Fluorescent and bioluminescent bovine H5N1 influenza viruses for evaluation of antiviral interventions.

In early 2024, a clade 2.3.4.4b high pathogenic H5N1 avian influenza virus was detected in dairy cows and humans in the United States. Since then, it has spread to herds in at least 13 states and caused symptomatic disease in at least fifteen people. To facilitate rapid testing of existing and novel countermeasures, here, we report the development of an H5N1 viral reverse genetic system, its use to produce fluorescent and bioluminescent variant strains, and their utility in high-throughput evaluation of antiviral interventions.

Proteomic analysis and protein structure prediction of Shigella phage Sfk20 based on a comparative study using structure prediction approaches.

Bacteriophages are the natural predators of bacteria and are available abundantly everywhere in nature. Lytic phages can specifically infect their bacterial host (through attachment to the receptor) and use their host replication machinery to replicate rapidly, a feature that enables them to kill a disease-causing bacteria. Hence, phage attachment to the host bacteria is the first important step of the infection process. It is reported in this study that the receptor could be an LPS which is responsible for the attachment of the Sfk20 phage to its host (Shigella flexneri 2a). Phage Sfk20 bacteriolytic activity was examined for preliminary optimization of phage titer. The phage Sfk20 viability at different saline conditions was conducted. The LC-MS/MS technique used here for detecting and identifying 40 Sfk20 phage proteins helped us to get an initial understanding of the structural landscape of phage Sfk20. From the identified proteins, six structurally significant proteins were selected for structure prediction using two neural network systems: AlphaFold2 and ESMFold, and one homology modeling software: Phyre2. Later the performance of these modeling systems was compared using various metrics. We conclude from the available and generated information that AlphaFold2 and Phyre2 perform better than ESMFold for predicting Sfk20 phage protein structures.

Rotavirus activates MLKL-mediated host cellular necroptosis concomitantly with apoptosis to facilitate dissemination of viral progeny.

Reprogramming the host cellular environment is an obligatory facet of viral pathogens to foster their replication and perpetuation. One of such reprogramming events is the dynamic cross-talk between viruses and host cellular death signaling pathways. Rotaviruses (RVs) have been reported to develop multiple mechanisms to induce apoptotic programmed cell death for maximizing viral spread and pathogenicity. However, the importance of non-apoptotic programmed death events has remained elusive in context of RV infection. Here, we report that RV-induced apoptosis accompanies another non-apoptotic mode of programmed cell death pathway called necroptosis to promote host cellular demise at late phase of infection. Phosphorylation of mixed lineage kinase domain-like (MLKL) protein indicative of necroptosis was observed to concur with caspase-cleavage (apoptotic marker) beyond 6 hr of RV infection. Subsequent studies demonstrated phosphorylated-MLKL to oligomerize and to translocate to plasma membrane in RV infected cells, resulting in loss of plasma membrane integrity and release of alarmin molecules e.g., high mobility group box protein 1 (HMGB1) in the extracellular media. Moreover, inhibiting caspase-cleavage and apoptosis could not fully rescue virus-induced cell death but rather potentiated the necroptotic trigger. Interestingly, preventing both apoptosis and necroptosis by small molecules significantly rescued virus-induced host cytopathy by inhibiting viral dissemination.

Controlling the bacterial load of Salmonella Typhi in an experimental mouse model by a lytic Salmonella phage STWB21: a phage therapy approach.

BACKGROUND: Salmonella enterica serotype Typhi is one of the major pathogens causing typhoid fever and a public health burden worldwide. Recently, the increasing number of multidrug-resistant strains of Salmonella spp. has made this utmost necessary to consider bacteriophages as a potential alternative to antibiotics for S. Typhi infection treatment. Salmonella phage STWB21, isolated from environmental water, has earlier been reported to be effective as a safe biocontrol agent by our group. In this study, we evaluated the efficacy of phage STWB21 in reducing the burden of salmonellosis in a mammalian host by inhibiting Salmonella Typhi invasion into the liver and spleen tissue. RESULTS: Phage treatment significantly improved the survival percentage of infected mice. This study also demonstrated that oral administration of phage treatment could be beneficial in both preventive and therapeutic treatment of salmonellosis caused by S. Typhi. Altogether the result showed that the phage treatment could control tissue inflammation in mice before and after Salmonella infection. CONCLUSIONS: To the best of our knowledge, this is the first report of phage therapy in a mouse model against a clinically isolated Salmonella Typhi strain that includes direct visualization of histopathology and ultrathin section microscopy images from the liver and spleen sections.

Pregnane X Receptor and the Gut-Liver Axis: A Recent Update.

It is well-known that the pregnane X receptor (PXR)/Nr1i2 is a critical xenobiotic-sensing nuclear receptor enriched in liver and intestine and is responsible for drug-drug interactions, due to its versatile ligand binding domain (LBD) and target genes involved in xenobiotic biotransformation. PXR can be modulated by various xenobiotics including pharmaceuticals, nutraceuticals, dietary factors, and environmental chemicals. Microbial metabolites such as certain secondary bile acids (BAs) and the tryptophan metabolite indole-3-propionic acid (IPA) are endogenous PXR activators. Gut microbiome is increasingly recognized as an important regulator for host xenobiotic biotransformation and intermediary metabolism. PXR regulates and is regulated by the gut-liver axis. This review summarizes recent research advancements leveraging pharmaco- and toxico-metagenomic approaches that have redefined the previous understanding of PXR. Key topics covered in this review include: (1) genome-wide investigations on novel PXR-target genes, novel PXR-DNA interaction patterns, and novel PXR-targeted intestinal bacteria; (2) key PXR-modulating activators and suppressors of exogenous and endogenous sources; (3) novel bidirectional interactions between PXR and gut microbiome under physiologic, pathophysiological, pharmacological, and toxicological conditions; and (4) modifying factors of PXR-signaling including species and sex differences and time (age, critical windows of exposure, and circadian rhythm). The review also discusses critical knowledge gaps and important future research topics centering around PXR. SIGNIFICANCE STATEMENT: This review summarizes recent research advancements leveraging O'mics approaches that have redefined the previous understanding of the xenobiotic-sensing nuclear receptor pregnane X receptor (PXR). Key topics include: (1) genome-wide investigations on novel PXR-targeted host genes and intestinal bacteria as well as novel PXR-DNA interaction patterns; (2) key PXR modulators including microbial metabolites under physiological, pathophysiological, pharmacological, and toxicological conditions; and (3) modifying factors including species, sex, and time.

Synergistic action of organophosphates and COVID-19 on inflammation, oxidative stress, and renin-angiotensin system can amplify the risk of cardiovascular maladies.

Organophosphates (OPs) are ubiquitous environmental contaminants, widely used as pesticides in agricultural fields. In addition, they serve as flame-retardants, plasticizers, antifoaming or antiwear agents in lacquers, hydraulic fluids, and floor polishing agents. Therefore, world-wide and massive application of these compounds have increased the risk of unintentional exposure to non-targets including the human beings. OPs are neurotoxic agents as they inhibit the activity of acetylcholinesterase at synaptic cleft. Moreover, they can fuel cardiovascular issues in the form of myocardities, cardiac oedema, arrhythmia, systolic malfunction, infarction, and altered electrophysiology. Such pathological outcomes might increase the severity of cardiovascular diseases which are the leading cause of mortality in the developing world. Coronavirus disease-19 (COVID-19) is the ongoing global health emergency caused by SARS-CoV-2 infection. Similar to OPs, SARS-CoV-2 disrupts cytokine homeostasis, redox-balance, and angiotensin-II/AT1R axis to promote cardiovascular injuries. Therefore, during the current pandemic milieu, unintentional exposure to OPs through several environmental sources could escalate cardiac maladies in patients with COVID-19.

Rational design of pincer-nickel complexes for catalytic cyanomethylation of benzaldehyde: A systematic DFT study.

The current study dwells upon the efforts to computationally probe a phosphine-free pincer-nickel complex that would demonstrate an efficiency better than the reported phosphine-based pincer-nickel complex (iPr2 POCNEt2 )Ni(CH2 CN) for cyanomethylation reaction. For this purpose, the mechanism of cyanomethylation of benzaldehyde was studied quantum mechanically for a series of 11 pincer-nickel complexes. The energetics of various intermediates and transition states involved in the catalytic cycle for each catalyst was compared with the corresponding energetics of the Miller's catalyst (iPr2 POCNEt2 )Ni(CH2 CN) that is reported to accomplish the cyanomethylation at room temperature. While pincer complexes (iPr4 NNN)Ni(CH2 CN) and (iPr4 NCN)Ni(CH2 CN) containing strong σ-donating amines were found to fare poorly, pincer-nickel complexes (iPr2 NCN)Ni(CH2 CN) and (dm PheboxNCN)Ni(CH2 CN) based on weaker σ-donating imines had energetics more favorable than the reported efficient catalyst (iPr2 POCNEt2 )Ni(CH2 CN). While strong trans-influencing C as the pincer central atom was found to be pivotal for lowering the cyanomethylation kinetics, presence of a poor trans-influencing N proved to be detrimental on the overall energetics.

Segmentation by classification: A novel and reliable approach for semi-automatic selection of HIV/SIV envelope spikes.

We describe a semiautomated approach to segment Env spikes from the membrane envelope of Simian Immunodeficiency Virus visualized by cryoelectron tomography of frozen-hydrated specimens. Multivariate data analysis is applied to a large set of overlapping subvolumes extracted semiautomatically from the viral envelope and does not utilize a template of the target structure. The major manual step used in the method involves determination of six points that define an ellipsoid approximating the virion shape. The approach is robust to departures of the actual virion from this starting ellipsoid. A point cage of sufficient density is generated to ensure that any spike-like protein is identified multiple times. Subsequently translational alignment of class averages to a cylindrical reference on a curved surface separates subvolumes with spikes from those without. Spike containing subvolumes identified multiple times are removed by proximity analysis. Slightly different procedures segment spikes in the equatorial and the polar regions. Once all spikes are segmented, further alignment of class averages using separately the polar and spin angles produces recognizable spike images. Our approach localized 96% of the equatorial spikes and 85% of all spikes identified manually; it identifies a significant number of additional spikes missed by manual selection. Two types of spike shapes were segmented, one with near 3-fold symmetry resembling the conventional spike, the other had a T-shape resembling the spike structure obtained when antibodies such as PG9 bind to HIV Env. The approach should be applicable to segmentation of any protein spikes extending from a cellular or virion envelope.

The exosome encapsulated microRNAs as circulating diagnostic marker for hepatocellular carcinoma with low alpha-fetoprotein.

Diagnosis of hepatocellular carcinoma (HCC) remains challenging to clinicians, particularly in a patient with low alpha-fetoprotein. Here, in silico, ex vivo and in vitro data were combined to identify liver-specific exosomal miRNAs as an early diagnostic marker for HCC. Transcriptome profiling for mRNA and small RNA in same HCV-HCC and normal liver tissues followed by cross-validation of 41 deregulated miRNAs (log2 FoldChange > 1.5, Padj < .1) with GEO/TCGA datasets of HCV/HBV related HCC vs normal/adjacent tissue revealed three miRNAs were commonly deregulated (miR-10b/miR-21/miR-182) among all HCC irrespective of viral etiology. Targets of top deregulated miRNAs were identified by TargetScan/miRwalk and validated in mRNA transcriptome data followed by Panther/Gene Ontology enrichment/Cytoscape analysis suggested that targets were mostly from carcinogenesis pathways. Hence, those miRNAs were validated in normal and HCV-HCC tissues by qRT-PCR and subsequently in plasma-derived-exosomes of both HBV/HCV infected non-HCC (chronic hepatitis [CH]/liver cirrhosis [LC]) and HCC samples, and in liver-specific Anti-Asgr2 immuno-enriched exosomes. Exosomes were verified using Nanosight/TEM/immune-blotting with anti-Alix/anti-GRP78/anti-Asgr2. Along with miR-21-5p, miR-10b-5p/miR-221-3p/miR-223-3p was found significantly upregulated in the exosome of HCC patients than CH/non-HCC. The comparable expression pattern was seen in anti-Asgr2 immuno-precipitated exosomes. Interestingly, the AFP level was found below 250 ng/mL in about 94% of HCV-HCC and 62% of HBV-HCC patients. ROC analysis showed that miR-10b-5p + miR-221-3p + miR-223-3p + miR-21-5p could differentiate CH/non-HCC(CH + LC) from HCC with AUROC: 0.86 (97.5% CI: 0.77-0.94)/0.80 (97.5% CI: 0.70-0.89), sensitivity: 74%/58% and specificity: 86%/95% while miR-10b-5p + miR-221-3p + miR-223-3p showed AUROC: 0.84 (97.5% CI: 0.74-0.94)/0.74 (97.5% CI: 0.63-0.84), sensitivity: 86%/86% and specificity:66%/53% for low AFP-HCC vs CH/non-HCC, respectively, having better sensitivity than the combination of four miRNAs. Multivariate analysis further revealed low Albumin and high miR-21-5p as probable independent risk factor for HCC.

Multifunctional transcription factor CytR of Vibrio cholerae is important for pathogenesis.

Vibrio cholerae, the Gram-negative facultative pathogen, resides in the aquatic environment and infects humans and causes diarrhoeagenic cholera. Although the environment differs drastically, V. cholerae thrives in both of these conditions aptly and chitinases play a vital role in their persistence and nutrient acquisition. Chitinases also play a role in V. cholerae pathogenesis. Chitinases and its downstream chitin utilization genes are regulated by sensor histidine kinase ChiS, which also plays a significant role in pathogenesis. Recent exploration suggests that CytR, a transcription factor of the LacI family in V. cholerae, also regulates chitinase secretion in environmental conditions. Since chitinases and chitinase regulator ChiS is involved in pathogenesis, CytR might also play a significant role in pathogenicity. However, the role of CytR in pathogenesis is yet to be known. This study explores the regulation of CytR on the activation of ChiS in the presence of mucin and its role in pathogenesis. Therefore, we created a CytR isogenic mutant strain of V. cholerae (CytR¯) and found considerably less ß-hexosaminidase enzyme production, which is an indicator of ChiS activity. The CytR¯ strain greatly reduced the expression of chitinases chiA1 and chiA2 in mucin-supplemented media. Electron microscopy showed that the CytR¯ strain was aflagellate. The expression of flagellar-synthesis regulatory genes flrB, flrC and class III flagellar-synthesis genes were reduced in the CytR¯ strain. The isogenic CytR mutant showed less growth compared to the wild-type in mucin-supplemented media as well as demonstrated highly retarded motility and reduced mucin-layer penetration. The CytR mutant revealed decreased adherence to the HT-29 cell line. In animal models, reduced fluid accumulation and colonization were observed during infection with the CytR¯ strain due to reduced expression of ctxB, toxT and tcpA. Collectively these data suggest that CytR plays an important role in V. cholerae pathogenesis.

Impact of Microbiome on Hepatic Metabolizing Enzymes and Transporters in Mice during Pregnancy.

The microbiome and pregnancy are known to alter drug disposition, yet the interplay of the two physiologic factors on the expression and/or activity of drug metabolizing enzymes and transporters (DMETs) is unknown. This study investigated the effects of microbiome on host hepatic DMETs in mice during pregnancy by comparing four groups of conventional (CV) and germ-free (GF) female mice and pregnancy status, namely, CV nonpregnant, GF non-pregnant, CV pregnant, and GF pregnant mice. Transcriptomic and targeted proteomics of hepatic DMETs were profiled by using multiomics. Plasma bile acid and steroid hormone levels were quantified by liquid chromatography tandem mass spectrometry. CYP3A activities were measured by mouse liver microsome incubations. The trend of pregnancy-induced changes in the expression or activity of hepatic DMETs in CV and GF mice was similar; however, the magnitude of change was noticeably different. For certain DMETs, pregnancy status had paradoxical effects on mRNA and protein expression in both CV and GF mice. For instance, the mRNA levels of Cyp3a11, the murine homolog of human CYP3A4, were decreased by 1.7-fold and 3.3-fold by pregnancy in CV and GF mice, respectively; however, the protein levels of CYP3A11 were increased similarly ∼twofold by pregnancy in both CV and GF mice. Microsome incubations revealed a marked induction of CYP3A activity by pregnancy that was 10-fold greater in CV mice than that in GF mice. This is the first study to show that the microbiome can alter the expression and/or activity of hepatic DMETs in pregnancy. SIGNIFICANCE STATEMENT: We demonstrated for the first time that microbiome and pregnancy can interplay to alter the expression and/or activity of hepatic drug metabolizing enzymes and transporters. Though the trend of pregnancy-induced changes in the expression or activity of hepatic drug metabolizing enzymes and transporters in conventional and germ-free mice was similar, the magnitude of change was noticeably different.

The protective role of metformin in autophagic status in peripheral blood mononuclear cells of type 2 diabetic patients.

Autophagy plays an important role in the pathophysiology of type 2 diabetes (T2D). Metformin is the most common antidiabetic drug. The main objective of this study was to explore the molecular mechanism of metformin in starvation-induced autophagy in peripheral blood mononuclear cells (PBMCs) of type 2 diabetic patients. PBMCs were isolated from 10 diabetic patients and 7 non-diabetic healthy volunteers. The autophagic puncta and markers were measured with the help of monodansylcadaverine staining and western blot. Additionally, transmission electron microscopy was also performed. No significant changes were observed in the initial autophagy marker protein levels in PBMCs of T2D after metformin treatment though diabetic PBMCs showed a high level of phospho-mammalian target of rapamycin, p62 and reduced expression of phospho-AMP-activated protein kinase and lysosomal membrane-associated protein 2, indicating a defect in autophagy. Also, induction of autophagy by tunicamycin resulted in apoptosis in diabetic PBMCs as observed by caspase-3 cleavage and reduced expression of Bcl2. Inhibition of autophagy by bafilomycin rendered consistent expression of p62 indicating a defect in the final process of autophagy. Further, electron microscopic studies also confirmed massive vacuole overload and a sign of apoptotic cell death in PBMCs of diabetic patients, whereas metformin treatment reduced the number of autophagic vacuoles perhaps by lysosomal fusion. Thus, our results indicate that defective autophagy in T2D is associated with the fusion process of lysosomes which could be overcome by metformin.

Structural analysis and proteomics studies on the Myoviridae vibriophage M4.

Bacteriophages play a crucial role in tracking the spread of bacterial epidemics. The frequent emergence of antibiotic-resistant bacterial strains throughout the world has motivated studies on bacteriophages that can potentially be used in phage therapy as an alternative to conventional antibiotic treatment. A recent outbreak of cholera in Haiti took many lives due to a rapid development of resistance to the available antibiotics. The properties of vibriophages, bacteriophages that infect Vibrio cholerae, are therefore of practical interest. A detailed understanding of the structure and assembly of a vibriophage is potentially useful in developing phage therapy against cholera as well as for fabricating artificial nanocontainers. Therefore, the aim of the present study was to determine the three-dimensional organization of vibriophage M4 at sub-nanometer resolution by electron microscopy and single-particle analysis techniques to facilitate its use as a therapeutic agent. We found that M4 has a large capsid with T = 13 icosahedral symmetry and a long contractile tail. This double-stranded DNA phage also contains a head-to-tail connector protein complex that joins the capsid to the tail and a prominent baseplate at the end of the tail. This study also provides information regarding the proteome of this phage, which is proteins similar to that of other Myoviridae phages, and most of the encoded proteins are structural proteins that form the exquisite architecture of this bacteriophage.

Correction to: Structural analysis and proteomics studies on the Myoviridae vibriophage M4.

Unfortunately, the original article was published with an incorrect figure. Figure 11 contains errors and needs to be withdrawn.

Structure of a Spumaretrovirus Gag Central Domain Reveals an Ancient Retroviral Capsid.

The Spumaretrovirinae, or foamy viruses (FVs) are complex retroviruses that infect many species of monkey and ape. Despite little sequence homology, FV and orthoretroviral Gag proteins perform equivalent functions, including genome packaging, virion assembly, trafficking and membrane targeting. However, there is a paucity of structural information for FVs and it is unclear how disparate FV and orthoretroviral Gag molecules share the same function. To probe the functional overlap of FV and orthoretroviral Gag we have determined the structure of a central region of Gag from the Prototype FV (PFV). The structure comprises two all α-helical domains NtDCEN and CtDCEN that although they have no sequence similarity, we show they share the same core fold as the N- (NtDCA) and C-terminal domains (CtDCA) of archetypal orthoretroviral capsid protein (CA). Moreover, structural comparisons with orthoretroviral CA align PFV NtDCEN and CtDCEN with NtDCA and CtDCA respectively. Further in vitro and functional virological assays reveal that residues making inter-domain NtDCEN-CtDCEN interactions are required for PFV capsid assembly and that intact capsid is required for PFV reverse transcription. These data provide the first information that relates the Gag proteins of Spuma and Orthoretrovirinae and suggests a common ancestor for both lineages containing an ancient CA fold.

IgE epitope proximity determines immune complex shape and effector cell activation capacity.

BACKGROUND: IgE-allergen complexes induce mast cell and basophil activation and thus immediate allergic inflammation. They are also important for IgE-facilitated allergen presentation to T cells by antigen-presenting cells. OBJECTIVE: To investigate whether the proximity of IgE binding sites on an allergen affects immune complex shape and subsequent effector cell activation in vitro and in vivo. METHODS: We constructed artificial allergens by grafting IgE epitopes in different numbers and proximity onto a scaffold protein. The shape of immune complexes formed between artificial allergens and the corresponding IgE was studied by negative-stain electron microscopy. Allergenic activity was determined using basophil activation assays. Mice were primed with IgE, followed by injection of artificial allergens to evaluate their in vivo allergenic activity. Severity of systemic anaphylaxis was measured by changes in body temperature. RESULTS: We could demonstrate simultaneous binding of 4 IgE antibodies in close vicinity to each other. The proximity of IgE binding sites on allergens influenced the shape of the resulting immune complexes and the magnitude of effector cell activation and in vivo inflammation. CONCLUSIONS: Our results demonstrate that the proximity of IgE epitopes on an allergen affects its allergenic activity. We thus identified a novel mechanism by which IgE-allergen complexes regulate allergic inflammation. This mechanism should be important for allergy and other immune complex-mediated diseases.

Visualization of retroviral envelope spikes in complex with the V3 loop antibody 447-52D on intact viruses by cryo-electron tomography.

UNLABELLED: The gp120 portion of the envelope spike on human immunodeficiency virus type 1 (HIV-1) plays a critical role in viral entry into host cells and is a key target for the humoral immune response, and yet many structural details remain elusive. We have used cryoelectron tomography to visualize the binding of the broadly neutralizing monoclonal antibody (MAb) 447-52D to intact envelope spikes on virions of HIV-1 MN strain. Antibody 447-52D has previously been shown to bind to the tip of the V3 loop. Our results show antibody arms radiating from the sides of the gp120 protomers at a range of angles and place the antibody-bound V3 loop in an orientation that differs from that predicted by most current models but consistent with the idea that antibody binding dislodges the V3 loop from its location in the Env spike, making it flexible and disordered. These data reveal information on the position of the V3 loop and its relative flexibility and suggest that 447-52D neutralizes HIV-1 MN by capturing the V3 loop, blocking its interaction with the coreceptor and altering the structure of the envelope spike. IMPORTANCE: Antibody neutralization is one of the primary ways that the body fights infection with HIV. Because HIV is a highly mutable virus, the body must constantly produce new antibodies to counter new strains of HIV that the body itself is producing. Consequently, antibodies capable of neutralizing multiple HIV strains are comparatively few. An improved understanding of the mechanism of antibody neutralization might advance the development of immunogens. Most neutralizing antibodies target the Env glycoprotein spikes found on the virus surface. The broadly neutralizing antibody 447-52D targets the highly conserved ß-turn of variable loop 3 (V3) of gp120. The importance of V3 lies in its contribution to the coreceptor binding site on the target cell. We show here that 447-52D binding to V3 converts the Env conformation from closed to open and makes the V3 loop highly flexible, implying disruption of coreceptor binding and attachment to the target cell.

Cryo-electron microscopy in the study of virus entry and infection.

Viruses have been responsible for many epidemics and pandemics that have impacted human life globally. The COVID-19 pandemic highlighted both our vulnerability to viral outbreaks, as well as the mobilization of the scientific community to come together to combat the unprecedented threat to humanity. Cryo-electron microscopy (cryo-EM) played a central role in our understanding of SARS-CoV-2 during the pandemic and continues to inform about this evolving pathogen. Cryo-EM with its two popular imaging modalities, single particle analysis (SPA) and cryo-electron tomography (cryo-ET), has contributed immensely to understanding the structure of viruses and interactions that define their life cycles and pathogenicity. Here, we review how cryo-EM has informed our understanding of three distinct viruses, of which two - HIV-1 and SARS-CoV-2 infect humans, and the third, bacteriophages, infect bacteria. For HIV-1 and SARS-CoV-2 our focus is on the surface glycoproteins that are responsible for mediating host receptor binding, and host and cell membrane fusion, while for bacteriophages, we review their structure, capsid maturation, attachment to the bacterial cell surface and infection initiation mechanism.

Binding of anti-membrane-proximal gp41 monoclonal antibodies to CD4-liganded and -unliganded human immunodeficiency virus type 1 and simian immunodeficiency virus virions.

The broadly neutralizing monoclonal antibodies (MAbs) 4E10, 2F5, and Z13e1 target membrane-proximal external region (MPER) epitopes of HIV-1 gp41 in a manner that remains controversial. The requirements for initial lipid bilayer binding and/or CD4 ligation have been proposed. To further investigate these issues, we probed for binding of these MAbs to human immunodeficiency virus type 1 (HIV-1) and simian immunodeficiency virus (SIV) virions with protein A-conjugated gold (PAG) nanoparticles using negative-stain electron microscopy. We found moderate levels of PAG associated with unliganded HIV-1 and SIV virions incubated with the three MAbs. Significantly higher levels of PAG were associated with CD4-liganded HIV-1 (epitope-positive) but not SIV (epitope-negative) virions. A chimeric SIV virion displaying the HIV-1 4E10 epitope also showed significantly higher PAG association after CD4 ligation and incubation with 4E10. MAbs accumulated rapidly on CD4-liganded virions and slowly on unliganded virions, although both reached similar levels in time. Anti-MPER epitope-specific binding was stable to washout. Virions incubated with an irrelevant MAb or CD4-only (no MAb) showed negligible PAG association, as did a vesicle-rich fraction devoid of virions. Preincubation with Fab 4E10 inhibited both specific and nonspecific 4E10 IgG binding. Our data provide evidence for moderate association of anti-MPER MAbs to viral surfaces but not lipid vesicles, even in the absence of cognate epitopes. Significantly greater MAb interaction occurs in epitope-positive virions following long incubation or CD4 ligation. These findings are consistent with a two-stage binding model where these anti-MPER MAbs bind first to the viral lipid bilayer and then to the MPER epitopes following spontaneous or induced exposure.