Heterologous Complex Research Articles

Subcellular imaging of MGAT1/MGAT2 homo- and heteromers in living cells using bioluminescence microscopy

Protein-protein interactions (PPIs) play fundamental roles in many biological processes including the functioning of glycosylation machineries present in the endoplasmic reticulum (ER) and Golgi apparatus of mammalian cells. For the last couple of years, we have been successfully employing the most advanced version of the split luciferase complementation assay, termed NanoBiT, to demonstrate PPIs between solute carrier 35 (SLC35) family members with nucleotide sugar transporting activity and functionally related glycosyltransferases. NanoBiT has several unmatched advantages as compared with other strategies for studying PPIs. Firstly, the tendency of the free luciferase fragments to spontaneously associate is strongly reduced. As a consequence, the fragments of the reconstituted luciferase may dissociate upon the disruption of the PPI of interest. Secondly, the recombinant fusion proteins are expressed at low (near-endogenous) levels. Both of these features significantly minimize the possibility of obtaining false positive results. In this study we pushed the boundaries of this already powerful technique even further by coupling it with bioluminescence imaging of PPIs. Specifically, we visualized homo- and heterologous complexes formed by MGAT1 and MGAT2 glycosylation enzymes tagged with NanoBiT fragments and demonstrated ER-to-Golgi transitions between enzyme homo- and heteromers.

Human Bocavirus 1 NP1 acts as an ssDNA-binding protein to help AAV2 DNA replication and cooperates with RPA to regulate AAV2 capsid expression.

Adeno-associated virus (AAV) requires co-infection with helper virus for efficient replication. We previously reported that Human Bocavirus 1 (HBoV1) genes, including NP1, NS2, and BocaSR, were critical for AAV2 replication. Here, we first demonstrate the essential roles of the NP1 protein in AAV2 DNA replication and protein expression. We show that NP1 binds to single-strand DNA (ssDNA) at least 30 nucleotides (nt) in length in a sequence-independent manner. Furthermore, NP1 colocalized with the BrdU-labeled AAV2 DNA replication center, and the loss of the ssDNA-binding ability of NP1 by site-directed mutation completely abolished AAV2 DNA replication. We used affinity-tagged NP1 protein to identify host cellular proteins associated with NP1 in cells cotransfected with the HBoV1 helper genes and AAV2 duplex genome. Of the identified proteins, we demonstrate that NP1 directly binds to the DBD-F domain of the RPA70 subunit with a high affinity through the residues 101-121. By reconstituting the heterotrimer protein RPA in vitro using gel filtration, we demonstrate that NP1 physically associates with RPA to form a heterologous complex characterized by typical fast-on/fast-off kinetics. Following a dominant-negative strategy, we found that NP1-RPA complex mainly plays a role in expressing AAV2 capsid protein by enhancing the transcriptional activity of the p40 promoter. Our study revealed a novel mechanism by which HBoV1 NP1 protein supports AAV2 DNA replication and capsid protein expression through its ssDNA-binding ability and direct interaction with RPA, respectively.IMPORTANCERecombinant adeno-associated virus (rAAV) vectors have been extensively used in clinical gene therapy strategies. However, a limitation of these gene therapy strategies is the efficient production of the required vectors, as AAV alone is replication-deficient in the host cells. HBoV1 provides the simplest AAV2 helper genes consisting of NP1, NS2, and BocaSR. An important question regarding the helper function of HBoV1 is whether it provides any direct function that supports AAV2 DNA replication and protein expression. Also of interest is how HBoV1 interplays with potential host factors to constitute a permissive environment for AAV2 replication. Our studies revealed that the multifunctional protein NP1 plays important roles in AAV2 DNA replication via its sequence-independent ssDNA-binding ability and in regulating AAV2 capsid protein expression by physically interacting with host protein RPA. Our findings present theoretical guidance for the future application of the HBoV1 helper genes in the rAAV vector production.

Construction of an Escherichia coli cell factory for de novo synthesis of tyrosol through semi-rational design based on phenylpyruvate decarboxylase ARO10 engineering

Tyrosol (2-(4-hydroxyphenyl) ethanol) is extensively used in the pharmaceutical industry as an important natural product from plants. In previous research, we constructed a recombinant Escherichia coli strain capable of de novo synthesis of tyrosol by integrating the phenylpyruvate decarboxylase ARO10 derived from Saccharomyces cerevisiae. Nevertheless, the insufficient catalytic efficiency of ARO10 required the insertion of multiple gene copies into the genome to attain enhanced tyrosol production. In this study, we constructed a mutation library of ARO10 based on a computer-aided semi-rational design strategy and developed a high-throughput screening method for selecting high-yield tyrosol mutants by introducing the heterologous hydroxylase complex HpaBC. Through multiple rounds of screening and site-saturation mutagenesis, we ultimately identified the two optimal ARO10 mutants, ARO10D331V and ARO10D331C, which respectively achieved a tyrosol titer of 2.02 g/L and 2.04 g/L in shake flasks, both representing more than 50 % improvement compared to the wild-type. Our study demonstrates the great potential of computer-based semi-rational enzyme design strategy in metabolic engineering. The high-throughput screening method for target compound derivative possesses a certain level of generality. Ultimately, we obtained promising mutants capable of achieving industrial-scale production of tyrosol, which also lays a solid foundation for the efficient synthesis of tyrosol derivatives.

Structural characterization of the type I-B CRISPR Cas7 from Thermobaculum terrenum

Clustered regularly interspaced short palindromic repeats (CRISPR) in many prokaryotes functions as an adaptive immune system against mobile genetic elements. A heterologous ribonucleoprotein silencing complex composed of CRISPR-associated (Cas) proteins and a CRISPR RNA (crRNA) neutralizes the incoming mobile genetic elements. The type I and III silencing complexes commonly include a protein-helical backbone of several copies of identical subunits, for example, Cas7 in the type I silencing complex.In this study, we structurally characterized type I-B Cas7 (Csh2 from Thermobaculum terrenum; TterCsh2). The revealed crystal structure of TterCsh2 shows a typical glove-like architecture of Cas7, which consists of a palm, a thumb, and a finger domain. Csh2 proteins have 5 conserved sequence motifs that are arranged to form a presumable crRNA-binding site in the TterCsh2 structure. This crRNA binding site of TterCsh2 is structurally and potentially comparable to those observed in helix-forming Cas7 structures in other sub-types. Analysis of the reported Cas7 structures and their sequences suggests that Cas7s can be divided into at least two sub-classes. These data will broaden our understanding on the Cascade complex of CRISPR/Cas systems.

An interaction between SLC35A1 and ST3Gal4 is differentially affected by CDG-causing mutations in the SLC35A1 gene

The sialylation of glycoconjugates is performed by a variety of sialyltransferases using CMP-sialic acid (CMP-Sia) as a substrate. Sialylation requires the translocation of CMP-Sia across the Golgi membranes. This function has been assigned to SLC35A1, the only CMP-Sia transporter identified to date. Mutations in the SLC35A1 gene cause a subtype of congenital disorder of glycosylation (CDG). Over the past several years, heterologous complexes formed in the Golgi membrane by some SLC35A subfamily members and functionally related glycosyltransferases have been reported. However, to date no interaction between SLC35A1 and a sialyltransferase has been identified. In this study we attempted to clarify the role of SLC35A1 in α2,3 sialylation of N-glycans. We showed that SLC35A1 associates with ST3Gal4, the main α2,3-sialyltransferase acting on N-glycans. This phenomenon is compromised by the E196K (but not T156R) mutation in the SLC35A1 gene. We also demonstrated that the E196K mutant is less efficient in restoring N-glycan sialylation upon expression in the SLC35A1 knockout cells. On the basis of our findings, we propose that the interaction between SLC35A1 and ST3Gal4 may be important for proper sialylation.

A comprehensive review and meta-analysis of recent advances in biotechnology for plant virus research and significant accomplishments in human health and the pharmaceutical industry

ABSTRACT Secondary metabolites made by plants and used through their metabolic routes are today’s most reliable and cost-effective way to make pharmaceuticals and improve health. The concept of genetic engineering is used for molecular pharming. As more people use plants as sources of nanotechnology systems, they are adding to this. These systems are made up of viruses-like particles (VLPs) and virus nanoparticles (VNPs). Due to their superior ability to be used as plant virus expression vectors, plant viruses are becoming more popular in pharmaceuticals. This has opened the door for them to be used in research, such as the production of medicinal peptides, antibodies, and other heterologous protein complexes. This is because biotechnological approaches have been linked with new bioinformatics tools. Because of the rise of high-throughput sequencing (HTS) and next-generation sequencing (NGS) techniques, it has become easier to use metagenomic studies to look for plant virus genomes that could be used in pharmaceutical research. A look at how bioinformatics can be used in pharmaceutical research is also covered in this article. It also talks about plant viruses and how new biotechnological tools and procedures have made progress in the field.

VI-116, A Novel Potent Inhibitor of VRAC with Minimal Effect on ANO1.

Volume-regulated anion channel (VRAC) is ubiquitously expressed and plays a pivotal role in vertebrate cell volume regulation. A heterologous complex of leucine-rich repeat containing 8A (LRRC8A) and LRRC8B-E constitutes the VRAC, which is involved in various processes such as cell proliferation, migration, differentiation, intercellular communication, and apoptosis. However, the lack of a potent and selective inhibitor of VRAC limits VRAC-related physiological and pathophysiological studies, and most previous VRAC inhibitors strongly blocked the calcium-activated chloride channel, anoctamin 1 (ANO1). In the present study, we performed a cell-based screening for the identification of potent and selective VRAC inhibitors. Screening of 55,000 drug-like small-molecules and subsequent chemical modification revealed 3,3′-((2-hydroxy-3-methoxyphenyl)methylene)bis(4-hydroxy-2H-chromen-2-one) (VI-116), a novel potent inhibitor of VRAC. VI-116 fully inhibited VRAC-mediated I− quenching with an IC50 of 1.27 ± 0.18 μM in LN215 cells and potently blocked endogenous VRAC activity in PC3, HT29 and HeLa cells in a dose-dependent manner. Notably, VI-116 had no effect on intracellular calcium signaling up to 10 μM, which completely inhibited VRAC, and showed high selectivity for VRAC compared to ANO1 and ANO2. However, DCPIB, a VRAC inhibitor, significantly affected ATP-induced increases in intracellular calcium levels and Eact-induced ANO1 activation. In addition, VI-116 showed minimal effect on hERG K+ channel activity up to 10 μM. These results indicate that VI-116 is a potent and selective VRAC inhibitor and a useful research tool for pharmacological dissection of VRAC.

Regulation of p53 Function by Formation of Non-Nuclear Heterologous Protein Complexes.

A transcription factor p53 is activated upon cellular exposure to endogenous and exogenous stresses, triggering either homeostatic correction or cell death. Depending on the stress level, often measurable as DNA damage, the dual outcome is supported by p53 binding to a number of regulatory and metabolic proteins. Apart from the nucleus, p53 localizes to mitochondria, endoplasmic reticulum and cytosol. We consider non-nuclear heterologous protein complexes of p53, their structural determinants, regulatory post-translational modifications and the role in intricate p53 functions. The p53 heterologous complexes regulate the folding, trafficking and/or action of interacting partners in cellular compartments. Some of them mainly sequester p53 (HSP proteins, G6PD, LONP1) or its partners (RRM2B, PRKN) in specific locations. Formation of other complexes (with ATP2A2, ATP5PO, BAX, BCL2L1, CHCHD4, PPIF, POLG, SOD2, SSBP1, TFAM) depends on p53 upregulation according to the stress level. The p53 complexes with SIRT2, MUL1, USP7, TXN, PIN1 and PPIF control regulation of p53 function through post-translational modifications, such as lysine acetylation or ubiquitination, cysteine/cystine redox transformation and peptidyl-prolyl cis-trans isomerization. Redox sensitivity of p53 functions is supported by (i) thioredoxin-dependent reduction of p53 disulfides, (ii) inhibition of the thioredoxin-dependent deoxyribonucleotide synthesis by p53 binding to RRM2B and (iii) changed intracellular distribution of p53 through its oxidation by CHCHD4 in the mitochondrial intermembrane space. Increasing knowledge on the structure, function and (patho)physiological significance of the p53 heterologous complexes will enable a fine tuning of the settings-dependent p53 programs, using small molecule regulators of specific protein–protein interactions of p53.

VgrG-dependent effectors and chaperones modulate the assembly of the type VI secretion system.

The type VI secretion system (T6SS) is a spear-like nanomachine found in gram-negative pathogens for delivery of toxic effectors to neighboring bacterial and host cells. Its assembly requires a tip spike complex consisting of a VgrG-trimer, a PAAR protein, and the interacting effectors. However, how the spike controls T6SS assembly remains elusive. Here we investigated the role of three VgrG-effector pairs in Aeromonas dhakensis strain SSU, a clinical isolate with a constitutively active T6SS. By swapping VgrG tail sequences, we demonstrate that the C-terminal ~30 amino-acid tail dictates effector specificity. Double deletion of vgrG1&2 genes (VgrG3+) abolished T6SS secretion, which can be rescued by ectopically expressing chimeric VgrG3 with a VgrG1/2-tail but not the wild type VgrG3. In addition, deletion of effector-specific chaperones also severely impaired T6SS secretion, despite the presence of intact VgrG and effector proteins, in both SSU and Vibrio cholerae V52. We further show that SSU could deliver a V. cholerae effector VasX when expressing a plasmid-borne chimeric VgrG with VasX-specific VgrG tail and chaperone sequences. Pull-down analyses show that two SSU effectors, TseP and TseC, could interact with their cognate VgrGs, the baseplate protein TssK, and the key assembly chaperone TssA. Effectors TseL and VasX could interact with TssF, TssK and TssA in V. cholerae. Collectively, we demonstrate that chimeric VgrG-effector pairs could bypass the requirement of heterologous VgrG complex and propose that effector-stuffing inside the baseplate complex, facilitated by chaperones and the interaction with structural proteins, serves as a crucial structural determinant for T6SS assembly.

Metabolic engineering of Saccharomyces cerevisiae for hydroxytyrosol overproduction directly from glucose.

SummaryHydroxytyrosol (HT) is one of the most powerful dietary antioxidants with numerous applications in different areas, including cosmetics, nutraceuticals and food. In the present work, heterologous hydroxylase complex HpaBC from Escherichia coli was integrated into the Saccharomyces cerevisiae genome in multiple copies. HT productivity was increased by redirecting the metabolic flux towards tyrosol synthesis to avoid exogenous tyrosol or tyrosine supplementation. After evaluating the potential of our selected strain as an HT producer from glucose, we adjusted the medium composition for HT production. The combination of the selected modifications in our engineered strain, combined with culture conditions optimization, resulted in a titre of approximately 375 mg l−1 of HT obtained from shake‐flask fermentation using a minimal synthetic‐defined medium with 160 g l−1 glucose as the sole carbon source. To the best of our knowledge, this is the highest HT concentration produced by an engineered S. cerevisiae strain.

Capture of carbon dioxide and hydrogen by engineered Escherichia coli: hydrogen-dependent CO2 reduction to formate

In times of global climate change and the fear of dwindling resources, we are facing different considerable challenges such as the replacement of fossil fuel–based energy carriers with the coincident maintenance of the increasing energy supply of our growing world population. Therefore, CO2 capturing and H2 storing solutions are urgently needed. In this study, we demonstrate the production of a functional and biotechnological interesting enzyme complex from acetogenic bacteria, the hydrogen-dependent CO2 reductase (HDCR), in the well-known model organism Escherichia coli. We identified the metabolic bottlenecks of the host organisms for the production of the HDCR enzyme complex. Here we show that the recombinant expression of a heterologous enzyme complex transforms E. coli into a whole-cell biocatalyst for hydrogen-driven CO2 reduction to formate without the need of any external co-factors or endogenous enzymes in the reaction process. This shifts the industrial platform organism E. coli more and more into the focus as biocatalyst for CO2-capturing and H2-storage.Key points• A functional HDCR enzyme complex was heterologously produced in E. coli.• The metabolic bottlenecks for HDCR production were identified.• HDCR enabled E. coli cell to capture and store H2and CO2in the form of formate.

Visualization and Analysis of the Dynamic Assembly of a Heterologous Lantibiotic Biosynthesis Complex in Bacillus subtilis.

ABSTRACTA membrane-associated lanthipeptide synthetase complex, consisting of the dehydratase NisB, the cyclase NisC, and the ABC transporter NisT, has been described for nisin biosynthesis in the coccoid bacterium Lactococcus lactis. Here, we used advanced fluorescence microscopy to visualize the functional nisin biosynthesis machinery in rod-shaped cells and analyzed its spatial distribution and dynamics employing a platform we developed for heterologous production of nisin in Bacillus subtilis. We observed that NisT, as well as NisB and NisC, were all distributed in a punctate pattern along the cell periphery, opposed to the situation in coccoid cells. NisBTC proteins were found to be highly colocalized, being visualized at the same spots by dual fluorescence microscopy. In conjunction with the successful isolation of the biosynthetic complex NisBTC from the cell membrane, this corroborated that the visual bright foci were the sites for nisin maturation and transportation. A strategy of differential timing of expression was employed to demonstrate the in vivo dynamic assembly of NisBTC, revealing the recruitment by NisT of NisBC to the membrane. Additionally, by use of mutated proteins, the nucleotide binding domain (NBD) of NisT was found to function as a membrane anchor for NisB and/or NisC. We also show that the nisin biosynthesis sites are static and likely associated with proteins residing in lipid rafts. Based on these data, we propose a model for a three-phase production of modified precursor nisin in rod-shaped bacteria, presenting the assembly dynamics of NisBTC and emphasizing the crucial role of NisBC, next to NisT, in the process of precursor nisin translocation.

Proposed mechanism for regulation of H2 O2 -induced programmed cell death in plants by binding of cytochrome c to 14-3-3 proteins.

Programmed cell death (PCD) is crucial for development and homeostasis of all multicellular organisms. In human cells, the double role of extra-mitochondrial cytochrome c in triggering apoptosis and inhibiting survival pathways is well reported. In plants, however, the specific role of cytochrome c upon release from the mitochondria remains in part veiled yet death stimuli do trigger cytochrome c translocation as well. Here, we identify an Arabidopsis thaliana 14-3-3ι isoform as a cytosolic cytochrome c target and inhibitor of caspase-like activity. This finding establishes the 14-3-3ι protein as a relevant factor at the onset of plant H2 O2 -induced PCD. The in vivo and in vitro studies herein reported reveal that the interaction between cytochrome c and 14-3-3ι exhibits noticeable similarities with the complex formed by their human orthologues. Further analysis of the heterologous complexes between human and plant cytochrome c with plant 14-3-3ι and human 14-3-3ε isoforms corroborated common features. These results suggest that cytochrome c blocks p14-3-3ι so as to inhibit caspase-like proteases, which in turn promote cell death upon H2 O2 treatment. Besides establishing common biochemical features between human and plant PCD, this work sheds light onto the signaling networks of plant cell death.

Cytoplasmic maturation in human oocytes: an ultrastructural study †.

Female fertility relies on successful egg development. Besides chromosome segregation, complex structural and biochemical changes in the cytoplasmic compartment are necessary to confer the female gamete the capacity to undergo normal fertilization and sustain embryonic development. Despite the profound impact on egg quality, morphological bases of cytoplasmic maturation remain largely unknown. Here, we report our findings from the ultrastructural analysis of 69 unfertilized human oocytes from 34 young and healthy egg donors. By comparison of samples fixed at three consecutive developmental stages, we explored how ooplasmic architecture changes during meiotic maturation in vitro. The morphometric image analysis supported observation that the major reorganization of cytoplasm occurs before polar body extrusion. The organelles initially concentrated around prophase nucleus were repositioned toward the periphery and evenly distributed throughout the ooplasm. As maturation progressed, distinct secretory apparatus appeared to transform into cortical granules that clustered underneath the oocyte's surface. The most prominent feature was the gradual formation of heterologous complexes composed of variable elements of endoplasmic reticulum and multiple mitochondria with primitive morphology. Based on the generated image dataset, we proposed a morphological map of cytoplasmic maturation, which may serve as a reference for future comparative studies. In conclusion, this work improves our understanding of human oocyte morphology, cytoplasmic maturation, and intracellular factors defining human egg quality. Although this analysis involved spare oocytes completing development in vitro, it provides essential insight into the enigmatic process by which human egg progenitors prepare for fertilization.

Cryo-EM Structure of Heterologous Protein Complex Loaded Thermotoga Maritima Encapsulin Capsid

Encapsulin is a class of nanocompartments that is unique in bacteria and archaea to confine enzymatic activities and sequester toxic reaction products. Here we present a 2.87 Å resolution cryo-EM structure of Thermotoga maritima encapsulin with heterologous protein complex loaded. It is the first successful case of expressing encapsulin and heterologous cargo protein in the insect cell system. Although we failed to reconstruct the cargo protein complex structure due to the signal interference of the capsid shell, we were able to observe some unique features of the cargo-loaded encapsulin shell, for example, an extra density at the fivefold pore that has not been reported before. These results would lead to a more complete understanding of the encapsulin cargo assembly process of T. maritima.

Demonstration of Heterologous Complexes formed by Golgi-Resident Type III Membrane Proteins using Split Luciferase Complementation Assay

Demonstration of Heterologous Complexes formed by Golgi-Resident Type III Membrane Proteins using Split Luciferase Complementation Assay

Demonstration of Heterologous Complexes formed by Golgi-Resident Type III Membrane Proteins using Split Luciferase Complementation Assay.

The goal of this protocol is to explore the applicability of the most recent variant of split luciferase complementation for demonstrating heterologous complexes formed by nucleotide sugar transporters (NSTs). These ER- and Golgi-resident multitransmembrane proteins carry the cytoplasmically synthesized nucleotide sugars across organelle membranes to supply enzymes that mediate glycosylation with their substrates. NSTs exist as dimers and/or higher oligomers. Heterologous interactions betweendifferent NSTs have also been reported. To verify whether the technique is suitable for studying the phenomenon of NST heteromerization, we tested it against a combination of the two Golgi-resident NSTs that have been previously shown to associate by several other means. Theluciferase complementation assay appears to be particularly suitable for studying interactions between Golgi-resident membrane proteins, as it does not require high expression levels, which often trigger protein mislocalization and increase the risk of false positives.

Molecular Functionality of Plant Proteins from Low- to High-Solid Systems with Ligand and Co-Solute.

In the food industry, proteins are regarded as multifunctional systems whose bioactive hetero-polymeric properties are affected by physicochemical interactions with the surrounding components in formulations. Due to their nutritional value, plant proteins are increasingly considered by the new product developer to provide three-dimensional assemblies of required structure, texture, solubility and interfacial/bulk stability with physical, chemical or enzymatic treatment. This molecular flexibility allows them to form systems for the preservation of fresh food, retention of good nutrition and interaction with a range of microconstituents. While, animal- and milk-based proteins have been widely discussed in the literature, the role of plant proteins in the development of functional foods with enhanced nutritional profile and targeted physiological effects can be further explored. This review aims to look into the molecular functionality of plant proteins in relation to the transport of bioactive ingredients and interaction with other ligands and proteins. In doing so, it will consider preparations from low- to high-solids and the effect of structural transformation via gelation, phase separation and vitrification on protein functionality as a delivery vehicle or heterologous complex. Applications for the design of novel functional foods and nutraceuticals will also be discussed.

Advanced Strategies for Production of Natural Products in Yeast.

Natural products account for more than 50% of all small-molecule pharmaceutical agents currently in clinical use. However, low availability often becomes problematic when a bioactive natural product is promising to become a pharmaceutical or leading compound. Advances in synthetic biology and metabolic engineering provide a feasible solution for sustainable supply of these compounds. In this review, we have summarized current progress in engineering yeast cell factories for production of natural products, including terpenoids, alkaloids, and phenylpropanoids. We then discuss advanced strategies in metabolic engineering at three different dimensions, including point, line, and plane (corresponding to the individual enzymes and cofactors, metabolic pathways, and the global cellular network). In particular, we comprehensively discuss how to engineer cofactor biosynthesis for enhancing the biosynthesis efficiency, other than the enzyme activity. Finally, current challenges and perspective are also discussed for future engineering direction.

Heterologous viral protein interactions within licensed seasonal influenza virus vaccines

Currently, licensed influenza virus vaccines are designed and tested only for their ability to elicit hemagglutinin (HA)-reactive, neutralizing antibodies. Despite this, the purification process in vaccine manufacturing often does not completely remove other virion components. In the studies reported here, we have examined the viral protein composition of a panel of licensed vaccines from different manufacturers and licensed in different years. Using western blotting, we found that, beyond HA proteins, there are detectable quantities of neuraminidase (NA), nucleoprotein (NP), and matrix proteins (M1) from both influenza A and influenza B viruses in the vaccines but that the composition differed by source and method of vaccine preparation. We also found that disparities in viral protein composition were associated with distinct patterns of elicited antibody specificities. Strikingly, our studies also revealed that many viral proteins contained in the vaccine form heterologous complexes. When H1 proteins were isolated by immunoprecipitation, NA (N1), M1 (M1-A), H3, and HA-B proteins were co-isolated with the H1. Further biochemical studies suggest that these interactions persist for at least 4 h at 37 °C and that the membrane/intracytoplasmic domains in the intact HA proteins are important for the intermolecular interactions detected. These studies indicate that, if such interactions persist after vaccines reach the draining lymph node, both dendritic cells and HA-specific B cells may take up multiple viral proteins simultaneously. Whether these interactions are beneficial or harmful to the developing immune response will depend on the functional potential of the elicited virus-specific CD4 T cells.