Supporting open science at PLOS Biology.

Open science is key to PLOS Biology's mission, both in its daily operations and in the role we aspire to have in the scholarly ecosystem. Here, we reflect on open science at the journal and discuss how and why we shall continue to hold it central to everything we do.

Promoting research quality.

Plenty of awards recognize scientific contributions, but a unique and important one honors those whose efforts significantly enhance the quality and robustness of research. We discuss why this is important to promote trust in science.

PLOS Biology at 20: Exploring possible futures.

Twenty years ago this month, PLOS Biology was launched, helping to catalyze a movement that has transformed publishing in the life sciences. In this issue, we explore how the community can continue innovating for positive change in the next decades.

PLOS Biology at 20: Ain't no mountain high enough.

PLOS began publishing influential open access science in 2003. As PLOS Biology enters its third decade, we reflect on our mission, what has changed, what remains to be done and our wishes for the future.

Premiering pre-registration at PLOS Biology.

Pre-registration promises to address some of the problems with traditional peer-review. As we publish our first Registered Report, we take stock of two years of submissions and the future possibilities of this approach.

The antimicrobial resistance crisis needs action now.

Antimicrobial resistance is a global problem of increasing proportions that we cannot afford to look away from. This World Antimicrobial Awareness Week, we shine a light on the crisis and ways we can all help to address it.

We need leaders that believe in scientific evidence.

In a world beset by attempts to undermine scientific evidence and evidence-based policy, we emphasize their important role in helping humanity rise to the challenges of our time.

In the family with ubiquitin.

The Cold Spring Harbor meeting on 'The Ubiquitin Family', held in May 2011, brought together scientists from a wide range of fields under the umbrella of ubiquitin and ubiquitin-like protein structure, function and regulation.

Inhibition of HIV-1 infection by a unique short hairpin RNA to chemokine receptor 5 delivered into macrophages through hematopoietic progenitor cell transduction.

BACKGROUND: We recently expressed a potent and noncytotoxic short hairpin (sh)RNA directed against chemokine (c-c motif) receptor 5 (CCR5) using lentiviral mediated transduction of CD34+ hematopoietic progenitor cells (HPCs) and demonstrated the stable reduction of CCR5 expression in T-lymphocytes. METHODS: In the present study, we further assessed the activity of the shRNA through HPC transduction and differentiation into macrophages derived from fetal liver CD34+ (FL-CD34+) HPCs. Transduced lentiviral vector encoding the human CCR5 shRNA was stably maintained in FL-CD34+ cells and in the terminally differentiated macrophages using macrophage colony-stimulating factor, granulocyte macrophage colony-stimulating factor, interleukin-3 and stem cell factor. RESULTS: Quantitative real-time polymerase chain reaction for CCR5 mRNA indicated over 90% reduction of CCR5 mRNA levels in CCR5 shRNA-transduced population. The cells with knockdown of CCR5 expression acquired resistance to R5 tropic HIV-1 NFN-SX strain. We also developed a novel approach utilizing a mCherry-CCR5 chimeric reporter to assess the effectiveness of CCR5 target down-regulation in macrophages directly. Both the shRNA and the reporter were maintained throughout HPC differentiation to macrophages without apparent cytotoxicity. CONCLUSIONS: The present study demonstrates a novel method to simply and directly assess the function of small interfering RNA and the effective inhibition of HIV-1 infection by a potential potent shRNA to CCR5 delivered into macrophages derived from HPCs.

Targeted transduction via CD4 by a lentiviral vector uses a clathrin-mediated entry pathway.

We recently developed a novel targeting Sindbis virus envelope pseudotyped lentiviral vector, 2.2ZZ, which acquires specific transduction capacity by antibody conjugation and binding with specific antigens on the surface of targeted cells. Here we characterize the virological properties of this vector by examining its targeting to CD4 antigen. Our results show that entry is dependent on CD4 cell surface density and occurs via the clathrin-mediated endocytic pathway. These findings provide insight into the mechanism of infection by a new viral vector with combined properties of Sindbis virus and lentiviruses and infectivity conferred by monoclonal antibody-ligand interactions.

Targeted transduction of CD34+ hematopoietic progenitor cells in nonpurified human mobilized peripheral blood mononuclear cells.

BACKGROUND: Conventional gene-therapy applications of hematopoietic stem cells (HSCs) involve purification of CD34+ progenitor cells from the mobilized peripheral blood, ex vivo transduction of the gene of interest into them, and reinfusion of the transduced CD34+ progenitor cells into patients. Eliminating the process of purification would save labor, time and money, while enhancing HSCs viability, transplantability and pluripotency. Lentiviral vectors have been widely used in gene therapy because they infect both dividing and nondividing cells and provide sustained transgene expression. One of the exceptions to this rule is quiescent primary lymphocytes, in which reverse transcription of viral DNA is not completed. METHODS: In the present study, we tested the possibility of targeting CD34+ progenitor cells within nonpurified human mobilized peripheral blood mononuclear cells (mPBMCs) utilizing vesicular stomatitis virus G (VSV-G) pseudotyped lentiviral vectors, based on the assumption that the CD34+ progenitor cells would be preferentially transduced. To further enhance the specificity of vector transduction, we also examined utilizing a modified Sindbis virus envelope (2.2) pseudotyped lentiviral vector, developed in our laboratory, that allows targeted transduction to specific cell receptors via antibody recognition. RESULTS: Both the VSV-G and 2.2 pseudotyped vectors achieved measurable results when they were used to target CD34+ progenitor cells in nonpurified mPBMCs. CONCLUSIONS: Overall, the data obtained demonstrate the potential of ex vivo targeting of CD34+ progenitor cells without purification.

Redirecting lentiviral vectors by insertion of integrin-tageting peptides into envelope proteins.

BACKGROUND: Targeting gene therapy vectors that can home in on desired cell and tissue types in vivo comprise the ultimate gene delivery system. We have previously developed targeting lentiviral vectors by pseudotyping vectors with modified Sindbis virus envelope proteins. The envelope protein contains the Fc-binding region of protein A (ZZ domain), so the virus can be conjugated with antibodies. The conjugated antibody mediates specific transduction of the cells and tissues expressing the target antigens, both in vitro and in vivo. However, more stable conjugation of targeting molecules would be optimal for use in immunocompetent animals, as well as in humans. METHODS: We inserted integrin-targeting peptides into two sites of the targeting envelope proteins and determined whether the peptides serve as receptor-binding regions of the envelope proteins and redirect the pseudotyped viruses. RESULTS: The integrin-targeting peptides can mediate binding to cells via the interaction with integrins on target cells and transduction. Peptides with a higher binding affinity increase titers of pseudotyped virus. We found two regions on the envelope protein that can accommodate insertion and serve as receptor-binding regions. Combining the peptides in two distinct regions increased the titers of the virus. CONCLUSIONS: Successful incorporation of targeting molecules into the envelope protein will broaden the application of targeting vectors for a wide variety of experimental and clinical settings.

Molecular basis for a lack of correlation between viral fitness and cell killing capacity.

The relationship between parasite fitness and virulence has been the object of experimental and theoretical studies often with conflicting conclusions. Here, we provide direct experimental evidence that viral fitness and virulence, both measured in the same biological environment provided by host cells in culture, can be two unrelated traits. A biological clone of foot-and-mouth disease virus acquired high fitness and virulence (cell killing capacity) upon large population passages in cell culture. However, subsequent plaque-to-plaque transfers resulted in profound fitness loss, but only a minimal decrease of virulence. While fitness-decreasing mutations have been mapped throughout the genome, virulence determinants-studied here with mutant and chimeric viruses-were multigenic, but concentrated on some genomic regions. Therefore, we propose a model in which viral virulence is more robust to mutation than viral fitness. As a consequence, depending on the passage regime, viral fitness and virulence can follow different evolutionary trajectories. This lack of correlation is relevant to current models of attenuation and virulence in that virus de-adaptation need not entail a decrease of virulence.

The quest for quasispecies.

This month marks 40 years since the publication of 'Nucleotide sequence heterogeneity of an RNA phage population' in Cell. We spoke with Esteban Domingo, leading author of this landmark study carried out during his postdoctoral work in Charles Weissman's lab, which proposed RNA viral populations to be quasispecies.

Action of mutagenic agents and antiviral inhibitors on foot-and-mouth disease virus.

Our current knowledge on foot-and-mouth disease virus (FMDV) entry into error catastrophe is reviewed. FMDV can establish cytolytic and persistent infections in the field and in cell culture. Both types of FMDV infection in cell culture can be treated with mutagens, with or without classical (non-mutagenic) antiviral inhibitors, to drive the virus to extinction. 5-Fluorouracil (FU) and 5-azacytidine (AZC) have been employed as mutagenic agents to treat cytolytic FMDV infections, and ribavirin (Rib) to treat persistent infections. Extinction is dependent on the relative fitness of the viral isolate, as well as on the viral load. In cytolytic infections, extinctions could be efficiently obtained with combinations of mutagens and inhibitors. High-fitness FMDV extinction could only be achieved with treatments that contained a mutagen, and not with combinations of inhibitors that exerted the same antiviral effect. Persistent infections could be cured with Rib treatment alone. The results presented here show entry into error catastrophe as a valid strategy for treatment of viral infections, although much work remains to be done before it can be implemented.