Antonio Mas

Unfinished Stories on Viral Quasispecies and Darwinian Views of Evolution

Experimental evidence that RNA virus populations consist of distributions of mutant genomes, termed quasispecies, was first published 31 years ago. This work provided the earliest experimental support for a theory to explain a system that replicated with limited fidelity and to understand the self-organization and adaptability of early life forms on Earth. High mutation rates and quasispecies dynamics of RNA viruses are intimately related to both viral disease and antiviral treatment strategies. Moreover, the quasispecies concept is being applied to other biological systems such as cancer research in which cellular mutant spectra can be also detected. This review addresses some of the unanswered questions regarding viral and theoretical quasispecies concepts as well as more practical aspects concerning resistance to antiviral treatments and pathogenesis.

Host Deadenylation-Dependent mRNA Decapping Factors Are Required for a Key Step in Brome Mosaic Virus RNA Replication

ABSTRACT 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.

Quasispecies as a matter of fact: Viruses and beyond

Abstract 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.

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

ABSTRACT 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-1BH10 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.

De Novo Polymerase Activity and Oligomerization of Hepatitis C Virus RNA-Dependent RNA-Polymerases from Genotypes 1 to 5

Hepatitis C virus (HCV) shows a great geographical diversity reflected in the high number of circulating genotypes and subtypes. The response to HCV treatment is genotype specific, with the predominant genotype 1 showing the lowest rate of sustained virological response. Virally encoded enzymes are candidate targets for intervention. In particular, promising antiviral molecules are being developed to target the viral NS3/4A protease and NS5B polymerase. Most of the studies with the NS5B polymerase have been done with genotypes 1b and 2a, whilst information about other genotypes is scarce. Here, we have characterized the de novo activity of NS5B from genotypes 1 to 5, with emphasis on conditions for optimum activity and kinetic constants. Polymerase cooperativity was determined by calculating the Hill coefficient and oligomerization through a new FRET-based method. The Vmax/Km ratios were statistically different between genotype 1 and the other genotypes (p<0.001), mainly due to differences in Vmax values, but differences in the Hill coefficient and NS5B oligomerization were noted. Analysis of sequence changes among the studied polymerases and crystal structures show the αF helix as a structural component probably involved in NS5B-NS5B interactions. The viability of the interaction of αF and αT helixes was confirmed by docking studies and calculation of electrostatic surface potentials for genotype 1 and point mutants corresponding to mutations from different genotypes. Results presented in this study reveal the existence of genotypic differences in NS5B de novo activity and oligomerization. Furthermore, these results allow us to define two regions, one consisting of residues Glu128, Asp129, and Glu248, and the other consisting of residues of αT helix possibly involved in NS5B-NS5B interactions.

Highly heterogeneous mutation rates in the hepatitis C virus genome.

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.

Xenopus Xp54 and Human RCK/p54 Helicases Functionally Replace Yeast Dhh1p in Brome Mosaic Virus RNA Replication

ABSTRACT By using a Brome mosaic virus (BMV)-Saccharomyces cerevisiae system, we previously showed that the cellular Lsm1p-7p/Pat1p/Dhh1p decapping-activator complex functions in BMV RNA translation and replication. As a first approach in investigating whether the corresponding human homologues play a similar role, we expressed human Lsm1p (hLsm1p) and RCK/p54 in yeast. Expression of RCK/p54 but not hLsm1p restored the defect in BMV RNA translation and replication observed in the dhh1Δ and lsm1Δ strains, respectively. This functional conservation, together with the common replication strategies of positive-stranded RNA viruses, suggests that RCK/p54 may also play a role in the replication of positive-stranded RNA viruses that infect humans.

Phylogeny of HIV type 1 group O isolates based on env gene sequences.

769 H UM A N IM M UNOD EFIC IEN CY VIRU S TY PE 1 (HIV-1) can be classified in two major groups, M (main) and O (outlier), although the emergence of a new cluster of equidistant isolates, termed group N, has been reported. 1 Whereas group M can be subdivided into 10 different subtypes (A±J), a possible distinction of clades within group O has been limited by the sequence information available in the current database. A subtype A-O has been proposed for a group of 11 HIV-1 group O isolates (ANT70, BCF01, BCF02, BCF03, BCF07, BCF08, BCF13, ESP1, ESP2, ESP3, and ESP4) on the basis of sequences belonging to the reverse transcriptase gene, whereas the other isolates analyzed (MVP5180, VAU, RUD, BCF06, and BCF11) did not form a defined cluster. Since its first description in 1990, HIV-1 group O infection has been reported in West Africa, Europe, and the United States, almost exclusively in native Cameroonians and their contacts. In this sequence note we describe env gene sequences corresponding to the C2V3-coding region of gp120 and to the immunodominant region of gp41. The sequences belong to four HIV-1 group O-infected patients identified in Spain up to November 1998. The sequences and a phylogenetic tree derived from all available sequences of these genomic regions support the existence of a defined subtype, termed A-O, within this highly heterogeneous group of viruses. Isolates ESP1, ESP2, ESP3, and ESP4 were obtained from patients at Hospital Carlos III in Madrid, Spain. ESP1 and ESP2 are from a heterosexual couple diagnosed with HIV-1 infection in 1995. ESP3 and ESP4 are from a 48-year-old man and a vertically infected 14-year-old boy, respectively, who were diagnosed with HIV-1 group O infection in 1997. Their main clinical and epidemiologica l features have been described elsewhere, and are summarized in Table 1. Peripheral blood mononuclear cells (PBMCs) were collected from these individuals using standard protocols. Proviral DNA was obtained from cryopreserved PBMCs (1-10 3 10 cells) by proteinase K digestion, phenol extraction and ethanol precipitation. DNA amplification of the C2V3-coding region of the env gene was carried out using a nested-PCR strategy as previously described. The gp41 immunodom inant coding region was amplified by nested PCR using two sets of primer pairs. The outer primers were gp41A (5 9 -CAACT ACAAA GTAGT AAGGG3 9 ), and gp41B (5 9 -CTACT AGTGC TCCTA CTATG-3 9 ), whose 5 9 -ends correspond to positions 7718 and 8352, respectively, according to the ANT70 isolate (GenBank accession number L20587). The inner primers were gp41C (5 9 -CAGTA GGATT GGGAA TGCTA-3 9 ), and gp41D (5 9 -CCATT TAGTT ATGTC AAGCC-3 9 ), whose 5 9 -ends correspond to positions 7813 and 8306, respectively, according to the ANT70 isolate. Consensus nucleotide sequences between positions 6849 and 7253, and 7814 and 8267 (numbering according to the HIV-1 ANT70 isolate), which correspond to the C2V3-coding and gp41-imm unodom inant regions, respectively, were determined as previously described. 11 Nucleotide sequences were compiled and translated by SEQED and TRANSLATE programs from the GCG package. 13 Multiple sequence alignments were carried out using the CLUSTALW program, 14 and sequence and phylogenetic analyses were performed using the PHYLIP software package version 3.5. Nucleotide sequence data from the C2V3-coding region and the gp41 immunodominant region reported in this sequence note have been deposited in the GenBank nucleotide sequence database (accession numbers AF081811 to AF081817). We have compared the sequences encoding the immunodominant region of gp41 determ ined for isolates ESP1, ESP2, ESP3, and ESP4 with sequences of the corresponding region of other representative HIV-1 group O isolates available in the current database. The comparison included isolates ANT70, MVP5180, VAU, vi686, and DUR (GenBank accession num-

Monitoring hepatitis C virus (HCV) RNA-dependent RNA polymerase oligomerization by a FRET-based in vitro system.

Hepatitis C virus (HCV) is a positive-strand RNA virus ((+)RNA) that replicates its genome in replication complexes (RC) associated to endoplasmic reticulum (ER)-derived micro-vesicles. One key protein in these complexes is NS5B, a viral enzyme that shows the RNA binding and RNA-dependent RNA polymerase (RdRp) activities. For this reason, NS5B protein has become one of the most important targets for designing new antiviral therapy compounds. Recently, it has been demonstrated that NS5B interacts itself forming oligomers, and mutations that disrupt these interactions are lethal for polymerase function. Therefore, NS5B oligomerization could be a new target for the design of anti-HCV compounds. In this study we describe a new accurate method to analyze NS5B-NS5B interactions by using Förster-resonance-energy transfer (FRET). This method allows analyses of the conditions, mainly ionic strength, driving the interactions between NS5B-cyan and NS5B-citrine constructs. Experiments using different combinations of point mutants rendered FRET values from zero to around 100%, suggesting the geometry of the interaction. Finally, oligomerization experiments in the presence of non-nucleoside inhibitor (NNI) PF-254027 gave a statistically significant reduction in the FRET signal, suggesting a new connection between NS5B oligomerization and NNI binding.

Hepatitis E Virus in Industrialized Countries: The Silent Threat.

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.