Differences in Oligomerization of the SARS-CoV-2 Envelope Protein, Poliovirus VP4, and HIV Vpu.

Viroporins constitute a class of viral membrane proteins with diverse roles in the viral life cycle. They can self-assemble and form pores within the bilayer that transport substrates, such as ions and genetic material, that are critical to the viral infection cycle. However, there is little known about the oligomeric state of most viroporins. Here, we use native mass spectrometry in detergent micelles to uncover the patterns of oligomerization of the full-length SARS-CoV-2 envelope (E) protein, poliovirus VP4, and HIV Vpu. Our data suggest that the E protein is a specific dimer, VP4 is exclusively monomeric, and Vpu assembles into a polydisperse mixture of oligomers under these conditions. Overall, these results revealed the diversity in the oligomerization of viroporins, which has implications for the mechanisms of their biological functions as well as their potential as therapeutic targets.

Insights into the oligomeric structure of the HIV-1 Vpu protein.

The HIV-1-encoded protein Vpu forms an oligomeric ion channel/pore in membranes and interacts with host proteins to support the virus lifecycle. However, Vpu molecular mechanisms are currently not well understood. Here, we report on the Vpu oligomeric organization under membrane and aqueous conditions and provide insights into how the Vpu environment affects the oligomer formation. For these studies, we designed a maltose-binding protein (MBP)-Vpu chimera protein and produced it in E. coli in soluble form. We analyzed this protein using analytical size-exclusion chromatography (SEC), negative staining electron microscopy (nsEM), and electron paramagnetic resonance (EPR) spectroscopy. Surprisingly, we found that MBP-Vpu formed stable oligomers in solution, seemingly driven by Vpu transmembrane domain self-association. A coarse modeling of nsEM data as well as SEC and EPR data suggests that these oligomers most likely are pentamers, similar to what was reported regarding membrane-bound Vpu. We also noticed reduced MBP-Vpu oligomer stability upon reconstitution of the protein in ß-DDM detergent and mixtures of lyso-PC/PG or DHPC/DHPG. In these cases, we observed greater oligomer heterogeneity, with MBP-Vpu oligomeric order generally lower than in solution; however, larger oligomers were also present. Notably, we found that in lyso-PC/PG, above a certain protein concentration, MBP-Vpu assembles into extended structures, which had not been reported for Vpu. Therefore, we captured various Vpu oligomeric forms, which can shed light on Vpu quaternary organization. Our findings could be useful in understanding Vpu organization and function in cellular membranes and could provide information regarding the biophysical properties of single-pass transmembrane proteins.

Unravelling the Immunomodulatory Effects of Viral Ion Channels, towards the Treatment of Disease.

The current COVID-19 pandemic has highlighted the need for the research community to develop a better understanding of viruses, in particular their modes of infection and replicative lifecycles, to aid in the development of novel vaccines and much needed anti-viral therapeutics. Several viruses express proteins capable of forming pores in host cellular membranes, termed "Viroporins". They are a family of small hydrophobic proteins, with at least one amphipathic domain, which characteristically form oligomeric structures with central hydrophilic domains. Consequently, they can facilitate the transport of ions through the hydrophilic core. Viroporins localise to host membranes such as the endoplasmic reticulum and regulate ion homeostasis creating a favourable environment for viral infection. Viroporins also contribute to viral immune evasion via several mechanisms. Given that viroporins are often essential for virion assembly and egress, and as their structural features tend to be evolutionarily conserved, they are attractive targets for anti-viral therapeutics. This review discusses the current knowledge of several viroporins, namely Influenza A virus (IAV) M2, Human Immunodeficiency Virus (HIV)-1 Viral protein U (Vpu), Hepatitis C Virus (HCV) p7, Human Papillomavirus (HPV)-16 E5, Severe Acute Respiratory Syndrome Coronavirus (SARS-CoV) Open Reading Frame (ORF)3a and Polyomavirus agnoprotein. We highlight the intricate but broad immunomodulatory effects of these viroporins and discuss the current antiviral therapies that target them; continually highlighting the need for future investigations to focus on novel therapeutics in the treatment of existing and future emergent viruses.

Can Glycosylation Mask the Detection of MHC Expressing p53 Peptides by T Cell Receptors?

Proteins of the major histocompatibility complex (MHC) class I, or human leukocyte antigen (HLA) in humans interact with endogenous peptides and present them to T cell receptors (TCR), which in turn tune the immune system to recognize and discriminate between self and foreign (non-self) peptides. Of especial importance are peptides derived from tumor-associated antigens. T cells recognizing these peptides are found in cancer patients, but not in cancer-free individuals. What stimulates this recognition, which is vital for the success of checkpoint based therapy? A peptide derived from the protein p53 (residues 161-169 or p161) was reported to show this behavior. T cells recognizing this unmodified peptide could be further stimulated in vitro to create effective cancer killing CTLs (cytotoxic T lymphocytes). We hypothesize that the underlying difference may arise from post-translational glycosylation of p161 in normal individuals, likely masking it against recognition by TCR. Defects in glycosylation in cancer cells may allow the presentation of the native peptide. We investigate the structural consequences of such peptide glycosylation by investigating the associated structural dynamics.

Phlorotannins as HIV Vpu inhibitors, an in silico virtual screening study of marine natural products.

The importance of new effective treatment methodologies for human immunodeficiency virus (HIV) is undeniable for the medical society. Viral protein U (Vpu), one of the disparaged accessory proteins of HIV, is responsible for the dissemination of viral particles, and HIV mutants lacking Vpu protein have remarkably reduced pathogenicity. Here, we explored the marine natural products to find the leading structures which can potentially inhibit the activity of Vpu in silico. To fulfill this goal, we set up a virtual screening based on molecular docking to evaluate the binding capacity of different marine products to Vpu. For validation, we used molecular dynamics simulation and monitored the root mean square deviation value and binding interactions. The results were intriguing when we realized that the hit compounds (phlorotannins) had previously been identified as reverse transcriptase and HIV protease inhibitors. This research inaugurates a new road to combat HIV by multifaceted mode of action of these marine natural products without putting the normal cells in jeopardy (with their safe toxicological profile).

Performance of rotation forest ensemble classifier and feature extractor in predicting protein interactions using amino acid sequences.

BACKGROUND: There are two significant problems associated with predicting protein-protein interactions using the sequences of amino acids. The first problem is representing each sequence as a feature vector, and the second is designing a model that can identify the protein interactions. Thus, effective feature extraction methods can lead to improved model performance. In this study, we used two types of feature extraction methods-global encoding and pseudo-substitution matrix representation (PseudoSMR)-to represent the sequences of amino acids in human proteins and Human Immunodeficiency Virus type 1 (HIV-1) to address the classification problem of predicting protein-protein interactions. We also compared principal component analysis (PCA) with independent principal component analysis (IPCA) as methods for transforming Rotation Forest. RESULTS: The results show that using global encoding and PseudoSMR as a feature extraction method successfully represents the amino acid sequence for the Rotation Forest classifier with PCA or with IPCA. This can be seen from the comparison of the results of evaluation metrics, which were >73% across the six different parameters. The accuracy of both methods was >74%. The results for the other model performance criteria, such as sensitivity, specificity, precision, and F1-score, were all >73%. The data used in this study can be accessed using the following link: https://www.dsc.ui.ac.id/research/amino-acid-pred/. CONCLUSIONS: Both global encoding and PseudoSMR can successfully represent the sequences of amino acids. Rotation Forest (PCA) performed better than Rotation Forest (IPCA) in terms of predicting protein-protein interactions between HIV-1 and human proteins. Both the Rotation Forest (PCA) classifier and the Rotation Forest IPCA classifier performed better than other classifiers, such as Gradient Boosting, K-Nearest Neighbor, Logistic Regression, Random Forest, and Support Vector Machine (SVM). Rotation Forest (PCA) and Rotation Forest (IPCA) have accuracy, sensitivity, specificity, precision, and F1-score values >70% while the other classifiers have values <70%.

Hydrophobic matching of HIV-1 Vpu transmembrane helix-helix interactions is optimized for subcellular location.

The HIV-1 accessory protein Vpu mediates the downregulation of several host cell proteins, an activity that is critical for viral replication in vivo. As the first step in directing cell-surface proteins to internal cellular compartments, and in many cases degradation, Vpu binds a subset of its target proteins through their transmembrane domains. Each of the known targets of Vpu are synthesized in the ER, and must traverse the different membrane environments found along the secretory pathway, thus it is important to consider how membrane composition might influence the interactions between Vpu and its targets. We have used Förster resonance energy transfer (FRET) to measure the oligomerization of Vpu with the transmembrane domains of target proteins in model membranes of varying lipid composition. Our data show that both lipid bilayer thickness and acyl chain order can significantly influence monomer-oligomer equilibria within the Vpu-target system. Changes in oligomerization levels were found to be non-specific with no single Vpu-target interaction being favored under any condition. Our analysis of the influence of the membrane environment on the strength of helix-helix interactions between Vpu and its targets in vitro suggests that the strength of Vpu-target interactions in vivo will be partially dependent on the membrane environment found in specific membrane compartments.

Human T-cell leukemia virus type 1 Gag domains have distinct RNA-binding specificities with implications for RNA packaging and dimerization.

Human T-cell leukemia virus type 1 (HTLV-1) is the first retrovirus that has conclusively been shown to cause human diseases. In HIV-1, specific interactions between the nucleocapsid (NC) domain of the Gag protein and genomic RNA (gRNA) mediate gRNA dimerization and selective packaging; however, the mechanism for gRNA packaging in HTLV-1, a deltaretrovirus, is unclear. In other deltaretroviruses, the matrix (MA) and NC domains of Gag are both involved in gRNA packaging, but MA binds nucleic acids with higher affinity and has more robust chaperone activity, suggesting that this domain may play a primary role. Here, we show that the MA domain of HTLV-1, but not the NC domain, binds short hairpin RNAs derived from the putative gRNA packaging signal. RNA probing of the HTLV-1 5' leader and cross-linking studies revealed that the primer-binding site and a region within the putative packaging signal form stable hairpins that interact with MA. In addition to a previously identified palindromic dimerization initiation site (DIS), we identified a new DIS in HTLV-1 gRNA and found that both palindromic sequences bind specifically the NC domain. Surprisingly, a mutant partially defective in dimer formation in vitro exhibited a significant increase in RNA packaging into HTLV-1-like particles, suggesting that efficient RNA dimerization may not be strictly required for RNA packaging in HTLV-1. Moreover, the lifecycle of HTLV-1 and other deltaretroviruses may be characterized by NC and MA functions that are distinct from those of the corresponding HIV-1 proteins, but together provide the functions required for viral replication.

HLA-C downregulation by HIV-1 adapts to host HLA genotype.

HIV-1 can downregulate HLA-C on infected cells, using the viral protein Vpu, and the magnitude of this downregulation varies widely between primary HIV-1 variants. The selection pressures that result in viral downregulation of HLA-C in some individuals, but preservation of surface HLA-C in others are not clear. To better understand viral immune evasion targeting HLA-C, we have characterized HLA-C downregulation by a range of primary HIV-1 viruses. 128 replication competent viral isolates from 19 individuals with effective anti-retroviral therapy, show that a substantial minority of individuals harbor latent reservoir virus which strongly downregulates HLA-C. Untreated infections display no change in HLA-C downregulation during the first 6 months of infection, but variation between viral quasispecies can be detected in chronic infection. Vpu molecules cloned from plasma of 195 treatment naïve individuals in chronic infection demonstrate that downregulation of HLA-C adapts to host HLA genotype. HLA-C alleles differ in the pressure they exert for downregulation, and individuals with higher levels of HLA-C expression favor greater viral downregulation of HLA-C. Studies of primary and mutant molecules identify 5 residues in the transmembrane region of Vpu, and 4 residues in the transmembrane domain of HLA-C, which determine interactions between Vpu and HLA. The observed adaptation of Vpu-mediated downregulation to host genotype indicates that HLA-C alleles differ in likelihood of mediating a CTL response that is subverted by viral downregulation, and that preservation of HLA-C expression is favored in the absence of these responses. Finding that latent reservoir viruses can downregulate HLA-C could have implications for HIV-1 cure therapy approaches in some individuals.

Phosphoserine acidic cluster motifs bind distinct basic regions on the µ subunits of clathrin adaptor protein complexes.

Protein trafficking in the endosomal system involves the recognition of specific signals within the cytoplasmic domains (CDs) of transmembrane proteins by clathrin adaptors. One such signal is the phosphoserine acidic cluster (PSAC), the prototype of which is in the endoprotease furin. How PSACs are recognized by clathrin adaptors has been controversial. We reported previously that HIV-1 Vpu, which modulates cellular immunoreceptors, contains a PSAC that binds to the µ subunits of clathrin adaptor protein (AP) complexes. Here, we show that the CD of furin binds the µ subunits of AP-1 and AP-2 in a phosphorylation-dependent manner. Moreover, we identify a potential PSAC in a cytoplasmic loop of the cellular transmembrane Serinc3, an inhibitor of the infectivity of retroviruses. The two serines within the PSAC of Serinc3 are phosphorylated by casein kinase II and mediate interaction with the µ subunits in vitro The sites of these serines vary among mammals in a manner suggesting host-pathogen conflict, yet the Serinc3 PSAC seems dispensable for anti-HIV activity and for counteraction by HIV-1 Nef. The CDs of Vpu and furin and the PSAC-containing loop of Serinc3 each bind the µ subunit of AP-2 (µ2) with similar affinities, but they appear to utilize different basic regions on µ2. The Serinc3 loop requires a region previously reported to bind the acidic plasma membrane lipid phosphatidylinositol 4,5-bisphosphate. These data suggest that the PSACs within different proteins recognize different basic regions on the µ surface, providing the potential to inhibit the activity of viral proteins without necessarily affecting cellular protein trafficking.

Structural discordance in HIV-1 Vpu from brain isolate alarms amyloid fibril forming behavior- a computational perspective.

HIV-1 being the most widespread type worldwide, its accounts for almost 95% of all infections including HIV associated dementia (HAD) that triggers neurological dysfunction and neurodegeneration in patients. The common features associated with HAD and other neurodegenerative diseases are accumulation of amyloid plaques, neuronal loss and deterioration of cognitive abilities, amongst which amyloid fibrillation is considered to be a hallmark. The success of effective therapeutics lies in the understanding of mechanisms leading to neurotoxicity. Few viral proteins like gp-120 are known to be involved in aggregation and enhancement of viral infectivity while comprehending the neurotoxic role of some other proteins is still underway. In the current study, amyloidogenic potential of HIV-1 Vpu protein from brain isolate is investigated through computational approaches. The aggregation propensity of brain derived HIV-1 Vpu was assessed by several amyloid prediction servers that projected the region 4-35 to be amyloidogenic. The protein structure was modeled and subjected to 70 ns molecular dynamics (MD) simulation to investigate the transformation of α-helical conformation of the predicted aggregate region into ß-sheet, proposing the protein's ability to initiate fibril formation that is central to amyloidogenic proteins. The structural features of brain derived HIV-1 Vpu were consistent with the in silico amyloid prediction results that depicts the conformational change in the region 8-28 of which residues Ala8, Ile9, Val10, Ala19, Ile20 and Val21 constitutes ß-sheet formation. The α-helix/ß-sheet discordance of the predicted region was reflected in the simulation study highlighting the possible structural transition associated with HIV-1 Vpu protein of brain isolate.

Prediction of HIV-1 and HIV-2 proteins by using Chou's pseudo amino acid compositions and different classifiers.

Human immunodeficiency virus (HIV) is the retroviral agent that causes acquired immune deficiency syndrome (AIDS). The number of HIV caused deaths was about 4 million in 2016 alone; it was estimated that about 33 million to 46 million people worldwide living with HIV. The HIV disease is especially harmful because the progressive destruction of the immune system prevents the ability of forming specific antibodies and to maintain an efficacious killer T cell activity. Successful prediction of HIV protein has important significance for the biological and pharmacological functions. In this study, based on the concept of Chou's pseudo amino acid (PseAA) composition and increment of diversity (ID), support vector machine (SVM), logisitic regression (LR), and multilayer perceptron (MP) were presented to predict HIV-1 proteins and HIV-2 proteins. The results of the jackknife test indicated that the highest prediction accuracy and CC values were obtained by the SVM and MP were 0.9909 and 0.9763, respectively, indicating that the classifiers presented in this study were suitable for predicting two groups of HIV proteins.

The HIV-1 accessory proteins Nef and Vpu downregulate total and cell surface CD28 in CD4+ T cells.

BACKGROUND: The HIV-1 accessory proteins Nef and Vpu alter cell surface levels of multiple host proteins to modify the immune response and increase viral persistence. Nef and Vpu can downregulate cell surface levels of the co-stimulatory molecule CD28, however the mechanism of this function has not been completely elucidated. RESULTS: Here, we provide evidence that Nef and Vpu decrease cell surface and total cellular levels of CD28. Moreover, using inhibitors we implicate the cellular degradation machinery in the downregulation of CD28. We shed light on the mechanisms of CD28 downregulation by implicating the Nef LL165 and DD175 motifs in decreasing cell surface CD28 and Nef DD175 in decreasing total cellular CD28. Moreover, the Vpu LV64 and S52/56 motifs were required for cell surface CD28 downregulation, while, unlike for CD4 downregulation, Vpu W22 was dispensable. The Vpu S52/56 motif was also critical for Vpu-mediated decreases in total CD28 protein level. Finally, the ability of Vpu to downregulate CD28 is conserved between multiple group M Vpu proteins and infection with viruses encoding or lacking Nef and Vpu have differential effects on activation upon stimulation. CONCLUSIONS: We report that Nef and Vpu downregulate cell surface and total cellular CD28 levels. We identified inhibitors and mutations within Nef and Vpu that disrupt downregulation, shedding light on the mechanisms utilized to downregulate CD28. The conservation and redundancy between the abilities of two HIV-1 proteins to downregulate CD28 highlight the importance of this function, which may contribute to the development of latently infected cells.

FRET Analysis of the Promiscuous yet Specific Interactions of the HIV-1 Vpu Transmembrane Domain.

The Vpu protein of HIV-1 functions to downregulate cell surface localization of host proteins involved in the innate immune response to viral infection. For several target proteins, including the NTB-A and PVR receptors and the host restriction factor tetherin, this antagonism is carried out via direct interactions between the transmembrane domains (TMDs) of Vpu and the target. The Vpu TMD also modulates homooligomerization of this protein, and the tetherin TMD forms homodimers. The mechanism through which a single transmembrane helix is able to recognize and interact with a wide range of select targets that do not share known interaction motifs is poorly understood. Here we use Förster resonance energy transfer to characterize the energetics of homo- and heterooligomer interactions between the Vpu TMD and several target proteins. Our data show that target TMDs compete for interaction with Vpu, and that formation of each heterooligomer has a similar dissociation constant (Kd) and free energy of association to the Vpu homooligomer. This leads to a model in which Vpu monomers, Vpu homooligomers, and Vpu-target heterooligomers coexist, and suggests that the conserved binding surface of Vpu TMD has been selected for weak binding to multiple targets.

Human Immunodeficiency Virus Proteins Mimic Human T Cell Receptors Inducing Cross-Reactive Antibodies.

Human immunodeficiency virus (HIV) hides from the immune system in part by mimicking host antigens, including human leukocyte antigens. It is demonstrated here that HIV also mimics the V-ß-D-J-ß of approximately seventy percent of about 600 randomly selected human T cell receptors (TCR). This degree of mimicry is greater than any other human pathogen, commensal or symbiotic organism studied. These data suggest that HIV may be evolving into a commensal organism just as simian immunodeficiency virus has done in some types of monkeys. The gp120 envelope protein, Nef protein and Pol protein are particularly similar to host TCR, camouflaging HIV from the immune system and creating serious barriers to the development of safe HIV vaccines. One consequence of HIV mimicry of host TCR is that antibodies against HIV proteins have a significant probability of recognizing the corresponding TCR as antigenic targets, explaining the widespread observation of lymphocytotoxic autoantibodies in acquired immunodeficiency syndrome (AIDS). Quantitative enzyme-linked immunoadsorption assays (ELISA) demonstrated that every HIV antibody tested recognized at least one of twelve TCR, and as many as seven, with a binding constant in the 10-8 to 10-9 m range. HIV immunity also affects microbiome tolerance in ways that correlate with susceptibility to specific opportunistic infections.

How to win the HIV-1 drug resistance hurdle race: running faster or jumping higher?

Infections by the human immunodeficiency virus type 1 (HIV-1), the causative agent of the acquired immunodeficiency syndrome (AIDS), are still totaling an appalling 36.7 millions worldwide, with 1.1 million AIDS deaths/year and a similar number of yearly new infections. All this, in spite of the discovery of HIV-1 as the AIDS etiological agent more than 30 years ago and the introduction of an effective combinatorial antiretroviral therapy (cART), able to control disease progression, more than 20 years ago. Although very effective, current cART is plagued by the emergence of drug-resistant viral variants and most of the efforts in the development of novel direct-acting antiviral agents (DAAs) against HIV-1 have been devoted toward the fighting of resistance. In this review, rather than providing a detailed listing of all the drugs and the corresponding resistance mutations, we aim, through relevant examples, at presenting to the general reader the conceptual shift in the approaches that are being taken to overcome the viral resistance hurdle. From the classic 'running faster' strategy, based on the development of novel DAAs active against the mutant viruses selected by the previous drugs and/or presenting to the virus a high genetic barrier toward the development of resilience, to a 'jumping higher' approach, which looks at the cell, rather than the virus, as a source of valuable drug targets, in order to make the cellular environment non-permissive toward the replication of both wild-type and mutated viruses.

Herb-target interaction network analysis helps to disclose molecular mechanism of traditional Chinese medicine.

Though many studies have been performed to elucidate molecular mechanism of traditional Chinese medicines (TCMs) by identifying protein-compound interactions, no systematic analysis at herb level was reported. TCMs are prescribed by herbs and all compounds from a certain herb should be considered as a whole, thus studies at herb level may provide comprehensive understanding of TCMs. Here, we proposed a computational strategy to study molecular mechanism of TCM at herb level and used it to analyze a TCM anti-HIV formula. Herb-target network analysis was carried out between 17 HIV-related proteins and SH formula as well as three control groups based on systematic docking. Inhibitory herbs were identified and active compounds enrichment was found to contribute to the therapeutic effectiveness of herbs. Our study demonstrates that computational analysis of TCMs at herb level can catch the rationale of TCM formulation and serve as guidance for novel TCM formula design.

LC3C Contributes to Vpu-Mediated Antagonism of BST2/Tetherin Restriction on HIV-1 Release through a Non-canonical Autophagy Pathway.

BST2 (bone marrow stromal antigen 2)/tetherin is a restriction factor of enveloped viruses, which blocks the release of viral particles. HIV-1 encodes proteins that antagonize this innate barrier, including the accessory protein Vpu. Here, we investigate whether the autophagy pathway and/or ATG proteins are hijacked by HIV-1 Vpu to circumvent BST2 restriction of viral release. We report that BST2 and Vpu are present in LC3-positive compartments. We found that Vpu selectively interacts with the ATG8 ortholog LC3C through the Vpu L63VEM66 sequence. This sequence is required for Vpu to antagonize BST2 restriction. LC3C expression favors the removal of BST2 from the HIV-1 budding site, and thus HIV-1 release in BST2-expressing cells. Additionally, ATG5 and beclin 1/ATG6, but not all the components of the autophagy pathway, act with LC3C to facilitate Vpu antagonism of BST2 restriction. Altogether, our data support the view that a non-canonical autophagy pathway reminiscent of LC3-associated phagocytosis contributes to Vpu counteraction of BST2 restriction.

Expression, purification and characterization of a full-length recombinant HIV-1 Vpu from inclusion bodies.

Vpu is one of four accessory proteins encoded by human immunodeficiency virus type I (HIV-1). Vpu modulates the expression of several cellular restriction factors within the HIV-1 infected cell including CD4, CD74, the bone marrow stromal antigen 2 (BST-2) and NK-T-and-B antigen. The interaction of HIV-1 Vpu with these proteins interferes with the innate immune response directed against HIV-1; thereby promoting viral persistence. The involvement of HIV-1 Vpu in manipulating the cellular environment in ways that favor viral replication makes it an attractive target for anti-HIV drug intervention. This paper describes the over-expression and purification of a soluble HIV-1 Vpu from inclusion bodies by ion-exchange chromatography, allowing production of 6 mg of highly purified protein (>95% purity) per 10 mg of pelleted cells obtained from 1 L of bacterial culture. Far-UV circular dichroism showed that the recombinant protein is folded and retained its secondary structure. Moreover, using ELISA, known HIV-1 Vpu binding partners, BST-2 and CD74, showed that the refolded purified protein is functional or at least assumes a conformation that is capable of binding these putative binding partners. To our knowledge, this is the first report of the purification and successful solubilization of full-length, wild-type HIV-1 Vpu from inclusion bodies in Escherichia coli.