Entry Efficiencies Research Articles

Bismuth-Cyclized Cell-Penetrating Peptides.

Intracellular delivery of biological cargos, which would yield new research tools and novel therapeutics, remains an active area of research. A convenient and potentially general approach involves the conjugation of a cell-penetrating peptide to a cargo of interest. However, linear CPPs lack sufficient cytosolic entry efficiency and metabolic stability, while previous backbone cyclized CPPs have several drawbacks including the necessity for chemical synthesis and posttranslational conjugation to peptide/protein cargos and epimerization during cyclization. We report here a new class of bismuth cyclized CPPs with excellent cytosolic entry efficiencies, proteolytic stability, and potential compatibility with genetic encoding and recombinant production.

Gutless Helper-Dependent and First-Generation HAdV5 Vectors Have Similar Mechanical Properties and Common Transduction Mechanisms.

Delivering vectorized information into cells with the help of viruses has been of high interest to fundamental and applied science, and bears significant therapeutic promise. Human adenoviruses (HAdVs) have been at the forefront of gene delivery for many years, and the subject of intensive development resulting in several generations of agents, including replication-competent, -defective or retargeted vectors, and recently also helper-dependent (HD), so-called gutless vectors lacking any viral protein coding information. While it is possible to produce HD-AdVs in significant amounts, physical properties of these virus-like particles and their efficiency of transduction have not been addressed. Here, we used single-cell and single virus particle assays to probe the effect of genome length on HAdV-C5 vector transduction. Our results demonstrate that first-generation C5 vectors lacking the E1/E3 regions of the viral genome as well as HD-AdV-C5 particles with a wild type (wt) ∼36 kbp or an undersized double-strand DNA genome are similar to human adenovirus C5 (HAdV-C5) wt regarding attachment to human lung epithelial cells, endocytic uptake, endosome penetration and dependency on the E3 RING ubiquitin ligase Mind Bomb 1 for DNA uncoating at the nuclear pore complex. Atomic force microscopy measurements of single virus particles indicated that small changes in the genome length from 94% to 103% of HAdV-C5 have no major impact on physical and mechanical features of AdV vectors. In contrast, an HD-AdV-C5 with ∼30 kbp genome was slightly stiffer and less heat-resistant than the other particles, despite comparable entry and transduction efficiencies in tissue culture cell lines, including murine alveolar macrophage-like Max-Planck-Institute (MPI)-2 cells. Together, our in vitro studies reinforce the use of HD-AdV vectors for effective single round gene delivery. The results illustrate how physical properties and cell entry behavior of single virus particles can provide functional information for anticipated therapeutic vector applications.

Programming Aggregate States of DNA Nanorods with Sub-10 nm Hydrophobic Patterns for Tunable Cell Entry.

The intracellular application of DNA nanodevices is challenged by their inadequate cellular entry efficiency, which may be addressed by the development of amphiphilic DNA nanostructures. However, the impact of the spatial distribution of hydrophobicity in cell entry has not been fully explored. Here, we program a spectrum of amphiphilic DNA nanostructures displaying diverse sub-10 nm patterns of cholesterol, which result in distinct aggregate states in the aqueous solution and thus varied cell entry efficiencies. We find that the hydrophobic patterns can lead to discrete aggregate states, from monomers to low-number oligomers (n = 1-6). We demonstrate that the monomers or oligomers with moderate hydrophobic density are preferred for cell entry, with up to ∼174-fold improvement relative to unmodified ones. Our study provides a new clue for the rational design of amphiphilic DNA nanostructures for intracellular applications.

SARS-CoV-2 variant Alpha has a spike-dependent replication advantage over the ancestral B.1 strain in human cells with low ACE2 expression.

Epidemiological data demonstrate that Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) variants of concern (VOCs) Alpha and Delta are more transmissible, infectious, and pathogenic than previous variants. Phenotypic properties of VOC remain understudied. Here, we provide an extensive functional study of VOC Alpha replication and cell entry phenotypes assisted by reverse genetics, mutational mapping of spike in lentiviral pseudotypes, viral and cellular gene expression studies, and infectivity stability assays in an enhanced range of cell and epithelial culture models. In almost all models, VOC Alpha spread less or equally efficiently as ancestral (B.1) SARS-CoV-2. B.1. and VOC Alpha shared similar susceptibility to serum neutralization. Despite increased relative abundance of specific sgRNAs in the context of VOC Alpha infection, immune gene expression in infected cells did not differ between VOC Alpha and B.1. However, inferior spreading and entry efficiencies of VOC Alpha corresponded to lower abundance of proteolytically cleaved spike products presumably linked to the T716I mutation. In addition, we identified a bronchial cell line, NCI-H1299, which supported 24-fold increased growth of VOC Alpha and is to our knowledge the only cell line to recapitulate the fitness advantage of VOC Alpha compared to B.1. Interestingly, also VOC Delta showed a strong (595-fold) fitness advantage over B.1 in these cells. Comparative analysis of chimeric viruses expressing VOC Alpha spike in the backbone of B.1, and vice versa, showed that the specific replication phenotype of VOC Alpha in NCI-H1299 cells is largely determined by its spike protein. Despite undetectable ACE2 protein expression in NCI-H1299 cells, CRISPR/Cas9 knock-out and antibody-mediated blocking experiments revealed that multicycle spread of B.1 and VOC Alpha required ACE2 expression. Interestingly, entry of VOC Alpha, as opposed to B.1 virions, was largely unaffected by treatment with exogenous trypsin or saliva prior to infection, suggesting enhanced resistance of VOC Alpha spike to premature proteolytic cleavage in the extracellular environment of the human respiratory tract. This property may result in delayed degradation of VOC Alpha particle infectivity in conditions typical of mucosal fluids of the upper respiratory tract that may be recapitulated in NCI-H1299 cells closer than in highly ACE2-expressing cell lines and models. Our study highlights the importance of cell model evaluation and comparison for in-depth characterization of virus variant-specific phenotypes and uncovers a fine-tuned interrelationship between VOC Alpha- and host cell-specific determinants that may underlie the increased and prolonged virus shedding detected in patients infected with VOC Alpha.

Malnutrition Disrupts T Cell Migration

Abstract Malnutrition is associated with reductions in the number and function of T lymphocytes. Previous studies in the lab suggest that malnutrition may also impart a “super-quiescent” phenotype to T cells, perhaps affecting the efficiency of their migration within and between lymph nodes. Thus, the purpose of this study is to evaluate the effect of malnutrition on T cell migration in vivo and to characterize malnutrition-induced changes in the expression of proteins known to be important for T cell migration. To determine if malnourishment alters T cell migration in vivo, we compared lymph node entry rates of adoptively-transferred malnourished and control T cells in malnourished and control recipients. In agreement with other studies, control CD4+ T cells were more efficient than control CD8+ T cells at entering the lymph nodes. Interestingly, regardless of recipient diet, malnourished CD4+ and CD8+ T cells entered the lymph nodes at equivalent rates, suggesting that malnourishment eliminates distinct lymph node entry efficiencies for CD8+ and CD4+ T cells. We also found important differences in the expression of key proteins involved in T cell migration between malnourished and control mice. Overall, we found that malnutrition disrupts T cell migration including the distinct migration efficiencies of CD4+ and CD8+ T cells. An improved understanding of T cell-intrinsic changes that occur during malnourishment should enhance our knowledge of CD4+ and CD8+ T cell migration and shed light on how organisms adapt to malnutrition. Supported by NSF-MRI [DBI- 1920116] NSF-RUI [IOS-1951881]

T cell migration potentiates HIV infection by enhancing viral fusion and integration.

T cells actively migrate along reticular networks within lymphoid organs in search for cognate antigen, but how these behaviors impact HIV entry and infection is unclear. Here, we show that migratory Tcells in 3D collagen matrix display significantly enhanced infection and integration by cell-free R5-tropic lab adapted and transmitted/founder molecular HIV clones in the absence of exogenous cytokines or cationic polymers. Using two different collagen matrices that either support or restrict Tcell migration, we observe high levels of HIV fusion in migratory Tcells, whereas non-motile Tcells display low viral entry and integration. Motile Tcells were less sensitive to combination antiretroviral drugs and were able to freely migrate into regions with high HIV densities, resulting in high infection rates. Together, our studies indicate that the environmental context in which initial HIV-T cell encounters occur modulates HIV-1 entry and integration efficiencies.

The interactions of ZDHHC5/GOLGA7 with SARS-CoV-2 spike (S) protein and their effects on S protein\u2019s subcellular localization, palmitoylation and pseudovirus entry

BackgroundSevere acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike (S) protein determines virus entry and the palmitoylation of S protein affects virus infection. An acyltransferase complex ZDHHC5/GOGAL7 that interacts with S protein was detected by affinity purification mass spectrometry (AP-MS). However, the palmitoylated cysteine residues of S protein, the effects of ZDHHC5 or GOLGA7 knockout on S protein’s subcellular localization, palmitoylation, pseudovirus entry and the enzyme for depalmitoylation of S protein are not clear.MethodsThe palmitoylated cysteine residues of S protein were identified by acyl-biotin exchange (ABE) assays. The interactions between S protein and host proteins were analyzed by co-immunoprecipitation (co-IP) assays. Subcellular localizations of S protein and host proteins were analyzed by fluorescence microscopy. ZDHHC5 or GOGAL7 gene was edited by CRISPR-Cas9. The entry efficiencies of SARS-CoV-2 pseudovirus into A549 and Hela cells were analyzed by measuring the activity of Renilla luciferase.ResultsIn this investigation, all ten cysteine residues in the endodomain of S protein were palmitoylated. The interaction of S protein with ZDHHC5 or GOLGA7 was confirmed. The interaction and colocalization of S protein with ZDHHC5 or GOLGA7 were independent of the ten cysteine residues in the endodomain of S protein. The interaction between S protein and ZDHHC5 was independent of the enzymatic activity and the PDZ-binding domain of ZDHHC5. Three cell lines HEK293T, A549 and Hela lacking ZDHHC5 or GOLGA7 were constructed. Furthermore, S proteins still interacted with one host protein in HEK293T cells lacking the other. ZDHHC5 or GOLGA7 knockout had no significant effect on S protein’s subcellular localization or palmitoylation, but significantly decreased the entry efficiencies of SARS-CoV-2 pseudovirus into A549 and Hela cells, while varying degrees of entry efficiencies may be linked to the cell types. Additionally, the S protein interacted with the depalmitoylase APT2.ConclusionsZDHHC5 and GOLGA7 played important roles in SARS-CoV-2 pseudovirus entry, but the reason why the two host proteins affected pseudovirus entry remains to be further explored. This study extends the knowledge about the interactions between SARS-CoV-2 S protein and host proteins and probably provides a reference for the corresponding antiviral methods.

Assessing the Cellular Uptake, Endosomal Escape, and Cytosolic Entry Efficiencies of Cyclic Peptides.

Intracellular biologics such as cyclic peptides are an emerging class of macromolecular drugs that are either intrinsically cell permeable or can be effectively delivered into the cell interior to modulate the activity of previously intractable drug targets. They generally enter the mammalian cell by endocytosis mechanisms and are initially localized inside the endosomes. They subsequently escape from the endosomes (and/or lysosomes) into the cytosol with varying efficiencies. In this chapter, we provide the detailed protocol for a flow cytometry-based assay method to quantitate the overall cellular uptake, endosomal escape, and cytosolic entry efficiencies of biomolecules (e.g., linear and cyclic peptides, proteins, and nucleic acids), by using cell-penetrating peptides as an example. The scope of applicability, strengths, and weaknesses of this assay are also discussed.

Strong alkaline electrolyzed water efficiently inactivates SARS-CoV-2, other viruses, and Gram-negative bacteria

Air spaces and material surfaces in a pathogen-contaminated environment can often be a source of infection to humans, and disinfection has become a common intervention focused on reducing the contamination levels. In this study, we examined the efficacy of SAIW, a unique electrolyzed water with chlorine-free, high pH, high concentration of dissolved hydrogen, and low oxygen reduction potential, for the inactivation of several viruses and bacteria. Infectivity assays revealed that initial viral titers of enveloped and non-enveloped viruses, including severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), influenza A virus, herpes simplex virus type 1, human coronavirus, feline calicivirus, and canine parvovirus, were reduced by 2.9- to 5.5-log10 within 30 s of SAIW exposure. Similarly, the culturability of three Gram-negative bacteria (Escherichia coli, Salmonella, and Legionella) dropped down by 1.9- to 4.9-log10 within 30 s of SAIW treatment. Mechanistically, treatment with SAIW was found to significantly decrease the binding and subsequent entry efficiencies of SARS-CoV-2 on Vero cells. Finally, we showed that this chlorine-free electrolytic ion water had no acute inhalation toxicity in mice, demonstrating that SAIW holds promise for a safer antiviral and antibacterial disinfectant.

Distinct lymph node entry efficiencies for CD8+ and CD4+ T cells are eliminated during malnourishment.

Abstract Previous work suggests that glucocorticoids upregulate expression of CD127, an IL-7 receptor component, on peripheral naïve T cells (NT), even though the total number of peripheral NT declines dramatically during malnourishment. We proposed that CD127 up-regulation contributes to peripheral NT reduction by increasing the scavenge rate of IL-7, providing a mechanism to rapidly adjust the total number of NTs during malnutrition. Each malnourished NT would then receive a larger dose of IL-7 than control NTs. We next wondered if this larger dose might confer additional energy-saving behaviors. NT migration across HEV’s may be a high energy-consuming activity. Thus, we compared lymph node entry rates of adoptively-transferred malnourished and control NT in malnourished and control recipients. Control CD4+ NTs migrated more efficiently than control CD8+ NT, but malnourished CD4+ and CD8+ NT migrated at equivalent rates, regardless of recipient diet. We next analyzed expression of proteins known to be involved in NT migration to uncover the currently unknown mechanisms responsible for these various migration patterns. Flow cytometry analysis revealed malnutrition significantly reduces expression of both components of LFA-1 (CD18 and CD11a), CD49d (a VLA-4 component), and S1PR1 in CD4+ and CD8+ NT. We also compared expression levels of ICAM-1 (CD54), a protein expressed in HEV’s which binds to LFA-1 on migrating NT, in malnourished and control lymph node tissue via confocal microscopy. Improved understanding of altered migration molecule expression during malnourishment should enhance our knowledge of the energy-conserving behavior of NT, as well as uncover strategies to improve vaccination responses in malnourished children.

Monitoring Viral Entry in Real-Time Using a Luciferase Recombinant Vesicular Stomatitis Virus Producing SARS-CoV-2, EBOV, LASV, CHIKV, and VSV Glycoproteins.

Viral entry is the first stage in the virus replication cycle and, for enveloped viruses, is mediated by virally encoded glycoproteins. Viral glycoproteins have different receptor affinities and triggering mechanisms. We employed vesicular stomatitis virus (VSV), a BSL-2 enveloped virus that can incorporate non-native glycoproteins, to examine the entry efficiencies of diverse viral glycoproteins. To compare the glycoprotein-mediated entry efficiencies of VSV glycoprotein (G), Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike (S), Ebola (EBOV) glycoprotein (GP), Lassa (LASV) GP, and Chikungunya (CHIKV) envelope (E) protein, we produced recombinant VSV (rVSV) viruses that produce the five glycoproteins. The rVSV virions encoded a nano luciferase (NLucP) reporter gene fused to a destabilization domain (PEST), which we used in combination with the live-cell substrate EndurazineTM to monitor viral entry kinetics in real time. Our data indicate that rVSV particles with glycoproteins that require more post-internalization priming typically demonstrate delayed entry in comparison to VSV G. In addition to determining the time required for each virus to complete entry, we also used our system to evaluate viral cell surface receptor preferences, monitor fusion, and elucidate endocytosis mechanisms. This system can be rapidly employed to examine diverse viral glycoproteins and their entry requirements.

Entry of Scotophilus Bat Coronavirus-512 and Severe Acute Respiratory Syndrome Coronavirus in Human and Multiple Animal Cells.

Bats are natural reservoirs of severe acute respiratory syndrome coronavirus (SARS-CoV) and Middle East respiratory syndrome CoV (MERS-CoV). Scotophilus bat CoV-512 demonstrates potential for cross-species transmission because its viral RNA and specific antibodies have been detected in three bat species of Taiwan. Understanding the cell tropism of Scotophilus bat CoV-512 is the first step for studying the mechanism of cross-species transmission. In this study, a lentivirus-based pseudovirus was produced using the spike (S) protein of Scotophilus bat CoV-512 or SARS-CoV as a surface protein to test the interaction between coronaviral S protein and its cell receptor on 11 different cells. Susceptible cells expressed red fluorescence protein (RFP) after the entry of RFP-bound green fluorescence protein (GFP)-fused S protein of Scotophilus bat CoV-512 (RFP-Sco-S-eGFP) or RFP-SARS-S pseudovirus, and firefly luciferase (FLuc) activity expressed by cells infected with FLuc-Sco-S-eGFP or FLuc-SARS-S pseudovirus was quantified. Scotophilus bat CoV-512 pseudovirus had significantly higher entry efficiencies in Madin Darby dog kidney epithelial cells (MDCK), black flying fox brain cells (Pabr), and rat small intestine epithelial cells (IEC-6). SARS-CoV pseudovirus had significantly higher entry efficiencies in human embryonic kidney epithelial cells (HEK-293T), pig kidney epithelial cells (PK15), and MDCK cells. These findings demonstrated that Scotophilus bat CoV-512 had a broad host range for cross-species transmission like SARS-CoV.

Distinct lymph node entry efficiencies for CD8+ and CD4+ T cells are eliminated during malnourishment

Abstract Previous work has suggested that glucocorticoids upregulate expression of the IL-7 receptor component, CD127 on peripheral T cells, even though the total number of peripheral T cells declines dramatically during malnourishment. We have proposed that CD127 up-regulation contributes to peripheral T cell diminishment by increasing the scavenge rate of IL-7 and thereby providing a mechanism to rapidly adjust the total number of T cells during malnutrition. As such, each malnourished T cell would receive a higher dose of IL-7 than control T cells. We next wondered if increased exposure to IL-7 during malnourishment might confer additional energy-saving behaviors. Thus, we compared lymph node entry rates of adoptively-transferred malnourished and control T cells in malnourished and control recipients. As expected, control CD4+ T cells were more efficient than control CD8+ T cells at entering the lymph nodes. Interestingly, regardless of recipient diet, malnourished CD4+ and CD8+T cells entered the lymph nodes at equivalent rates. The molecular events that enable faster CD4+ T cell entry into lymph nodes in control mice is currently unknown. An improved understanding of T cell-intrinsic changes that occur during malnourishment should enhance our knowledge of CD4+ and CD8+ T cell migration, as well as uncover strategies to improve vaccination responses in malnourished children.

Distinct lymph node entry efficiencies for CD8+ and CD4+ T cells are eliminated during malnourishment.

Abstract Previous work has suggested that glucocorticoids upregulate expression of the IL-7 receptor component, CD127 on peripheral T cells, even though the total number of peripheral T cells declines dramatically during malnourishment. We have proposed that CD127 up-regulation contributes to peripheral T cell diminishment by increasing the scavenge rate of IL-7 and thereby providing a mechanism to rapidly adjust the total number of T cells during malnutrition. As such, each malnourished T cell would receive a higher dose of IL-7 than control T cells. We next wondered if increased exposure to IL-7 during malnourishment might confer additional energy-saving behaviors. Thus, we compared lymph node entry rates of adoptively-transferred malnourished and control T cells in malnourished and control recipients. As expected, control CD4+ T cells were more efficient than control CD8+ T cells at entering the lymph nodes. Interestingly, regardless of recipient diet, malnourished CD4+ and CD8+T cells entered the lymph nodes at equivalent rates. The molecular events that enable faster CD4+ T cell entry into lymph nodes in control mice is currently unknown. An improved understanding of T cell-intrinsic changes that occur during malnourishment should enhance our knowledge of CD4+ and CD8+ T cell migration, as well as uncover strategies to improve vaccination responses in malnourished children.

In silico Analysis of HIV-1 Env-gp120 Reveals Structural Bases for Viral Adaptation in Growth-Restrictive Cells.

Variable V1/V2 and V3 loops on human immunodeficiency virus type 1 (HIV-1) envelope-gp120 core play key roles in modulating viral competence to recognize two infection receptors, CD4 and chemokine-receptors. However, molecular bases for the modulation largely remain unclear. To address these issues, we constructed structural models for a full-length gp120 in CD4-free and -bound states. The models showed topologies of gp120 surface loop that agree with those in reported structural data. Molecular dynamics simulation showed that in the unliganded state, V1/V2 loop settled into a thermodynamically stable arrangement near V3 loop for conformational masking of V3 tip, a potent neutralization epitope. In the CD4-bound state, however, V1/V2 loop was rearranged near the bound CD4 to support CD4 binding. In parallel, cell-based adaptation in the absence of anti-viral antibody pressures led to the identification of amino acid substitutions that individually enhance viral entry and growth efficiencies in association with reduced sensitivity to CCR5 antagonist TAK-779. Notably, all these substitutions were positioned on the receptors binding surfaces in V1/V2 or V3 loop. In silico structural studies predicted some physical changes of gp120 by substitutions with alterations in viral replication phenotypes. These data suggest that V1/V2 loop is critical for creating a gp120 structure that masks co-receptor binding site compatible with maintenance of viral infectivity, and for tuning a functional balance of gp120 between immune escape ability and infectivity to optimize HIV-1 replication fitness.

Assessment of Inhibitors of Pathogenic Crimean-Congo Hemorrhagic Fever Virus Strains Using Virus-Like Particles.

Crimean-Congo hemorrhagic fever (CCHF) is an often lethal, acute inflammatory illness that affects a large geographic area. The disease is caused by infection with CCHF virus (CCHFV), a nairovirus from the Bunyaviridae family. Basic research on CCHFV has been severely hampered by biosafety requirements and lack of available strains and molecular tools. We report the development of a CCHF transcription- and entry-competent virus-like particle (tecVLP) system that can be used to study cell entry and viral transcription/replication over a broad dynamic range (~4 orders of magnitude). The tecVLPs are morphologically similar to authentic CCHFV. Incubation of immortalized and primary human cells with tecVLPs results in a strong reporter signal that is sensitive to treatment with neutralizing monoclonal antibodies and by small molecule inhibitors of CCHFV. We used glycoproteins and minigenomes from divergent CCHFV strains to generate tecVLPs, and in doing so, we identified a monoclonal antibody that can prevent cell entry of tecVLPs containing glycoproteins from 3 pathogenic CCHFV strains. In addition, our data suggest that different glycoprotein moieties confer different cellular entry efficiencies, and that glycoproteins from the commonly used strain IbAr10200 have up to 100-fold lower ability to enter primary human cells compared to glycoproteins from pathogenic CCHFV strains.

Phenotypic characterisation of Human Immunodeficiency Virus type 1 Envelope entry efficiency of transmitted/founder variants circulating in Mbeya, Tanzania

Background: Altered fitness of transmitted founder (T/F) HIV-1 variants has been linked to slowed disease progression, yet almost all of these studies have focussed on the role of attenuating immune escape mutations in T/F Gag or entry efficiency of Envelope (Env) in long-term non-progressors (LTNP) and elite suppressors (ES) during chronic stages of infection. As Env could also play an important role in viral fitness during early infection, we investigated if T/F Env entry efficiency, prior to immune selection, affected viral loads of progressors in vivo. Findings: Functional Env clones from eight progressors and one LTNP, representing the T/F virus responsible for clinical infection, were constructed. Subsequently Env pseudoviruses (PSVs) were generated, and confirmed to be all R5 but not macrophage tropic. PSVs had a ~80 fold range in entry efficiencies using TZM-bl cells with the LTNP being the poorest enterer. Enhanced entry was associated with increased viral load at 3 months although this association was reduced by 12 months. Finally, we show that entry efficiency was influenced by fusion capacity, CCR5/CD4 dependency, and/or incorporation of gp120 into pseudovirions.Conclusion: The variation in TZM-bl entry efficiency of T/F viruses was due to one or more advantageous Env attributes. Viruses with high entry efficiency may have a replication advantage subsequent to transmission, leading to increased viral loads during early infection of progressors.

Distinct HIV-1 entry phenotypes are associated with transmission, subtype specificity, and resistance to broadly neutralizing antibodies.

BackgroundThe efficiency of CD4/CCR5 mediated HIV-1 entry has important implications for pathogenesis and transmission. The HIV-1 receptor affinity profiling (Affinofile) system analyzes and quantifies the infectivity of HIV-1 envelopes (Envs) across a spectrum of CD4/CCR5 expression levels and distills these data into a set of Affinofile metrics. The Affinofile system has shed light on how differential CD4/CCR5 usage efficiencies contributes to an array of Env phenotypes associated with cellular tropism, viral pathogenesis, and CCR5 inhibitor resistance. To facilitate more rapid, convenient, and robust analysis of HIV-1 entry phenotypes, we engineered a reporter Affinofile system containing a Tat- and Rev-dependent Gaussia luciferase-eGFP-Reporter (GGR) that is compatible with the use of pseudotyped or replication competent viruses with or without a virally encoded reporter gene. This GGR Affinofile system enabled a higher throughput characterization of CD4/CCR5 usage efficiencies associated with differential Env phenotypes.ResultsWe first validated our GGR Affinofile system on isogenic JR-CSF Env mutants that differ in their affinity for CD4 and/or CCR5. We established that their GGR Affinofile metrics reflected their differential entry phenotypes on primary PBMCs and CD4+ T-cell subsets. We then applied GGR Affinofile profiling to reveal distinct entry phenotypes associated with transmission, subtype specificity, and resistance to broadly neutralizing antibodies (BNAbs). First, we profiled a panel of reference subtype B transmitted/founder (T/F) and chronic Envs (n = 12) by analyzing the infectivity of each Env across 25 distinct combinations of CD4/CCR5 expression levels. Affinofile metrics revealed that at low CCR5 levels, our panel of subtype B T/F Envs was more dependent on high levels of CD4 for HIV-1 entry compared to chronic Envs. Next, we analyzed a reference panel of 28 acute/early subtype A-D Envs, and noted that subtype C Envs could be distinguished from the other subtypes based on their infectivity profiles and relevant Affinofile metrics. Lastly, mutations known to confer resistance to VRC01 or PG6/PG19 BNAbs, when engineered into subtypes A-D Envs, resulted in significantly decreased CD4/CCR5 usage efficiency.ConclusionsGGR Affinofile profiling reveals pathophysiological phenotypes associated with varying HIV-1 entry efficiencies, and highlight the fitness costs associated with resistance to some broadly neutralizing antibodies.

Improving promiscuous mammalian cell entry by the baculovirus Autographa californica multiple nuclear polyhedrosis virus.

The insect baculovirus AcMNPV (Autographa californica multiple nuclear polyhedrosis virus) enters many mammalian cell lines, prompting its application as a general eukaryotic gene delivery agent, but the basis of entry is poorly understood. For adherent mammalian cells, we show that entry is favoured by low pH and by increasing the available cell-surface area through a transient release from the substratum. Low pH also stimulated baculovirus entry into mammalian cells grown in suspension which, optimally, could reach 90% of the transduced population. The basic loop, residues 268–281, of the viral surface glycoprotein gp64 was required for entry and a tetra mutant with increasing basicity increased entry into a range of mammalian cells. The same mutant failed to plaque in Sf9 cells, instead showing individual cell entry and minimal cell-to-cell spread, consistent with an altered fusion phenotype. Viruses grown in different insect cells showed different mammalian cell entry efficiencies, suggesting that additional factors also govern entry.

A Quantitative Affinity-Profiling System That Reveals Distinct CD4/CCR5 Usage Patterns among Human Immunodeficiency Virus Type 1 and Simian Immunodeficiency Virus Strains

The affinity of human immunodeficiency virus (HIV) envelope for CD4 and CCR5 appears to be associated with aspects of R5 virus (virus using the CCR5 coreceptor) pathogenicity. However, entry efficiency results from complex interactions between the viral envelope glycoprotein and both CD4 and CCR5, which limits attempts to correlate viral pathogenicity with surrogate measures of envelope CD4 and CCR5 affinities. Here, we present a system that provides a quantitative and comprehensive characterization of viral entry efficiency as a direct interdependent function of both CD4 and CCR5 levels. This receptor affinity profiling system also revealed heretofore unappreciated complexities underlying CD4/CCR5 usage. We first developed a dually inducible cell line in which CD4 and CCR5 could be simultaneously and independently regulated within a physiologic range of surface expression. Infection by multiple HIV type 1 (HIV-1) and simian immunodeficiency virus isolates could be examined simultaneously for up to 48 different combinations of CD4/CCR5 expression levels, resulting in a distinct usage pattern for each virus. Thus, each virus generated a unique three-dimensional surface plot in which viral infectivity varied as a function of both CD4 and CCR5 expression. From this functional form, we obtained a sensitivity vector along with corresponding metrics that quantified an isolate's overall efficiency of CD4/CCR5 usage. When applied to viral isolates with well-characterized sensitivities to entry/fusion inhibitors, the vector metrics were able to encapsulate their known biological phenotypes. The application of the vector metrics also indicated that envelopes derived from elite suppressors had overall-reduced entry efficiencies compared to those of envelopes derived from chronically infected viremic progressors. Our affinity-profiling system may help to refine studies of R5 virus tropism and pathogenesis.