The HIV/SIV envelope glycoprotein (Env) cytoplasmic domain contains a highly conserved Tyr-based trafficking signal that mediates both clathrin-dependent endocytosis and polarized sorting. Despite extensive analysis, the role of these functions in viral infection and pathogenesis is unclear. An SIV molecular clone (SIVmac239) in which this signal is inactivated by deletion of Gly-720 and Tyr-721 (SIVmac239ΔGY), replicates acutely to high levels in pigtail macaques (PTM) but is rapidly controlled. However, we previously reported that rhesus macaques and PTM can progress to AIDS following SIVmac239ΔGY infection in association with novel amino acid changes in the Env cytoplasmic domain. These included an R722G flanking the ΔGY deletion and a nine nucleotide deletion encoding amino acids 734-736 (ΔQTH) that overlaps the rev and tat open reading frames. We show that molecular clones containing these mutations reconstitute signals for both endocytosis and polarized sorting. In one PTM, a novel genotype was selected that generated a new signal for polarized sorting but not endocytosis. This genotype, together with the ΔGY mutation, was conserved in association with high viral loads for several months when introduced into naïve PTMs. For the first time, our findings reveal strong selection pressure for Env endocytosis and particularly for polarized sorting during pathogenic SIV infection in vivo.

Nanoparticles (NP) are attractive options for the therapeutic delivery of active pharmaceutical drugs, proteins and nucleic acids into cells, tissues and organs. Research into the development and application of NP most often starts with a diverse group of scientists, including chemists, bioengineers and material and pharmaceutical scientists, who design, fabricate and characterize NP in vitro (Stage 1). The next step (Stage 2) generally investigates cell toxicity as well as the processes by which NP bind, are internalized and deliver their cargo to appropriate model tissue culture cells. Subsequently, in Stage 3, selected NP are tested in animal systems, mostly mouse. Whereas the chemistry-based development and analysis in Stage 1 is increasingly sophisticated, the investigations in Stage 2 are not what could be regarded as 'state-of-the-art' for the cell biology field and the quality of research into NP interactions with cells is often sub-standard. In this review we describe our current understanding of the mechanisms by which particles gain entry into mammalian cells via endocytosis. We summarize the most important areas for concern, highlight some of the most common mis-conceptions, and identify areas where NP scientists could engage with trained cell biologists. Our survey of the different mechanisms of uptake into cells makes us suspect that claims for roles for caveolae, as well as macropinocytosis, in NP uptake into cells have been exaggerated, whereas phagocytosis has been under-appreciated.

The first step of cellular entry for the human immunodeficiency virus type-1 (HIV-1) occurs through the binding of its envelope protein (Env) with the plasma membrane receptor CD4 and co-receptor CCR5 or CXCR4 on susceptible cells, primarily CD4+ T cells and macrophages. Although there is considerable knowledge of the molecular interactions between Env and host cell receptors that lead to successful fusion, the precise way in which HIV-1 receptors redistribute to sites of virus binding at the nanoscale remains unknown. Here, we quantitatively examine changes in the nanoscale organisation of CD4 on the surface of CD4+ T cells following HIV-1 binding. Using single-molecule super-resolution imaging, we show that CD4 molecules are distributed mostly as either individual molecules or small clusters of up to 4 molecules. Following virus binding, we observe a local 3-to-10-fold increase in cluster diameter and molecule number for virus-associated CD4 clusters. Moreover, a similar but smaller magnitude reorganisation of CD4 was also observed with recombinant gp120. For one of the first times, our results quantify the nanoscale CD4 reorganisation triggered by HIV-1 on host CD4+ T cells. Our quantitative approach provides a robust methodology for characterising the nanoscale organisation of plasma membrane receptors in general with the potential to link spatial organisation to function.

Understanding the molecular mechanisms involved in the assembly of viruses is essential for discerning how viruses transmit from cell to cell and host to host. Although molecular aspects of assembly have been studied for many viruses, we still have little information about these events in real time. Enveloped viruses such as HIV that assemble at, and bud from, the plasma membrane have been studied in some detail using live cell fluorescence imaging techniques; however, these approaches provide little information about the real-time morphological changes that take place as viral components come together to form individual virus particles. Here we used correlative scanning ion conductance microscopy and fluorescence confocal microscopy to measure the topological changes, together with the recruitment of fluorescently labeled viral proteins such as Gag and Vpr, during the assembly and release of individual HIV virus-like particles (VLPs) from the top, nonadherent surfaces of living cells. We show that 1) labeling of viral proteins with green fluorescent protein affects particle formation, 2) the kinetics of particle assembly on different plasma membrane domains can vary, possibly as a consequence of differences in membrane biophysical properties, and 3) VLPs budding from the top, unimpeded surface of cells can reach full size in 20 s and disappear from the budding site in 0.5 to 3 min from the moment curvature is initially detected, significantly faster than has been previously reported.

Localization based super-resolution microscopy relies on the detection of individual molecules cycling between fluorescent and non-fluorescent states. These transitions are commonly regulated by high-intensity illumination, imposing constrains to imaging hardware and producing sample photodamage. Here, we propose single-molecule self-quenching as a mechanism to generate spontaneous photoswitching independent of illumination. To demonstrate this principle, we developed a new class of DNA-based open-source Super-Resolution probes named Super-Beacons, with photoswitching kinetics that can be tuned structurally, thermally and chemically. The potential of these probes for live-cell friendly Super-Resolution Microscopy without high-illumination or toxic imaging buffers is revealed by imaging Interferon Inducible Transmembrane proteins (IFITMs) at sub-100nm resolutions.

Traffic was launched 20 years ago by Frances Brodsky, Mark Marsh, Sandy Schmid and Thomas Kreis with the goal of creating a journal for scientists run by scientists. Frances and Mark previously documented the history of Traffic through its first 10 years.1 Now, we are celebrating the end of our second decade and beginning of the third. A key feature of the journal has been the shared commitment of all past and current editors to work together to produce a journal that serves the community and maintains a rapid and constructive review process. Over the years, Sandy, Frances and Mark have stepped down as editors, and Trina Schroer, Tom Stevens, Gillian Griffiths, Gerrit van Meer, Mickey Marks, Sharon Tooze and Rob Parton have, in turn, stepped in. They were joined by an impressive panel of associate editors (Bruno Goud, Chris Hawes, Scott Emr, Ted Hackstadt, Carolyn Machamer, Dominique Soldati-Favre, Murray Stewart, Mark von Zastrow, Sergio Grinstein, Alexander Sorkin, Christian Ungermann, Anita Corbett and Elizabeth Miller). All of the present and past editors and associate editors have contributed significant amounts of time and talent, each has upheld the founding principles of Traffic, and each has left their mark on the character of the journal. Throughout, the journal has been sustained by a fantastic Managing Editor, Lisa Hannan, who on many occasions has held us all to task and ensured timely publication of each issue of the journal. We are also indebted to all of our referees without whom Traffic would not be what it has become. Traffic is now a mainstay of the international community of cell biologists interested in the molecular control of intracellular traffic and of cellular organization and how these contribute to physiological responses. We thank all of our colleagues for your continued trust in us to publish your important research findings and for supporting Traffic. The last 20 years have witnessed remarkable advances in our understanding of the molecular regulation of intracellular membrane trafficking, particularly within the endomembrane system. Exploiting technological advances in microscopy, genetics, computational biology, and innovative cell culture, plant and animal models, we have extended the pioneering work of Nobel laureates Palade, Claude, De Duve, Blobel, Rothman, Schekman, Südhof and Ohsumi to uncover detailed molecular mechanisms underlying secretory traffic, endocytosis, organelle biogenesis, the transfer of material between organelles, membrane motility, cell stress responses and the behaviors of intracellular pathogens. In turn, these studies have led to the discovery of new phenomena that were unimaginable 20 years ago, including the roles of organelle: organelle contacts in regulating intracellular dynamics, the interplay between membrane traffic and metabolism, and the roles of phosphoinositides and other phospholipids in shaping membrane dynamics. Many of these mechanisms are altered in unique ways in specialized cell types to effect specific physiological responses, such as immune responses or insulin-responsive glucose uptake, and by pathogens to promote their entry into, survival within and release from host cells. Many of these advances have been chronicled in Traffic. We have revisited some of the most influential research papers and reviews from the past 20 years in two special anniversary issues online. This has been a challenging task as there are so many wonderful contributions from our community. As anniversaries are not just about looking back, some of the leaders in our field have written commentaries for the special issue to celebrate both the advances in cell biology that have been published in Traffic over the past 20 years and the advances to come. We remain committed to the principle of scientist editors handling submissions by scientists; to fast, fair and constructive review; and to the responsible publishing practices as outlined by DORA. We very much look forward to the next decade of transformative traffic!

Two-pore channels (TPCs) are a ubiquitous class of Ca2+- and Na+-permeable ion channels expressed within the endolysosomal system. They have emerged as central regulators of a wide array of physiological processes intimately linked to information processing. In this short review, we highlight how molecular and chemical strategies have uncovered multiple roles for TPCs in regulating various aspects of endo-lysosomal trafficking associated with disease. We summarise advances in the identification of new small molecules to pharmacologically target TPCs for medical benefit. Lastly, we discuss possible underpinning molecular mechanism(s) that translate TPC-mediated ionic fluxes to function.

Bone Marrow Stromal Cell Antigen 2 (BST-2)/tetherin inhibits the release of numerous enveloped viruses by physically tethering nascent particles to infected cells during the process of viral budding from the cell surface. Tetherin also restricts human immunodeficiency virus (HIV), and pandemic main (M) group HIV type 1s (HIV-1s) are thought to rely exclusively on their Vpu proteins to overcome tetherin-mediated restriction of virus release. However, at least one M group HIV-1 strain, the macrophage-tropic primary AD8 isolate, is unable to express Vpu due to a mutation in its translation initiation codon. Here, using primary monocyte-derived macrophages (MDMs), we show that AD8 Nef protein can compensate for the absence of Vpu and restore virus release to wild type levels. We demonstrate that HIV-1 AD8 Nef reduces endogenous cell surface tetherin levels, physically separating it from the site of viral budding, thus preventing HIV retention. Mechanistically, AD8 Nef enhances internalisation of the long isoform of human tetherin, leading to perinuclear accumulation of the restriction factor. Finally, we show that Nef proteins from other HIV strains also display varying degrees of tetherin antagonism. Overall, we show that M group HIV-1s can use an accessory protein other than Vpu to antagonise human tetherin.

Twenty-two years ago, four leading investigators in the membrane trafficking community, each with distinct expertise, initiated a bold experiment to test a new publishing model. The idea was to create a journal run by scientists in which editors and referees were encouraged to provide positive and rapid peer review to support fair policies in publishing. Working collaboratively, the co-Editors, together with a PhD-trained scientist as a Managing Editor, recruited an outstanding group of well-respected and like-minded scientists to an editorial board that would be used as a primary source of advice and review. A goal was to work constructively with authors to improve papers so that the majority could be published and results communicated in a timely fashion. The events surrounding the founding of Traffic have been well documented in previously published editorials.1, 2 This endeavor was a close partnership with Peter Hartmann and Pernille Hammelsoe at Munksgaard, the publisher at the time, who were responsive to the goals and needs of Traffic's co-Editors (Frances Brodsky, Thomas Kreis*, Mark Marsh and Sandra Schmid), and to the scientific community served by the journal. The co-Editors and publisher set the highest goals for Traffic, implementing new practices and resources to realize these ambitions. Traffic was the first Munksgaard journal with a website and the ability to publish on-line video content, and the first to adopt an e-based referee process before other electronic editorial offices were available. We established high standards for data integrity, mandating the review of all figures for inappropriate modification prior to acceptance. We were the first to accept manuscript reviews from other journals (now widely practiced). We encouraged the publication of reviews, methods, commentaries and meeting reports that advanced research in our fields. We insisted on open access after 6 months, well before open access publication was mandated. In addition to these initiatives, Traffic has been a lead influencer in shaping the policy of publication and peer-review. We were among the initiators of DORA, the San Francisco Declaration on Research Assessment, pointing out the failures of the Impact Factor as a measure of publication quality,3 and have eschewed the attachment of an Impact Factor to the journal. After 3900 submissions (including some on vehicular traffic, drug trafficking, and even one or two on human trafficking), 2200 have been accepted for publication in Traffic. In total, our papers have been cited nearly 100 000 times. The experiment worked. Traffic became a leading journal in the field. Co-Editors and editorial board members have come and gone, and our publisher changed from Munksgaard to Blackwell and ultimately to Wiley, but the basic principles on which the journal was founded have remained the same. We are proud of our rapid and efficient review process and the high caliber of the papers we have published over the years. Many are highly cited. Several of the most influential papers published in the first 20 years of Traffic were highlighted as part of our virtual 20th anniversary celebration. As we embark on our 22nd year of publication, our publisher has opted to explore a different publication model. Our Managing Editor for the first 21 years, Dr. Lisa Hannan, will no longer be part of Traffic. We - past and present co-Editors, editorial board members, and authors who served at Traffic—recognize Lisa's essential and foundational contribution to the journal with heartfelt thanks. As a consequence of the publisher's decision regarding the Managing Editor position, the current co-Editors have decided to step down, as have many Editorial Board members, to make way for an entirely new editorial team. We hope that Wiley will continue to embrace the principles of constructive and fair review and a positive attitude to publishing high caliber science. Details about how Traffic will be managed in the future will be forthcoming. We wish the new leadership every success as they oversee the next era of Traffic.

Single-molecule localization microscopy (SMLM) techniques allow near molecular scale resolution (~ 20nm) as well as precise and robust analysis of protein organization at different scales. SMLM hardware, analytics and probes have been the focus of a variety of studies and are now commonly used in laboratories across the world. Protocol reliability and artefact identification are increasingly seen as important aspects of super-resolution microscopy. The reliability of these approaches thus requires in-depth evaluation so that biological findings are based on solid foundations. Here we explore how different fixation approaches that disrupt or preserve the actin cytoskeleton affect membrane protein organization. Using CD4 as a model, we show that fixation-mediated disruption of the actin cytoskeleton correlates with changes in CD4 membrane organization. We highlight how these artefacts are easy to overlook and how careful sample preparation is essential for extracting meaningful results from super-resolution microscopy.

Host interferon-induced transmembrane proteins (IFITMs) are broad-spectrum antiviral restriction factors. Of these, IFITM3 potently inhibits viruses that enter cells through acidic endosomes, many of which are zoonotic and emerging viruses with bats (order Chiroptera) as natural hosts. We previously demonstrated that microbat IFITM3 is antiviral. Here we show that bat IFITMs are characterized by strong adaptive evolution and identify a highly variable and functionally important site - codon 70 - within the conserved CD225 domain of IFITMs. Mutation of this residue in microbat IFITM3 impairs restriction of four different virus families that enter cells via endosomes. This mutant shows altered subcellular localization and reduced S-palmitoylation, a phenotype copied by mutation of conserved cysteine residues in microbat IFITM3. Furthermore, we show that microbat IFITM3 is S-palmitoylated on cysteine residues C71, C72 and C105, mutation of each cysteine residue individually impairs virus restriction, and a triple C71-C72-C105 mutant loses all restriction, concomitant with subcellular re-localization of microbat IFITM3 to Golgi-associated sites. Thus, we propose that S-palmitoylation is critical for Chiropteran IFITM3 function and identify a key molecular determinant of IFITM3 S-palmitoylation.

The acute antiviral response is mediated by a family of interferon-stimulated genes (ISGs), providing cell-intrinsic immunity. Mutations in genes encoding these proteins are often associated with increased susceptibility to viral infections. One family of ISGs with antiviral function is the interferon-inducible transmembrane proteins (IFITMs), of which IFITM3 has been studied extensively. In contrast, IFITM1 has not been studied in detail. Since IFITM1 can localize to the plasma membrane, we investigated its function with a range of enveloped viruses thought to infect cells by fusion with the plasma membrane. Overexpression of IFITM1 prevented infection by a number of Paramyxoviridae and Pneumoviridae, including respiratory syncytial virus (RSV), mumps virus, and human metapneumovirus (HMPV). IFITM1 also restricted infection with an enveloped DNA virus that can enter via the plasma membrane, herpes simplex virus 1 (HSV-1). To test the importance of plasma membrane localization for IFITM1 function, we identified blocks of amino acids in the conserved intracellular loop (CIL) domain that altered the subcellular localization of the protein and reduced antiviral activity. By screening reported data sets, 12 rare nonsynonymous single nucleotide polymorphisms (SNPs) were identified in human IFITM1, some of which are in the CIL domain. Using an Ifitm1(-/-) mouse, we show that RSV infection was more severe, thereby extending the range of viruses restricted in vivo by IFITM proteins and suggesting overall that IFITM1 is broadly antiviral and that this antiviral function is associated with cell surface localization. IMPORTANCE Host susceptibility to viral infection is multifactorial, but early control of viruses not previously encountered is predominantly mediated by the interferon-stimulated gene (ISG) family. There are upwards of 300 of these genes, the majority of which do not have a clearly defined function or mechanism of action. The cellular location of these proteins may have an important effect on their function. One ISG located at the plasma membrane is interferon-inducible transmembrane protein 1 (IFITM1). Here we demonstrate that IFITM1 can inhibit infection with a range of viruses that enter via the plasma membrane. Mutant IFITM1 proteins that were unable to localize to the plasma membrane did not restrict viral infection. We also observed for the first time that IFITM1 plays a role in vivo, and Ifitm1(-/-) mice were more susceptible to viral lung infection. These data contribute to our understanding of how ISGs prevent viral infections.

The process of entry into a host cell is a key step in the life cycle of most viruses. In recent years, there has been a significant increase in our understanding of the routes and mechanisms of entry for a number of these viruses. This has led to the development of novel broad-spectrum antiviral approaches that target host cell proteins and pathways, in addition to strategies focused on individual viruses or virus families. Here we consider a number of these approaches and their broad-spectrum potential.