminated primers and extend the unblocked primer, compared with wild-type HIV-1 RT (BH10 isolate). Insertion of two serines in the sequence context of BH10 RT did not affect the ATP-dependent phosphorolytic activity of the enzyme, and had no in¯uence in resistance to RT inhibitors. However, SS RT mutants lacking the dipeptide insertion or bearing a four-serine insertion showed reduced ATP-dependent phosphorolytic activity that correlated with increased AZT sensitivity, as determined using a recombinant virus assay. Therefore, the insertion appears to be critical to enhance AZT resistance in the sequence context of multidrug-resistant HIV-1 RT. Keywords: ATP-dependent phosphorolysis/AZT resistance/HIV-1/pyrophosphorolysis/reverse transcriptase IntroductionHuman immunode®ciency virus type 1 (HIV-1) reverse transcriptase (RT) is a heterodimeric enzyme composed of two subunits of 66 and 51 kDa, respectively, which is required to convert the viral genomic RNA into double-stranded DNA that then integrates into the host cell genome. HIV-1 RT is an important target for antiretroviral therapy (for recent reviews see Balzarini, 1999;Jonckheere et al., 2000). Several nucleoside analog RT inhibitors have been approved for clinical treatment of HIV-1-infected patients: 3¢-azido-3¢de-oxythymidine (AZT; zidovudine), 2¢,3¢dideoxyinosine (ddI; didanosine), 2¢,3¢-dideoxycytidine (ddC; zalcitabine), 2¢,3¢-dideoxy-3¢-thiacytidine (3TC; lamivudine), 2¢,3¢-didehydro-2¢,3¢-dideoxythymidine (d4T; stavudine) and a dideoxyguanosine derivative (abacavir).Despite being the ®rst anti-HIV drug to be approved, AZT is still widely used in the clinical setting in combination with other antiretroviral drugs. Its mode of action involves its conversion to AZT triphosphate (AZTTP) in a process mediated by cellular kinases. AZTTP is a competitive inhibitor of RT and once incorporated into DNA it brings about chain termination due to its lack of a 3¢-hydroxyl group. Prolonged clinical use of AZT monotherapy invariably results in the appearance of virus resistant to the drug. High-level resistance to AZT is correlated with multiple mutations in the RT, including D67N, K70R, T215F or T215Y and K219Q, and in some cases, M41L and L210W. Virus having the quadruple mutant of RT (D67N, K70R, T215F/Y and K219Q) is >100-fold less sensitive to AZT than the wildtype (WT) virus in cell culture studies (Kellam et al., 1992). The biochemical mechanism of resistance has been dif®cult to investigate because...
Spontaneous mutations are the ultimate source of genetic variation and have a prominent role in evolution. RNA viruses such as hepatitis C virus (HCV) have extremely high mutation rates, but these rates have been inferred from a minute fraction of genome sites, limiting our view of how RNA viruses create diversity. Here, by applying high-fidelity ultradeep sequencing to a modified replicon system, we scored >15,000 spontaneous mutations, encompassing more than 90% of the HCV genome. This revealed >1,000-fold differences in mutability across genome sites, with extreme variations even between adjacent nucleotides. We identify base composition, the presence of high- and low-mutation clusters and transition/transversion biases as the main factors driving this heterogeneity. Furthermore, we find that mutability correlates with the ability of HCV to diversify in patients. These data provide a site-wise baseline for interrogating natural selection, genetic load and evolvability in HCV, as well as for evaluating drug resistance and immune evasion risks.
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.Positive-strand RNA [(ϩ)RNA] viruses include important plant, animal, and human pathogens such as the severe acute respiratory syndrome coronavirus and hepatitis C virus. This large group of viruses replicate in the cytoplasm through negative-strand intermediates and share some fundamental features in their replication processes. A key common feature is the function of (ϩ)RNA virus genomes as templates for both translation and replication. In contrast to other virus groups, (ϩ)RNA viruses do not encapsidate viral polymerases required for viral replication, so upon virus entry into the cell, the genomic RNA must first be translated to produce viral replication factors. These replication factors then specifically recognize the viral RNA and recruit it from translation into the RNA replication complex. These two genomic RNA functions are mutually exclusive because 5Ј-to-3Ј ribosome trafficking blocks 3Ј-to-5Ј polymerase copying of viral (ϩ)RNA (5, 17). Therefore, the switch from genomic RNA translation to replication must be highly regulated to allow sufficient translation but also efficient replication. The molecular features underlying this regulation are poorly understood. Other important common features in the replication of (ϩ)RNA viruses are the assembly of replication complexes on intracellular membranes (31) and the requirement for host factors in multiple steps of the replication process (2). The identification of such host factors is important for a better understanding of fundamental issues in (ϩ)RNA ...
Hepatitis E virus (HEV) is the main cause of acute viral hepatitis worldwide. Its presence in developing countries has been documented for decades. Developed countries were supposed to be virus-free and initially only imported cases were detected in those areas. However, sporadic and autochthonous cases of HEV infection have been identified and studies reveal that the virus is worldwide spread. Chronic hepatitis and multiple extrahepatic manifestations have also been associated with HEV. We review the data from European countries, where human, animal, and environmental data have been collected since the 90s. In Europe, autochthonous HEV strains were first detected in the late 90s and early 2000s. Since then, serological data have shown that the virus infects quite frequently the European population and that some species, such as pigs, wild boars, and deer, are reservoirs. HEV strains can be isolated from environmental samples and reach the food chain, as shown by the detection of the virus in mussels and in contaminated pork products as sausages or meat. All these data highlight the need of studies directed to control the sources of HEV to protect immunocompromised individuals that seem the weakest link of the HEV epidemiology in industrialized regions.
We review the origins of the quasispecies concept and its relevance for RNA virus evolution, viral pathogenesis and antiviral treatment strategies. We emphasize a critical point of quasispecies that refers to genome collectivities as the unit of selection, and establish parallels between RNA viruses and some cellular systems such as bacteria and tumor cells. We refer also to tantalizing new observations that suggest quasispecies behavior in prions, perhaps as a result of the same quantum-mechanical indeterminations that underlie protein conformation and error-prone replication in genetic systems. If substantiated, these observations with prions could lead to new research on the structure-function relationship of non-nucleic acid biological molecules.
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-DNA⅐DNA-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.Human immunodeficiency virus type 1 (HIV-1) 1 reverse transcriptase (RT) replicates the viral genomic RNA to synthesize a double-stranded DNA that integrates into the host genome. The viral enzyme is multifunctional, possessing RNAand DNA-dependent DNA polymerase, RNase H, strand transfer, and strand displacement activities (1). HIV-1 RT is a heterodimeric enzyme composed of two polypeptide chains of 66 and 51 kDa, with subdomains termed fingers, thumb, palm, and connection in both subunits and an RNase H domain in the larger subunit only.HIV-1 RT is an important target for chemotherapeutic intervention in the control of AIDS. Antiretroviral drugs targeting the viral polymerase include nucleoside analogue inhibitors (NRTIs), acyclic nucleoside phosphonates, and non-nucleoside RT inhibitors (NNRTIs) (reviewed in Ref. 2). Inside the cell, nucleoside derivatives are converted to their active triphosphate forms by host cell kinases and are then incorporated into the HIV-1 genome by the viral RT. Because nucleoside analogues lack a 3Ј-OH group, their incorporation blocks elongation of the growing DNA chain. On the other hand, NNRTIs bind to an allosteric site located 10 -15 Å away from the polymerase active site, distorting the geometry and/or the mobility...
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