Virus-like Particle System Research Articles

Novel transcription and replication-competent virus-like particles system modelling the Nipah virus life cycle

ABSTRACT Nipah virus (NiV), a highly pathogenic Henipavirus in humans, has been responsible for annual outbreaks in recent years. Experiments involving live NiV are highly restricted to biosafety level 4 (BSL-4) laboratories, which impedes NiV research. In this study, we developed transcription and replication-competent NiV-like particles (trVLP-NiV) lacking N, P, and L genes. This trVLP-NiV exhibited the ability to infect and continuously passage in cells ectopically expressing N, P, and L proteins while maintaining stable genetic characteristics. Moreover, the trVLP-NiV displayed a favourable safety profile in hamsters. Using the system, we found the NiV nucleoprotein residues interacting with viral RNA backbone affected viral replication in opposite patterns. This engineered system was sensitive to well-established antiviral drugs, innate host antiviral factors, and neutralizing antibodies. We then established a high-throughput screening platform utilizing the trVLP-NiV, leading to the identification of tunicamycin as a potential anti-NiV compound. Evidence showed that tunicamycin inhibited NiV replication by decreasing the infectivity of progeny virions. In conclusion, this trVLP-NiV system provided a convenient and versatile molecular tool for investigating NiV molecular biology and conducting antiviral drug screening under BSL-2 conditions. Its application will contribute to the development of medical countermeasures against NiV infections.

The RRE-REV module has no effect on the packaging efficiency of cas9 and Gag proteins into nanomedic virus-like particles

Delivery of ribonucleoprotein complexes of Cas9 nuclease and guide RNA into target cells with virus-like particles (VLP) is one of the novel methods of genome editing, suitable for gene therapy of human diseases in the future. Efficiency of genome editing with VLPs depends on the packaging of Cas9 into VLPs, that is mediated by viral Gag protein. To increase the packaging of Cas9 into NanoMEDIC system VLPs plasmid constructs for expression of Cas9 and Gag were modified by the addition of HIV RRE (Rev response element), that is expected to increase the nuclear export of RRE-containing transcripts to cytosol via accessory protein Rev, as described for Vpr-Cas9-based VLP system. Here we found that Cas9 and Gag protein levels in the cell lysates are increased upon cotransfection of either Rev-expressing plasmid or empty control plasmid. Moreover, this effect does not depend on the presence of RRE in the transcript. On the top of that, we showed that AP21967-induced dimerization of FRB and FKBP12, but not the modification of plasmids with RRE and/or cotransfection of Rev-expressing plasmid, plays the major role in packaging of Cas9 into NanoMEDIC system VLPs. These data suggest that it is impractical to use the RRE-Rev module to enhance the packing of Cas9 nuclease into VLPs.

The RRE-Rev Module Has No Effect on the Packaging Efficiency of Cas9 and Gag Proteins into NanoMEDIC Virus-like Particles.

Delivery of ribonucleoprotein complexes of Cas9 nuclease and guide RNA into target cells with virus-like particles (VLP) is one of the novel methods of genome editing and is suitable for gene therapy of human diseases in the future. The efficiency of genome editing with VLPs depends on the Cas9 packaging into VLPs, the process mediated by the viral Gag protein. To improve the packaging of Cas9 into NanoMEDIC VLPs, plasmid constructs for Cas9 and Gag expression were modified by adding the HIV Rev response element (RRE), which was expected to increase the nuclear export of RRE-containing transcripts into the cytosol via the Rev accessory protein, as described for a Vpr-Cas9-based VLP system. The Cas9 and Gag protein levels in cell lysates were found to increase upon cotransfection with either the Rev-expressing plasmid or the empty control plasmid. The effect was independent of the presence of RRE in the transcript. Moreover, AP21967-induced dimerization of FRB and FKBP12, but not plasmid modification with RRE and/or cotransfection with the Rev-expressing plasmid, was shown to play the major role in Cas9 packaging into NanoMEDIC VLPs. The data indicated that it is impractical to use the RRE-Rev module to enhance the packaging of Cas9 nuclease into VLPs.

A vaccine delivery system promotes strong immune responses against SARS-CoV-2 variants.

Global coronavirus disease 2019(COVID-19) pandemics highlight the need of developing vaccines with universal and durable protection against emerging SARS-CoV-2 variants. Here we developed an extended-release vaccine delivery system (GP-diABZI-RBD), consisting the original SARS-CoV-2 WA1 strain receptor-binding domain (RBD) as the antigen and diABZI stimulator of interferon genes(STING) agonist in conjunction with yeast β-glucan particles (GP-diABZI) as the platform. GP-diABZI-RBD could activate STING pathway and inhibit SARS-CoV-2 replication. Compared to diABZI-RBD, intraperitoneal injection of GP-diABZI-RBD elicited robust cellular and humoral immune responses in mice. Using SARS-CoV-2 GFP/ΔN transcription and replication-competent virus-like particle system (trVLP), we demonstrated that GP-diABZI-RBD-prototype vaccine exhibited the strongest and durable humoral immune responses and antiviral protection; whereas GP-diABZI-RBD-Omicron displayed minimum neutralization responses against trVLP. By using pseudotype virus (PsVs) neutralization assay, we found that GP-diABZI-RBD-Prototype, GP-diABZI-RBD-Delta, and GP-diABZI-RBD-Gamma immunized mice sera could efficiently neutralize Delta and Gamma PsVs, but had weak protection against Omicron PsVs. In contrast, GP-diABZI-RBD-Omicron immunized mice sera displayed the strongest neutralization response to Omicron PsVs. Taken together, the results suggest that GP-diABZI can serve as a promising vaccine delivery system for enhancing durable humoral and cellular immunity against broad SARS-CoV-2 variants. Our study provides important scientific basis for developing SARS-CoV-2 VOC-specific vaccines.

Interferon-Induced HERC5 Inhibits Ebola Virus Particle Production and Is Antagonized by Ebola Glycoprotein

Survival following Ebola virus (EBOV) infection correlates with the ability to mount an early and robust interferon (IFN) response. The host IFN-induced proteins that contribute to controlling EBOV replication are not fully known. Among the top genes with the strongest early increases in expression after infection in vivo is IFN-induced HERC5. Using a transcription- and replication-competent VLP system, we showed that HERC5 inhibits EBOV virus-like particle (VLP) replication by depleting EBOV mRNAs. The HERC5 RCC1-like domain was necessary and sufficient for this inhibition and did not require zinc finger antiviral protein (ZAP). Moreover, we showed that EBOV (Zaire) glycoprotein (GP) but not Marburg virus GP antagonized HERC5 early during infection. Our data identify a novel ‘protagonist–antagonistic’ relationship between HERC5 and GP in the early stages of EBOV infection that could be exploited for the development of novel antiviral therapeutics.

Assembly and Entry of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV2): Evaluation Using Virus-Like Particles.

Research on infectious severe acute respiratory syndrome coronavirus 2 (SARS-CoV2) is currently restricted to BSL-3 laboratories. SARS-CoV2 virus-like particles (VLPs) offer a BSL-1, replication-incompetent system that can be used to evaluate virus assembly and virus-cell entry processes in tractable cell culture conditions. Here, we describe a SARS-CoV2 VLP system that utilizes nanoluciferase (Nluc) fragment complementation to track assembly and entry. We utilized the system in two ways. Firstly, we investigated the requirements for VLP assembly. VLPs were produced by concomitant synthesis of three viral membrane proteins, spike (S), envelope (E), and matrix (M), along with the cytoplasmic nucleocapsid (N). We discovered that VLP production and secretion were highly dependent on N proteins. N proteins from related betacoronaviruses variably substituted for the homologous SARS-CoV2 N, and chimeric betacoronavirus N proteins effectively supported VLP production if they contained SARS-CoV2 N carboxy-terminal domains (CTD). This established the CTDs as critical features of virus particle assembly. Secondly, we utilized the system by investigating virus-cell entry. VLPs were produced with Nluc peptide fragments appended to E, M, or N proteins, with each subsequently inoculated into target cells expressing complementary Nluc fragments. Complementation into functional Nluc was used to assess virus-cell entry. We discovered that each of the VLPs were effective at monitoring virus-cell entry, to various extents, in ways that depended on host cell susceptibility factors. Overall, we have developed and utilized a VLP system that has proven useful in identifying SARS-CoV2 assembly and entry features.

Wrapping Up Viruses at Multiscale Resolution: Optimizing PACKMOL and SIRAH Execution for Simulating the Zika Virus.

Simulating huge biomolecular complexes of million atoms at relevant biological time scales is becoming accessible to the broad scientific community. That proves to be crucial for urgent responses against emergent diseases in real time. Yet, there are still issues to sort regarding the system setup so that molecular dynamics (MD) simulations can be run in a simple and standard way. Here, we introduce an optimized pipeline for building and simulating enveloped virus-like particles (VLP). First, the membrane packing problem is tackled with new features and optimized options in PACKMOL. This allows preparing accurate membrane models of thousands of lipids in the context of a VLP within a few hours using a single CPU. Then, the assembly of the VLP system is done within the multiscale framework of the coarse-grained SIRAH force field. Finally, the equilibration protocol provides a system ready for production MD simulations within a few days on broadly accessible GPU resources. The pipeline is applied to study the Zika virus as a test case for large biomolecular systems. The VLP stabilizes at approximately 0.5 μs of MD simulation, reproducing correlations greater than 0.90 against experimental density maps from cryo-electron microscopy. Detailed structural analysis of the protein envelope also shows very good agreement in root-mean-square deviations and B-factors with the experimental data. The level of details attained shows for the first time a possible role for anionic phospholipids in stabilizing the envelope. Combining an efficient and reliable setup procedure with an accurate coarse-grained force field provides a valuable pipeline for simulating arbitrary viral systems or subcellular compartments, paving the way toward whole-cell simulations.

Virus-Like Particle Systems for Vaccine Development against Viruses in the Flaviviridae Family.

Viruses in the Flaviviridae family are important human and animal pathogens that impose serious threats to global public health. This family of viruses includes emerging and re-emerging viruses, most of which are transmitted by infected mosquito or tick bites. Currently, there is no protective vaccine or effective antiviral treatment against the majority of these viruses, and due to their growing spread, several strategies have been employed to manufacture prophylactic vaccines against these infectious agents including virus-like particle (VLP) subunit vaccines. VLPs are genomeless viral particles that resemble authentic viruses and contain critical repetitive conformational structures on their surface that can trigger the induction of both humoral and cellular responses, making them safe and ideal vaccine candidates against these viruses. In this review, we focus on the potential of the VLP platform in the current vaccine development against the medically important viruses in the Flaviviridae family.

A proof-of-concept study for the design of a VLP-based combinatorial HPV and placental malaria vaccine

In Africa, cervical cancer and placental malaria (PM) are a major public health concern. There is currently no available PM vaccine and the marketed Human Papillomavirus (HPV) vaccines are prohibitively expensive. The idea of a combinatorial HPV and PM vaccine is attractive because the target population for vaccination against both diseases, adolescent girls, would be overlapping in Sub-Saharan Africa. Here we demonstrate proof-of-concept for a combinatorial vaccine utilizing the AP205 capsid-based virus-like particle (VLP) designed to simultaneously display two clinically relevant antigens (the HPV RG1 epitope and the VAR2CSA PM antigen). Three distinct combinatorial VLPs were produced displaying one, two or five concatenated RG1 epitopes without obstructing the VLP’s capacity to form. Co-display of VAR2CSA was achieved through a split-protein Tag/Catcher interaction without hampering the vaccine stability. Vaccination with the combinatorial vaccine(s) was able to reduce HPV infection in vivo and induce anti-VAR2CSA IgG antibodies, which inhibited binding between native VAR2CSA expressed on infected red blood cells and chondroitin sulfate A in an in vitro binding-inhibition assay. These results show that the Tag/Catcher AP205 VLP system can be exploited to make a combinatorial vaccine capable of eliciting antibodies with dual specificity.

Efficient mRNA delivery system utilizing chimeric VSVG-L7Ae virus-like particles

The delivery of mRNA is advantageous over DNA delivery as it is transient and does not carry the risk of genomic DNA integration. However, there are currently few efficient mRNA delivery options available, especially for hard-to-transfect cell types, and thus new delivery methods are needed. To this end, we have established a novel mRNA delivery system utilizing chimeric virus-like particles (VLPs). We generated a novel VLP by fusing protein G of Vesicular stomatitis virus (VSV-G) with a ribosomal protein L7Ae of Archeoglobus fulgidus. This system allowed the efficient delivery of EGFP mRNA which was independent from the presence of BoxC/D motif in the mRNA sequence. Our VSVG-L7Ae VLP system demonstrated high transduction efficacy in hard-to-transfect cell lines, such as human induced pluripotent stem cells (iPS cells) and monocytes. In summary, this platform may serve as an efficient and transient transgene delivery tool for an mRNA of interest.

High-throughput drug screening using the Ebola virus transcription- and replication-competent virus-like particle system

The massive epidemic of Ebola virus disease (EVD) in West Africa, followed in recent months by two outbreaks in the Democratic Republic of the Congo, underline the importance of this severe disease. Because Ebola virus (EBOV) must be manipulated under biosafety level 4 (BSL4) containment, the discovery and development of virus-specific therapies have been hampered. Recently, a transient transfection-based transcription- and replication competent virus-like particle (trVLP) system was described, enabling modeling of the entire EBOV life cycle under BSL2 conditions. Using this system, we optimized the condition for bulk co-transfection of multiple plasmids, developed a luciferase reporter-based assay in 384-well microtiter plates, and performed a high-throughput screening (HTS) campaign of an 8,354-compound collection consisting of U.S. Food & Drug Administration (FDA) -approved drugs, bioactives, kinase inhibitors, and natural products in duplicates. The HTS achieved a good signal-to-background ratio with a low percent coefficient of variation resulting in Z’ = 0.7, and data points were reproducible with R2 = 0.89, indicative of a robust assay. After applying stringent hit selection criteria of ≥70% EBOV trVLP inhibition and ≥70% cell viability, 381 hits were selected targeting early, entry, and replication steps and 49 hits targeting late, maturation, and secretion steps in the viral life cycle. Of the total 430 hits, 220 were confirmed by dose-response analysis in the primary HTS assay. They were subsequently triaged by time-of-addition assays, then clustered and ranked according to their chemical structures, biological functions, therapeutic index, and maximum inhibition. Several novel drugs have been identified to very efficiently inhibit EBOV. Interestingly, most showed pharmacological activity in treatments for central nervous system-related diseases. We developed and screened an HTS assay using the novel EBOV trVLP system. Newly identified inhibitors are useful tools to study the poorly understood EBOV life cycle. In addition, they also provide opportunities to either repurpose FDA-approved drugs or develop novel viral interventions to combat EVD.

Analysis of a Putative Late Domain Using an Ebola Virus Transcription and Replication-Competent Virus-Like Particle System.

The Ebola virus (EBOV) matrix protein VP40 drives budding of virions and encodes 2 overlapping late domain motifs at amino acid positions 7-13 (PTAPPEY). However, these motifs are not absolutely essential for replication in cell culture, and recently a potential third late domain motif (YPx(6)I) was proposed at amino acid positions 18-26 of VP40. To analyze the importance of this motif in viral budding, we used a transcription and replication-competent virus-like particle system. Using this system, we show that this motif does not contribute to EBOV budding or particle propagation.

A Rapid Screen for Host-Encoded miRNAs with Inhibitory Effects against Ebola Virus Using a Transcription- and Replication-Competent Virus-Like Particle System.

MicroRNAs (miRNAs) may become efficient antiviral agents against the Ebola virus (EBOV) targeting viral genomic RNAs or transcripts. We previously conducted a genome-wide search for differentially expressed miRNAs during viral replication and transcription. In this study, we established a rapid screen for miRNAs with inhibitory effects against EBOV using a tetracistronic transcription- and replication-competent virus-like particle (trVLP) system. This system uses a minigenome comprising an EBOV leader region, luciferase reporter, VP40, GP, VP24, EBOV trailer region, and three noncoding regions from the EBOV genome and can be used to model the life cycle of EBOV under biosafety level (BSL) 2 conditions. Informatic analysis was performed to select up-regulated miRNAs targeting the coding regions of the minigenome with the highest binding energy to perform inhibitory effect screening. Among these miRNAs, miR-150-3p had the most significant inhibitory effect. Reverse transcription polymerase chain reaction (RT-PCR), Western blot, and double fluorescence reporter experiments demonstrated that miR-150-3p inhibited the reproduction of trVLPs via the regulation of GP and VP40 expression by directly targeting the coding regions of GP and VP40. This novel, rapid, and convenient screening method will efficiently facilitate the exploration of miRNAs against EBOV under BSL-2 conditions.

Identification of broadly neutralizing monoclonal antibodies against Crimean-Congo hemorrhagic fever virus

Despite the serious public health impact of Crimean-Congo hemorrhagic fever (CCHF), the efficacy of antivirals targeting the causative agent, CCHF virus (CCHFV), remains debatable. Neutralizing monoclonal antibodies (MAbs) targeting the CCHFV glycoprotein Gc have been reported to protect mice against challenge with the prototype CCHFV strain, IbAr10200. However, due to extensive sequence diversity of CCHFV glycoproteins, it is unknown whether these MAbs neutralize other CCHFV strains. We initially used a CCHF virus-like particle (VLP) system to generate 11 VLP moieties, each possessing a glycoprotein from a genetically diverse CCHFV strain isolated in either Africa, Asia, the Middle East, or southeastern Europe. We used these VLPs in biosafety level 2 conditions to efficiently screen MAb cross-neutralization potency. Of the 16 MAbs tested, 3 (8A1, 11E7, and 30F7) demonstrated cross-neutralization activity with most CCHF VLPs, with 8A1 neutralizing all VLPs tested. Although binding studies suggest that none of the MAbs compete for the same epitope, combining 11E7, 30F7, or both 11E7 and 30F7 with 8A1 had no additive effect on increasing neutralization in this system. To confirm our findings from the VLP system, the 3 MAbs capable of strain cross-neutralization were confirmed to effectively neutralize 5 diverse CCHFV strains in vitro. Passaging CCHFV strains in the presence of sub-neutralizing concentrations of MAbs did not generate escape mutants resistant to subsequent neutralization. This study demonstrates the utility of the VLP system for screening neutralizing MAbs against multiple CCHFV strains, and provides the first evidence that a single MAb can effectively neutralize a number of diverse CCHFV strains in vitro, which may lead to development of future CCHF therapeutics.

Sortase-Mediated Ligation as a Modular Approach for the Covalent Attachment of Proteins to the Exterior of the Bacteriophage P22 Virus-like Particle.

Virus-like particles are unique platforms well suited for the construction of nanomaterials with broad-range applications. The research presented here describes the development of a modular approach for the covalent attachment of protein domains to the exterior of the versatile bacteriophage P22 virus-like particle (VLP) via a sortase-mediated ligation strategy. The bacteriophage P22 coat protein was genetically engineered to incorporate an LPETG amino acid sequence on the C-terminus, providing the peptide recognition sequence utilized by the sortase enzyme to catalyze peptide bond formation between the LPETG-tagged protein and a protein containing a polyglycine sequence on the N-terminus. Here we evaluate attachment of green fluorescent protein (GFP) and the head domain of the influenza hemagglutinin (HA) protein by genetically producing polyglycine tagged proteins. Attachment of both proteins to the exterior of the P22 VLP was found to be highly efficient as judged by SDS-PAGE densitometry. These results enlarge the tool kit for modifying the P22 VLP system and provide new insights for other VLPs that have an externally displayed C-terminus that can use the described strategy for the modular modification of their external surface for various applications.

Improving the malaria transmission-blocking activity of a Plasmodium falciparum 48/45 based vaccine antigen by SpyTag/SpyCatcher mediated virus-like display

Malaria is a devastating disease caused by Plasmodium parasites, resulting in almost 0.5 million deaths per year. The Pfs48/45 protein exposed on the P. falciparum sexual stages is one of the most advanced antigen candidates for a transmission-blocking (TB) vaccine in the clinical pipeline. However, it remains essential to identify an optimal vaccine formulation that can facilitate induction of a long-lasting TB anti-Pfs48/45 response. Here we report on the development and evaluation of two Pfs48/45-based virus-like particle (VLP) vaccines generated using the AP205 SpyTag/Catcher VLP system. Two different recombinant proteins (SpyCatcher-R0.6C and SpyCatcher-6C), comprising the Pfs48/45-6C region, were covalently attached to the surface of Spy-tagged Acinetobacter phage AP205 VLPs. Resulting Pfs48/45-VLP complexes appeared as non-aggregated particles of ∼30nm, each displaying an average of 216 (R0.6C) or 291 (6C) copies of the antigens. Both R0.6C and 6C VLP conjugates were strongly reactive with a monoclonal antibody (mAb45.1) targeting a conformational TB Pfs48/45 epitope, suggesting that the TB epitope is accessible for immune recognition on the particles. To select the most suitable vaccine formulation for downstream clinical studies the two VLP vaccines were tested in CD1 mice using different adjuvant formulations. The study demonstrates that VLP-display of R0.6C and 6C significantly increases antigen immunogenicity when using Montanide ISA 720 VG as extrinsic adjuvant.

Functional Characterization of Adaptive Mutations during the West African Ebola Virus Outbreak.

The dimension of the Ebola virus outbreak in West Africa was unprecedented. Amino acid substitutions in the viral L polymerase, surface glycoprotein GP, and nucleocapsid protein NP emerged, were fixed early in the outbreak, and were found in almost 90% of the sequences. Here we showed that these mutations affected the functional activity of viral proteins and improved viral growth in cell culture. Our results demonstrate emergence of adaptive changes in the Ebola virus genome during virus circulation in humans and prompt further studies on the potential role of these changes in virus transmissibility and pathogenicity.

The Respiratory Syncytial Virus Phosphoprotein, Matrix Protein, and Fusion Protein Carboxy-Terminal Domain Drive Efficient Filamentous Virus-Like Particle Formation.

hRSV is responsible for >100,000 deaths in children worldwide, and a vaccine is not available. Among the potential anti-hRSV approaches are virus-like particle (VLP) vaccines, which, based on resemblance to virus or viral components, can induce protective immunity. For hRSV, few reports are available concerning authentic VLP production or testing, in large part because VLP production is inefficient and the mechanisms underlying particle assembly are poorly understood. Here, we took advantage of the cell-associated nature of RSV particles and used high-resolution microscopy analyses to examine the viral proteins required for formation of wild-type-virus-resembling VLPs, the contributions of these proteins to morphology, and the domains involved in incorporation of the antigenically important viral F protein. The results provide new insights that will facilitate future production of hRSV VLPs with defined shapes and compositions and may translate into improved manufacture of live-attenuated hRSV vaccines.

Tuning Viral Capsid Nanoparticle Stability with Symmetrical Morphogenesis.

Virus-like particles (VLPs) provide engineering platforms for the design and implementation of protein-based nanostructures. These capsids are comprised of protein subunits whose precise arrangement and mutual interactions determine their stability, responsiveness to destabilizing environments, and ability to undergo morphological transitions. The precise interplay between subunit contacts and the overall stability of the bulk capsid population remains poorly resolved. Approaching this relationship requires a combination of techniques capable of accessing nanoscale properties, such as the mechanics of individual capsids, and bulk biochemical procedures capable of interrogating the stability of the VLP ensemble. To establish such connection, a VLP system is required where the subunit interactions can be manipulated in a controlled fashion. The P22 VLP is a promising platform for the design of nanomaterials and understanding how nanomanipulation of the particle affects bulk behavior. By contrasting single-particle atomic force microscopy and bulk chemical perturbations, we have related symmetry-specific anisotropic mechanical properties to the bulk ensemble behavior of the VLPs. Our results show that the expulsion of pentons at the vertices of the VLP induces a concomitant chemical and mechanical destabilization of the capsid and implicates the capsid edges as the points of mechanical fracture. Subsequent binding of a decoration protein at these critical edge regions restores both chemical and mechanical stability. The agreement between our single molecule and bulk techniques suggests that the same structural determinants govern both destabilizing and restorative mechanisms, unveiling a phenomenological coupling between the chemical and mechanical behavior of self-assembled cages and laying a framework for the analysis and manipulation of other VLPs and symmetric self-assembled structures.

Tetherin Sensitivity of Influenza A Viruses Is Strain Specific: Role of Hemagglutinin and Neuraminidase.

The expression of the antiviral host cell factor tetherin is induced by interferon and can inhibit the release of enveloped viruses from infected cells. The Vpu protein of HIV-1 antagonizes the antiviral activity of tetherin, and tetherin antagonists with Vpu-like activity have been identified in other viruses. In contrast, it is incompletely understood whether tetherin inhibits influenza A virus (FLUAV) release and whether FLUAV encodes tetherin antagonists. Here, we show that release of several laboratory-adapted FLUAV strains and a seasonal FLUAV strain is inhibited by tetherin, while pandemic FLUAV A/Hamburg/4/2009 is resistant. Studies with a virus-like particle system and analysis of reassortant viruses provided evidence that the viral hemagglutinin (HA) is an important determinant of tetherin antagonism but requires the presence of its cognate neuraminidase (NA) to inhibit tetherin. Finally, tetherin antagonism by FLUAV was dependent on the virion context, since retrovirus release from tetherin-positive cells was not rescued, and correlated with an HA- and NA-dependent reduction in tetherin expression. In sum, our study identifies HA and NA proteins of certain pandemic FLUAV as tetherin antagonists, which has important implications for understanding FLUAV pathogenesis. Influenza A virus (FLUAV) infection is responsible for substantial global morbidity and mortality, and understanding how the virus evades the immune defenses of the host may uncover novel targets for antiviral intervention. Tetherin is an antiviral effector molecule of the innate immune system which can contribute to control of viral invasion. However, it has been unclear whether FLUAV is inhibited by tetherin and whether these viruses encode tetherin-antagonizing proteins. Our observation that several pandemic FLUAV strains can counteract tetherin via their HA and NA proteins identifies these proteins as novel tetherin antagonists and indicates that HA/NA-dependent inactivation of innate defenses may contribute to the efficient spread of pandemic FLUAV.