Luis Menéndez-Arias

Viral quasispecies and the problem of vaccine-escape and drug-resistant mutants

Since a first version of this article on the relevance of quasispecies to viral disease control was published by one of us [1], an explosion of information on viral quasispecies has been gathered by several groups. For many viruses, extreme complexity at the population level has been documented by direct copying into cDNA of viral RNA extracted from biological specimens using reverse transcriptase and amplification by thermostable polymerases (RT-PCR).Two systems relevant to human health for which extensive population heterogeneity has been revealed are hepatitis C virus (HCV) [2-4] and the human immunodeficiency viruses (HIV) [5-7, and references therein]. Particularly dramatic has been the inability to produce effective vaccines against any of these viruses [8-11] in spite of an urgent need to stop the progression of the AIDS pandemic. Also, the systematic selection of HIV-1 mutants resistant to antiretroviral inhibitors [12-14] has greatly limited the efficacy of antiretroviral treatments until the recent encouraging results with combination therapy.

Mutation Rates and Intrinsic Fidelity of Retroviral Reverse Transcriptases

Retroviruses are RNA viruses that replicate through a DNA intermediate, in a process catalyzed by the viral reverse transcriptase (RT). Although cellular polymerases and host factors contribute to retroviral mutagenesis, the RT errors play a major role in retroviral mutation. RT mutations that affect the accuracy of the viral polymerase have been identified by in vitro analysis of the fidelity of DNA synthesis, by using enzymological (gel-based) and genetic assays (e.g., M13mp2 lacZ forward mutation assays). For several amino acid substitutions, these observations have been confirmed in cell culture using viral vectors. This review provides an update on studies leading to the identification of the major components of the fidelity center in retroviral RTs.

Viral Quasispecies and Fitness Variations

Publisher Summary The quasispecies model of molecular evolution was initially proposed to describe the error-prone replication, self-organization, and adaptability of primitive replicons such as those thought to have populated the earth some 4000 million years ago. Error-prone replication has been maintained as a stable trait in present-day RNA viruses. One of the critical features that distinguish cells from viruses is the extreme difference in the complexity of their genetic material, even after accounting for repeated DNA in animal and plant cells. The dynamic mutant distributions that compose replicating RNA viruses are termed viral quasispecies. When the relative fitness of the evolving quasispecies reaches a high value, even quite large population sizes can constitute an effective bottleneck and prevent continuing fitness increase. The experiments on fitness variation of viruses in cell culture have been instrumental in defining some basic influences presaging fitness evolution of viral quasispecies. However, in their replication in a natural setting, viruses encounter multiple changing environments and often have to cope with conflicting selective constraints. Even in a relatively constant biological and physical environment, such as vitro cell culture systems, the degree of adaptation of viral quasispecies may undergo remarkable quantitative variations.

Virus population dynamics, fitness variations and the control of viral disease: an update

Viral quasispecies dynamics and variations of viral fitness are reviewed in connection with viral disease control. Emphasis is put on resistance of human immunodeficiency virus and some human DNA viruses to antiviral inhibitors. Future trends in multiple target antiviral therapy and new approaches based on virus entry into error catastrophe (extinction mutagenesis) are discussed.

Effect of the Human Immunodeficiency Virus Type 1 Reverse Transcriptase Polymorphism Leu-214 on Replication Capacity and Drug Susceptibility

ABSTRACT A negative association between polymorphism Leu-214 and type-1 thymidine analogue mutations (TAM1) and a positive association with a clinically favorable virological response to thymidine analogue-based combination antiretroviral therapy have been described. In this study, the impact of Leu-214 on replication capacity and resistance to zidovudine (ZDV) of viruses containing TAM1 or TAM2 was determined. Leu-214 decreased the growth rate of viruses bearing Tyr-215, as well as their resistance to ZDV. This observation was confirmed by structural and molecular modeling data, suggesting a regulatory role for Leu-214 in the emergence and phenotypic resistance of TAM1.

HIV-1 Adapts To Replicate in Cells Expressing Common Marmoset APOBEC3G and BST2

ABSTRACT Previous studies have shown that a major block to HIV-1 replication in common marmosets operates at the level of viral entry and that this block can be overcome by adaptation of the virus in tissue-cultured cells. However, our current studies indicate that HIV-1 encounters additional postentry blocks in common marmoset peripheral blood mononuclear cells. Here, we show that the common marmoset APOBEC3G (A3G) and BST2 proteins block HIV-1 in cell cultures. Using a directed-evolution method that takes advantage of the natural ability of HIV-1 to mutate during replication, we have been able to overcome these blocks in tissue-cultured cells. In the adapted viruses, specific changes were observed in gag, vif, env, and nef. The contribution of these changes to virus replication in the presence of the A3G and BST2 restriction factors was studied. We found that certain amino acid changes in Vif and Env that arise during adaptation to marmoset A3G and BST2 allow the virus to replicate in the presence of these restriction factors. The changes in Vif reduce expression levels and encapsidation of marmoset APOBEC3G, while the changes in Env increase viral fitness and discretely favor cell-to-cell transmission of the virus, allowing viral escape from these restriction factors. IMPORTANCE HIV-1 can infect only humans and chimpanzees. The main reason for this narrow tropism is the presence in many species of dominant-acting factors, known as restriction factors, that block viral replication in a species-specific way. We have been exploring the blocks to HIV-1 in common marmosets, with the ultimate goal of developing a new animal model of HIV-1 infection in these monkeys. In this study, we observed that common marmoset APOBEC3G and BST2, two known restriction factors, are able to block HIV-1 in cell cultures. We have adapted HIV-1 to replicate in the presence of these restriction factors and have characterized the mechanisms of escape. These studies can help in the development of a novel animal model for in vivo infection of marmosets with HIV-1-like viruses.

Special issue: retroviral enzymes.

The retroviral RNA genome encodes for three enzymes essential for virus replication: (i) the viral protease (PR), that converts the immature virion into a mature virus through the cleavage of precursor polypeptides; (ii) the reverse transcriptase (RT), responsible for the conversion of the single-stranded genomic RNA into double-stranded proviral DNA; and (iii) the integrase (IN) that inserts the proviral DNA into the host cell genome. All of them are important targets for therapeutic intervention. This Special Issue provides authoritative reviews on the most recent research towards a better understanding of structure-function relationships in retroviral enzymes. The Issue includes three reviews on retroviral PRs, seven on RT and reverse transcription, and four dedicated to viral integration. [...]

CHAPTER 4 – Viral Quasispecies: Dynamics, Interactions, and Pathogenesis*

Quasispecies theory is providing a solid, evolving conceptual framework for insights into virus population dynamics, adaptive potential, and response to lethal mutagenesis. The complexity of mutant spectra can influence disease progression and viral pathogenesis, as demonstrated using virus variants selected for increased replicative fidelity. Complementation and interference exerted among components of a viral quasispecies can either reinforce or limit the replicative capacity and disease potential of the ensemble. In particular, a progressive enrichment of a replicating mutant spectrum with interfering mutant genomes prompted by enhanced mutagenesis may be a key event in the sharp transition of virus populations into error catastrophe that leads to virus extinction. Fitness variations are influenced by the passage regimes to which viral populations are subjected, notably average fitness decreases upon repeated bottleneck events and fitness gains upon competitive optimization of large viral populations. Evolving viral quasispecies respond to selective constraints by replication of subpopulations of variant genomes that display higher fitness than the parental population in the presence of the selective constraint. This has been profusely documented with fitness effects of mutations associated with resistance of pathogenic viruses to antiviral agents. In particular, selection of HIV-1 mutants resistant to one or multiple antiretroviral inhibitors, and the compensatory effect of mutations in the same genome, offers a compendium of the molecular intricacies that a virus can exploit for its survival. This chapter reviews the basic principles of quasispecies dynamics as they can serve to explain the behavior of viruses.n ABSTRACTn n n Quasispecies theory is providing a solid, evolving conceptual framework for insights into virus population dynamics, adaptive potential, and response to lethal mutagenesis. The complexity of mutant spectra can influence disease progression and viral pathogenesis, as demonstrated using virus variants selected for increased replicative fidelity. Complementation and interference exerted among components of a viral quasispecies can either reinforce or limit the replicative capacity and disease potential of the ensemble. In particular, a progressive enrichment of a replicating mutant spectrum with interfering mutant genomes prompted by enhanced mutagenesis may be a key event in the sharp transition of virus populations into error catastrophe that leads to virus extinction. Fitness variations are influenced by the passage regimes to which viral populations are subjected, notably average fitness decreases upon repeated bottleneck events and fitness gains upon competitive optimization of large viral populations. Evolving viral quasispecies respond to selective constraints by replication of subpopulations of variant genomes that display higher fitness than the parental population in the presence of the selective constraint. This has been profusely documented with fitness effects of mutations associated with resistance of pathogenic viruses to antiviral agents. In particular, selection of HIV-1 mutants resistant to one or multiple antiretroviral inhibitors, and the compensatory effect of mutations in the same genome, offers a compendium of the molecular intricacies that a virus can exploit for its survival. This chapter reviews the basic principles of quasispecies dynamics as they can serve to explain the behavior of viruses.n n

Viral Quasispecies: Dynamics, Interactions, and Pathogenesis**Dedicated to Manfred Eigen on the occasion of his 80th birthday, for the insights that his pioneer studies have represented for virology.

Quasispecies theory is providing a solid, evolving conceptual framework for insights into virus population dynamics, adaptive potential, and response to lethal mutagenesis. The complexity of mutant spectra can influence disease progression and viral pathogenesis, as demonstrated using virus variants selected for increased replicative fidelity. Complementation and interference exerted among components of a viral quasispecies can either reinforce or limit the replicative capacity and disease potential of the ensemble. In particular, a progressive enrichment of a replicating mutant spectrum with interfering mutant genomes prompted by enhanced mutagenesis may be a key event in the sharp transition of virus populations into error catastrophe that leads to virus extinction. Fitness variations are influenced by the passage regimes to which viral populations are subjected, notably average fitness decreases upon repeated bottleneck events and fitness gains upon competitive optimization of large viral populations. Evolving viral quasispecies respond to selective constraints by replication of subpopulations of variant genomes that display higher fitness than the parental population in the presence of the selective constraint. This has been profusely documented with fitness effects of mutations associated with resistance of pathogenic viruses to antiviral agents. In particular, selection of HIV-1 mutants resistant to one or multiple antiretroviral inhibitors, and the compensatory effect of mutations in the same genome, offers a compendium of the molecular intricacies that a virus can exploit for its survival. This chapter reviews the basic principles of quasispecies dynamics as they can serve to explain the behavior of viruses.n ABSTRACTn n n Quasispecies theory is providing a solid, evolving conceptual framework for insights into virus population dynamics, adaptive potential, and response to lethal mutagenesis. The complexity of mutant spectra can influence disease progression and viral pathogenesis, as demonstrated using virus variants selected for increased replicative fidelity. Complementation and interference exerted among components of a viral quasispecies can either reinforce or limit the replicative capacity and disease potential of the ensemble. In particular, a progressive enrichment of a replicating mutant spectrum with interfering mutant genomes prompted by enhanced mutagenesis may be a key event in the sharp transition of virus populations into error catastrophe that leads to virus extinction. Fitness variations are influenced by the passage regimes to which viral populations are subjected, notably average fitness decreases upon repeated bottleneck events and fitness gains upon competitive optimization of large viral populations. Evolving viral quasispecies respond to selective constraints by replication of subpopulations of variant genomes that display higher fitness than the parental population in the presence of the selective constraint. This has been profusely documented with fitness effects of mutations associated with resistance of pathogenic viruses to antiviral agents. In particular, selection of HIV-1 mutants resistant to one or multiple antiretroviral inhibitors, and the compensatory effect of mutations in the same genome, offers a compendium of the molecular intricacies that a virus can exploit for its survival. This chapter reviews the basic principles of quasispecies dynamics as they can serve to explain the behavior of viruses.n n

Ritonavir-boosted darunavir: a powerful option for treatment-experienced HIV-1-infected patients

Darunavir is a HIV protease inhibitor with potent activity inxa0vitro against a broad range of HIV-1 strains and isolates containing multiple protease inhibitor resistance-associated mutations. Its bioavailability increases when co-administered with low-dose ritonavir, or if taken with a meal. Darunavir (in combination with ritonavir) has been approved for treatment of antiretroviral drug-experienced patients with limited therapeutic options. Clinical trials demonstrated significant efficacy with darunavir/ritonavir 600/100xa0mg twice daily plus optimized background regimens, with sustained response after 48xa0weeks and no major safety and tolerability concerns. Clinical trials assessing its efficacy in earlier treatment failure have been favorable, however, its role in the treatment of naive patients has not yet been defined.