Plasma Membrane Of Infected Cells Research Articles

A novel hypothesis on mechanisms and potential role of host cell membrane proteins incorporated into the viral envelope in alloreactivity

Despite significant advances in transplantation medicine, allograft rejection remains a major challenge in clinical practice. The preexistence of alloreactive lymphocytes is a primary cause of allograft rejection. The forthcoming challenges in clinical transplantation will involve the suppression or eradication of allospecific memory T cells. Viral infections and allograft rejection interact in multiple ways that can compromise the survival of transplanted organs. Evidence is currently accumulating on the incorporation of HLA antigens and other host cell plasma membrane molecules into the viral envelope. A consequence of the viral envelope displaying the HLA signature acquired from the plasma membrane of infected cells is that when virions are transmitted to the next host, the newly infected person became exposed to allogeneic molecules stimulating alloimmunity and generating memory to alloantigens. Thus, successive exposure to viral infections throughout life could potentially contribute to the development of alloimmunity and the generation of numerous clones of memory alloreactive lymphocytes. In this paper a novel hypothesis is developed on the potential role and mechanisms of alloresponse induced by human spread of enveloped viruses. As this hypothesis could aid in the knowledge of preexisting alloreactivity, additional research into the intricacies of virion-incorporated host cell proteins is necessary. A deeper understanding of this dynamic interaction will help promote advances in the long-term acceptance of transplants.

Enforced Dimerization of CD45 by the Adenovirus E3/49K Protein Inhibits T Cell Receptor Signaling.

Human adenoviruses (HAdVs) are widespread pathogens that generally cause mild infections in immunocompetent individuals but severe or even fatal diseases in immunocompromised patients. In order to counteract the host immune defenses, HAdVs encode various immunomodulatory proteins in the early transcription unit 3 (E3). The E3/49K protein is a highly glycosylated type I transmembrane protein uniquely expressed by species D HAdVs. Its N-terminal ectodomain sec49K is released by metalloprotease-mediated shedding at the cell surface and binds to the receptor-like protein tyrosine phosphatase CD45, a critical regulator of leukocyte activation and functions. It remained elusive which domains of CD45 and E3/49K are involved in the interaction and whether such an interaction can also occur on the cell surface with membrane-anchored full-length E3/49K. Here, we show that the two extracellular domains R1 and R2 of E3/49K bind to the same site in the domain d3 of CD45. This interaction enforces the dimerization of CD45, causing the inhibition of T cell receptor signaling. Intriguingly, the membrane-anchored E3/49K appears to be designed like a "molecular fishing rod" using an extended disordered region of E3/49K as a "fishing line" to bridge the distance between the plasma membrane of infected cells and the CD45 binding site on T cells to effectively position the domains R1 and R2 as baits for CD45 binding. This design strongly suggests that both secreted sec49K as well as membrane-anchored full-length E3/49K have immunomodulatory functions. The forced dimerization of CD45 may be applied as a therapeutic strategy in chronic inflammatory disorders and cancer. IMPORTANCE The battle between viruses and their hosts is an ongoing arms race. Whereas the host tries to detect and eliminate the virus, the latter counteracts such antiviral measures to replicate and spread. Adenoviruses have evolved various mechanisms to evade the human immune response. The E3/49K protein of species D adenoviruses mediates the inhibition of immune cell function via binding to the protein tyrosine phosphatase CD45. Here, we show that E3/49K triggers the dimerization of CD45 and thereby inhibits its phosphatase activity. Intriguingly, the membrane-anchored E3/49K seems to be designed like a "molecular fishing rod" with the two CD45 binding domains of E3/49K as baits positioned at the end of an extended disordered region reminiscent of a fishing line. The adenoviral strategy to inhibit CD45 activity by forced dimerization may be used for therapeutic intervention in autoimmune diseases or to prevent graft rejection after transplantation.

NK-92-CD16A cell externalization of CD107a for detection of antigen-specific antibodies able to support ADCC: sensitive discrimination of antibody a-fucosylation

Abstract Antibody-dependent cell-mediated cytotoxicity (ADCC) by natural killer (NK) lymphocytes kills cells infected with enveloped viruses. Antiviral ADCC has three components: antigen-specific IgG1 or IgG3; effector cells with CD16A that recognize the Fc of IgG; and antigenic proteins in the plasma membranes of infected cells. Afucosylated antibodies support ADCC better than fucosylated antibodies; afucosylated fractions vary among individuals. Current assays that detect afucosylated antibodies to specific viruses involve antigen-specific antibody purification and mass spectrometry. We are developing clinical bioassays to detect antibodies that will support ADCC and assess their extents of afucosylation. Our pilot assay uses externalization of CD107a by NK-92-CD16A cells, this’ occurs when NK cells recognize cell-bound antibodies. We used two humanized monoclonal antibodies on CD20, GA101 WT or glycoengineered GE, 10% or ~50% afucosylated, with Raji cells. Cellular CD107a externalization was more sensitive than AF-647 donkey anti-IgG to detect total cell-bound antibody. The effective concentrations (EC50s) of the two antibodies for CD107a externalization differed by >15-fold. The more afucosylated GA101-GE was more detectable, at 0.2 ng/ml (=/< 700 Abs/Raji cell). CD107a externalization increased with excess antibody-bearing cells. Short 37 °C incubation (40 minutes) optimized detection of ADCC over intrinsic NK activity. Controls of GA101-GE alone and 56 °C heated human serum lacked effects on CD107a externalization. The assay appears compatible with formaldehyde treatment before NK cell staining. This sensitive bioassay appears suitable for assess serum samples for antiviral antibodies that will support ADCC. UNR Foundation C.E. Hudig Fund.

A Virus Genetic System to Analyze the Fusogenicity of Human Cytomegalovirus Glycoprotein B Variants.

Viruses can induce the fusion of infected and neighboring cells, leading to the formation of syncytia. Cell-cell fusion is mediated by viral fusion proteins on the plasma membrane of infected cells that interact with cellular receptors on neighboring cells. Viruses use this mechanism to spread rapidly to adjacent cells or escape host immunity. For some viruses, syncytium formation is a hallmark of infection and a known pathogenicity factor. For others, the role of syncytium formation in viral dissemination and pathogenicity remains poorly understood. Human cytomegalovirus (HCMV) is an important cause of morbidity and mortality in transplant patients and the leading cause of congenital infections. Clinical HCMV isolates have broad cell tropism but differ in their ability to induce cell-cell fusions, and little is known about the molecular determinants. We developed a system to analyze HCMV glycoprotein B (gB) variants in a defined genetic background. HCMV strains TB40/E and TR were used as vectors to compare the fusogenicity of six gB variants from congenitally infected fetuses with those from three laboratory strains. Five of them conferred the ability to induce the fusion of MRC-5 human embryonic lung fibroblasts to one or both backbone strains, as determined by a split GFP-luciferase reporter system. The same gB variants were not sufficient to induce syncytia in infected ARPE-19 epithelial cells, suggesting that additional factors are involved. The system described here allows a systematic comparison of the fusogenicity of viral envelope glycoproteins and may help to clarify whether fusion-promoting variants are associated with increased pathogenicity.

Conditional permeabilization of the P. falciparum plasma membrane in infected cells links cation influx to reduced membrane integrity.

The intracellular human malaria parasite, Plasmodium falciparum, uses the PfATP4 cation pump to maintain Na+ and H+ homeostasis in parasite cytosol. PfATP4 is the target of advanced antimalarial leads, which produce many poorly understood metabolic disturbances within infected erythrocytes. Here, we expressed the mammalian ligand-gated TRPV1 ion channel at the parasite plasma membrane to study ion regulation and examine the effects of cation leak. TRPV1 expression was well-tolerated, consistent with negligible ion flux through the nonactivated channel. TRPV1 ligands produced rapid parasite death in the transfectant line at their activating concentrations, but were harmless to the wild-type parent. Activation triggered cholesterol redistribution at the parasite plasma membrane, reproducing effects of PfATP4 inhibitors and directly implicating cation dysregulation in this process. In contrast to predictions, TRPV1 activation in low Na+ media accentuated parasite killing but a PfATP4 inhibitor had unchanged efficacy. Selection of a ligand-resistant mutant revealed a previously uncharacterized G683V mutation in TRPV1 that occludes the lower channel gate, implicating reduced permeability as a mechanism for parasite resistance to antimalarials targeting ion homeostasis. Our findings provide key insights into malaria parasite ion regulation and will guide mechanism-of-action studies for advanced antimalarial leads that act at the host-pathogen interface.

Backbone NMR resonance assignment of the apo human Tsg101-UEV domain.

The Endosomal Sorting Complex Required for Transport (ESCRT) pathway, through inverse topology membrane remodeling, is involved in many biological functions, such as ubiquitinated membrane receptor trafficking and degradation, multivesicular bodies (MVB) formation and cytokinesis. Dysfunctions in ESCRT pathway have been associated to several human pathologies, such as cancers and neurodegenerative diseases. The ESCRT machinery is also hijacked by many enveloped viruses to bud away from the plasma membrane of infected cells. Human tumor susceptibility gene 101 (Tsg101) protein is an important ESCRT-I complex component. The structure of the N-terminal ubiquitin E2 variant (UEV) domain of Tsg101 (Tsg101-UEV) comprises an ubiquitin binding pocket next to a late domain [P(S/T)AP] binding groove. These two binding sites have been shown to be involved both in the physiological roles of ESCRT-I and in the release of the viral particles, and thus are attractive targets for antivirals. The structure of the Tsg101-UEV domain has been characterized, using X-ray crystallography or NMR spectroscopy, either in its apo-state or bound to ubiquitin or late domains. In this study, we report the backbone NMR resonance assignments, including the proline signals, of the apo human Tsg101-UEV domain, that so far was not publicly available. These data, that are in good agreement with the crystallographic structure of Tsg101-UEV domain, can therefore be used for further NMR studies, including protein-protein interaction studies and drug discovery.

Inhibitory effects of antibody crosslinking on influenza virus assembly and release.

Influenza viruses bud from the plasma membrane of infected cells, where the viral surface proteins hemagglutinin (HA), neuraminidase (NA) and the ion channel M2 are vulnerable to antibodies in the extracellular environment. Although binding of multivalent antibodies is likely to perturb the dynamics and organization of the viral surface proteins, how this affects the assembly and release of new virions remains unclear. This is due in part to limitations of available techniques: while antibody neutralization of virus entry is routinely measured using hemagglutination inhibition and microneutralization assays, there are few approaches to specifically evaluate antibody inhibition of virus assembly and release that are quantitative and mechanistic. Motivated by this need, we developed fluorescence imaging-based assays to quantify virus shedding under antibody challenge and to dissect the underlying inhibition mechanisms. We find that HA-, NA-, and M2-specific monoclonal antibodies targeting a range of antigenic sites significantly decrease the number of viruses released during a single replication cycle. These antibodies inhibit virus release at concentrations ∼1-10-fold higher than their inhibitory concentrations for viral entry, reinforcing the idea that antibody function is multifaceted. Using fluorescence recovery after photobleaching, we observe that HA stem-binding IgG antibodies CR9114 and FI6v3 disrupt the diffusion of cell surface HAs by crosslinking adjacent HAs in cis, thus restricting their contributions to viral assembly. In contrast, head-binding antibodies S139 and C05 are unable to alter the HA diffusion rate but potently inhibit the release of fully formed virions by crosslinking HAs in trans, leading to the sequestration of virions at the cell surface. Collectively, these results suggest that antibody inhibition of virus assembly and release is a general feature of IgG antibodies, and that antibody crosslinking either in cis or in trans can contribute to the inhibition potency.

Phosphatidylinositol 4,5-Bisphosphate Mediates the Co-Distribution of Influenza A Hemagglutinin and Matrix Protein M1 at the Plasma Membrane.

The fully assembled influenza A virus (IAV) has on its surface the highest density of a single membrane protein found in nature-the glycoprotein hemagglutinin (HA) that mediates viral binding, entry, and assembly. HA clusters at the plasma membrane of infected cells, and the HA density (number of molecules per unit area) of these clusters correlates with the infectivity of the virus. Dense HA clusters are considered to mark the assembly site and ultimately lead to the budding of infectious IAV. The mechanism of spontaneous HA clustering, which occurs with or without other viral components, has not been elucidated. Using super-resolution fluorescence photoactivation localization microscopy (FPALM), we have previously shown that these HA clusters are interdependent on phosphatidylinositol 4,5-biphosphate (PIP2). Here, we show that the IAV matrix protein M1 co-clusters with PIP2, visualized using the pleckstrin homology domain. We find that cetylpyridinium chloride (CPC), which is a positively charged quaternary ammonium compound known for its antibacterial and antiviral properties at millimolar concentrations, disrupts M1 clustering and M1-PIP2 co-clustering at micromolar concentrations well below the critical micelle concentration (CMC). CPC also disrupts the co-clustering of M1 with HA at the plasma membrane, suggesting the role of host cell PIP2 clusters as scaffolds for gathering and concentrating M1 and HA to achieve their unusually high cluster densities in the IAV envelope.

High-speed AFM reveals the dynamics of virus budding

Retrovirus assembly is coordinated by the viral polyprotein Gag, and after assembly, the particles are released from the host cell in a process called budding. Assembly of one of the best-studied retroviruses, human immunodeficiency virus-1 (HIV-1), occurs at the plasma membrane of infected cells (Fig. 1). Driven by Gag, an almost complete viral shell is formed when the particle buds from the cell via membrane fission (2). As other retroviruses, HIV-1 hijacks the host endosomal complex required for transport (ESCRT) machinery for budding.

Analysis of individual HIV-1 budding event using fast AFM reveals a multiplexed role for VPS4

The assembly and budding of newly formed human immunodeficiency virus-1 (HIV-1) particles occur at the plasma membrane of infected cells. Although the molecular basis for viral budding has been studied extensively, investigation of its spatiotemporal characteristics has been limited by the small dimensions (∼100 nm) of HIV particles and the fast kinetics of the process (a few minutes from bud formation to virion release). Here we applied ultra-fast atomic force microscopy to achieve real-time visualization of individual HIV-1 budding events from wild-type (WT) cell lines as well as from mutated lines lacking vacuolar protein sorting-4 (VPS4) or visceral adipose tissue-1 protein (VTA1). Using single-particle analysis, we show that HIV-1 bud formation follows two kinetic pathways (fast and slow) with each composed of three distinct phases (growth, stationary, decay). Notably, approximately 38% of events did not result in viral release and were characterized by the formation of short (rather than tall) particles that slowly decayed back into the cell membrane. These non-productive events became more abundant in VPS4 knockout cell lines. Strikingly, the absence of VPS4B, rather than VPS4A, increased the production of short viral particles, suggesting a role for VPS4B in earlier stages of HIV-1 budding than traditionally thought.

Viral and Host Factors Regulating HIV-1 Envelope Protein Trafficking and Particle Incorporation.

The HIV-1 envelope glycoprotein (Env) is an essential structural component of the virus, serving as the receptor-binding protein and principal neutralizing determinant. Env trimers are incorporated into developing particles at the plasma membrane of infected cells. Incorporation of HIV-1 Env into particles in T cells and macrophages is regulated by the long Env cytoplasmic tail (CT) and the matrix region of Gag. The CT incorporates motifs that interact with cellular factors involved in endosomal trafficking. Env follows an unusual pathway to arrive at the site of particle assembly, first traversing the secretory pathway to the plasma membrane (PM), then undergoing endocytosis, followed by directed sorting to the site of particle assembly on the PM. Many aspects of Env trafficking remain to be defined, including the sequential events that occur following endocytosis, leading to productive recycling and particle incorporation. This review focuses on the host factors and pathways involved in Env trafficking, and discusses leading models of Env incorporation into particles.

A Reverse Mutation E143K within the PrM Protein of Zika Virus Asian Lineage Natal RGN Strain Increases Infectivity and Cytopathicity.

Zika virus (ZIKV) is a positive-sense single-stranded RNA virus in the Flaviviridae, which is classified into two different lineages Asian and African. The outbreak of ZIKV Asian lineage isolates in 2015–2016 is associated with the increase in cases with prenatal microcephaly and Guillain–Barré syndrome, and has sparked attention throughout the world. Genome sequence alignment and the analysis of Asian and African lineage isolates indicate that amino acid changes, particular in positively charged amino acid substitutions in the pr region of prM protein might involve a phenotypic change that links with the global outbreak of ZIKV Asian-lineage. The study generated and characterized the virological properties of wild type and mutants of single-round infectious particles (SRIPs) and infectious clones (i.c.s) of ZIKV Asian-lineage Natal RGN strain, and then identified the function of amino acid substitutions at the positions 139 [Asn139→Ser139 (N139S)] and 143 [Glu143→Lys143 (E143K)] in ZIKV polyproteins (located within the pr region of prM protein) in the infectivity and cytopathogenicity. The E143K SRIP and i.c. of Natal RGN strain exhibited relatively higher levels of cytopathic effect, EGFP reporter, viral RNA and protein synthesis, and virus yield in three types of human cell lines, TE617, SF268 and HMC3, compared to wild type (WT), N139S SRIPs and i.c.s, which displayed more efficiency in replication kinetics. Additionally, E143K Natal RGN i.c. had greater activities of virus attachment and entry, yielded higher titers of intracellular and extracellular virions, and assembled the E proteins near to the plasma membrane in infected cells than the other i.c.s. The results indicate that the positively charged amino acid residue Lys143, a conserved residue in the pr region of prM of ZIKV African lineages, plays a crucial role in viral replication kinetics, including viral attachment, entry, assembly and egress. Thus, the negatively charged amino acid residue Glu143 within the pr region of prM leads to an alteration of the phenotypes, in particular, a lower replication efficiency of ZIKV Asian-lineage isolates with the attenuation of infectivity and cytopathicity.

Advances in HIV-1 Assembly.

The assembly of HIV-1 particles is a concerted and dynamic process that takes place on the plasma membrane of infected cells. An abundance of recent discoveries has advanced our understanding of the complex sequence of events leading to HIV-1 particle assembly, budding, and release. Structural studies have illuminated key features of assembly and maturation, including the dramatic structural transition that occurs between the immature Gag lattice and the formation of the mature viral capsid core. The critical role of inositol hexakisphosphate (IP6) in the assembly of both the immature and mature Gag lattice has been elucidated. The structural basis for selective packaging of genomic RNA into virions has been revealed. This review will provide an overview of the HIV-1 assembly process, with a focus on recent advances in the field, and will point out areas where questions remain that can benefit from future investigation.

Interactions between AMOT PPxY motifs and NEDD4L WW domains function in HIV-1 release

Like most enveloped viruses, HIV must acquire a lipid membrane as it assembles and buds through the plasma membrane of infected cells to spread infection. Several sets of host cell machinery facilitate this process, including proteins of the endosomal sorting complexes required for transport pathway, which mediates the membrane fission reaction required to complete viral budding, as well as angiomotin (AMOT) and NEDD4L, which bind one another and promote virion membrane envelopment. AMOT and NEDD4L interact through the four NEDD4L WW domains and three different AMOT Pro-Pro-x (any amino acid)-Tyr (PPxY) motifs, but these interactions are not yet well defined. Here, we report that individual AMOT PPxY and NEDD4L WW domains interact with the following general affinity hierarchies: AMOT PPxY1>PPxY2>PPxY3 and NEDD4L WW3>WW2>WW1∼WW4. The unusually high-affinity of the AMOT PPxY1–NEDD4L WW3 interaction accounts for most of the AMOT–NEDD4L binding and is critical for stimulating HIV-1 release. Comparative structural, binding, and virological analyses reveal that complementary ionic and hydrophobic contacts on both sides of the WW–PPxY core interaction account for the unusually high affinity of the AMOT PPxY1–NEDD4L WW3 interaction. Taken together, our studies reveal how the first AMOT PPxY1 motif binds the third NEDD4L WW domain to stimulate HIV-1 viral envelopment and promote infectivity.

Design of Neuraminidase-Targeted Imaging and Therapeutic Agents for the Diagnosis and Treatment of Influenza Virus Infections.

The last step in influenza virus replication involves the assembly of viral components on the infected cell's plasma membrane followed by budding of intact virus from the host cell surface. Because viral neuraminidase and hemagglutinin are both inserted into the host cell's membrane during this process, influenza virus-infected cells are distinguished from uninfected cells by the presence of viral neuraminidase and hemagglutinin on their cell surfaces. In an effort to exploit this difference in cell surface markers for development of diagnostic and therapeutic agents, we have modified an influenza neuraminidase inhibitor, zanamivir, for targeting of attached imaging and therapeutic agents selectively to influenza viruses and virus-infected cells. We have designed here a zanamivir-conjugated rhodamine dye that allows visual monitoring of binding, internalization, and intracellular trafficking of the fluorescence-labeled neuraminidase in virus-infected cells. We also synthesize a zanamivir-99mTc radioimaging conjugate that permits whole body imaging of the virus's biodistribution and abundance in infected mice. Finally, we create both a zanamivir-targeted cytotoxic drug (i.e., zanamivir-tubulysin B) and a viral neuraminidase-targeted CAR T cell and demonstrate that they are both able to kill viral neuraminidase-expressing cells without damaging healthy cells. Taken together, these data suggest that the influenza virus neuraminidase inhibitor, zanamivir, can be exploited to improve the diagnosis, imaging, and treatment of influenza virus infections.

1635. Oral Delivery of Amikacin-Lipid NanoCrystal Formulations Safely and Effectively Treat Macrolide Resistant Mycobacteria Infections in a Mouse Model of Cystic Fibrosis

BackgroundIn the cystic fibrosis lung, infections by intracellular pathogens, such as Mycobacteria, are problematic to treat due to a thick buildup of mucous as well as the difficulty of many anti-microbial agents, such as amikacin, to penetrate across the plasma membranes of infected cells. Lipid NanoCrystals (LNCs), mediate oral bioavailability for injectable drugs, reduce toxicity, and significantly enhance targeting to mycobacterial infected cells followed by intracellular drug delivery.MethodsThe oral efficacy of Amikacin-LNC (AmK-LNC) was evaluated in the cystic fibrosis (B6CFTRtm1UNC/CFTRtm1UNC) chronic mouse model against each of the three NTM strains having high resistance to macrolide antibiotics (M. avium subsp intracellulare 25292, M. abscesus ssp abscessus 1513, and M. abscessus ssp bolletii 1948). Mice were infected with a pulmonary aerosol of 1x108 CFUs of the macrolide resistant strain and treated daily starting on day 28 for a total of 8 weeks with saline control, oral LP-4 CAmK Lyophilized 50 mg/kg BID, oral LP-4 CAMK Lyophilized 100 mg/kg BID, IP Amikacin (AMI) 150 mg/kg QD, or oral Clarithromycin 250 mg/kg QD. Bacterial burden was measured on day 1, 27, 42, 56 and 84 after infection by plating serial dilutions of organ homogenates on nutrient 7H11 and charcoal agar and counting CFUs after 25-30 days incubation at 32°C. Results represent the average of six experiments (n=5 mice per experiment) bacterial load was expressed as the average Log10 CFU (± SEM) cells (± SEM). ANOVA, saline control compared to drug-treated groups, * denotes the compound that resulted in the highest bacterial reduction, *p< 0.05.ResultsOral administration of AmK-LNCs safely and effectively treated all three macrolide resistant Mycobacteria infections. Colony counts showed that oral administration of AmK-LNC resulted in CFU lung, spleen and liver counts lower than treatment with IP amikacin or clarithromycin.Lung pathology showed that lesions were more numerous and larger in infected mice treated with clarithromycin or amikacin compared to the smaller lesions after treatment with oral AmK-LNC.Bacterial Counts in the Lungs (A), Spleens (B) and Livers (C) Lung Pathology ConclusionConclusionsOral administration of amikacin-LNCs safely and effectively treats macrolide resistant mycobacterial infections in a mouse model of Cystic Fibrosis.DisclosuresRuying Lu, n/a, Matinas BioPharma Inc. (Employee)Matinas BioPharma Inc. (Employee, Shareholder) Raphael J. Mannino, n/a, Matinas BioPharma Inc. (Employee, Shareholder)

Evolutionarily related small viral fusogens hijack distinct but modular actin nucleation pathways to drive cell-cell fusion

Fusion-associated small transmembrane (FAST) proteins are a diverse family of nonstructural viral proteins. Once expressed on the plasma membrane of infected cells, they drive fusion with neighboring cells, increasing viral spread and pathogenicity. Unlike viral fusogens with tall ectodomains that pull two membranes together through conformational changes, FAST proteins have short fusogenic ectodomains that cannot bridge the intermembrane gap between neighboring cells. One orthoreovirus FAST protein, p14, has been shown to hijack the actin cytoskeleton to drive cell-cell fusion, but the actin adaptor-binding motif identified in p14 is not found in any other FAST protein. Here, we report that an evolutionarily divergent FAST protein, p22 from aquareovirus, also hijacks the actin cytoskeleton but does so through different adaptor proteins, Intersectin-1 and Cdc42, that trigger N-WASP-mediated branched actin assembly. We show that despite using different pathways, the cytoplasmic tail of p22 can replace that of p14 to create a potent chimeric fusogen, suggesting they are modular and play similar functional roles. When we directly couple p22 with the parallel filament nucleator formin instead of the branched actin nucleation promoting factor N-WASP, its ability to drive fusion is maintained, suggesting that localized mechanical pressure on the plasma membrane coupled to a membrane-disruptive ectodomain is sufficient to drive cell-cell fusion. This work points to a common biophysical strategy used by FAST proteins to push rather than pull membranes together to drive fusion, one that may be harnessed by other short fusogens responsible for physiological cell-cell fusion.

Identification of the Cleavage Domain within Glycoprotein G of Herpes Simplex Virus Type 2.

Glycoprotein G (gG) from herpes simplex virus type 1 and 2 (HSV-1 and HSV-2, respectively) functions as a viral chemokine binding protein (vCKBP). Soluble recombinant forms of gG of HSV-1 and HSV-2 (SgG1 and SgG2, respectively) enhance chemokine-mediated leukocyte migration, in contrast to most known vCKBPs, including those from animal alpha-herpesviruses. Furthermore, both proteins bind to nerve growth factor (NGF), but only SgG2 enhances NGF-dependent neurite outgrowth. The basis and implications of this functional difference between the two proteins are still unknown. While gG1 and gG2 are positional homologues in the genome, they share very limited sequence homology. In fact, US4, the open reading frame encoding gG is the most divergent genetic locus between these viruses. Full-length gG1 and gG2 are type I transmembrane proteins located on the plasma membrane of infected cells and at the viral envelope. However, gG2 is larger than gG1 and is cleaved during protein maturation, secreting the N-terminal domain to the supernatant of infected cells, whereas gG1 is not. The enzyme involved in gG2 cleavage and the functional relevance of gG2 cleavage and secretion are unknown. We aim to identify the gG2 sequence required for cleavage to determine its functional role in future experiments. Our results prove the existence of at least two cleavage motifs in gG2 within the amino acid region 314-343. Transfer of this sequence to a fusion protein results in cleavage. Finally, we show that propeptide convertases like furin are responsible for gG2 cleavage.

Air pollution particulate matter as a potential carrier of SARS-CoV-2 to the nervous system and/or neurological symptom enhancer: arguments in favor.

Entry receptor for SARS-CoV-2 is expressed in nasal epithelial cells, and nasal delivery pathway can be a key feature of transmission. Here, a possibility of interaction of SARS-CoV-2 with air pollution particulate matter (PM) was considered. It was shown in our recent studies that water-suspended plastic and wood smoke aerosol PM and carbon-containing nanoparticles from burning organics can interact with the plasma membrane of brain nerve terminals presumably due to their lipid components. COVID-19 patients have neurological symptoms, viral particles were found in the brain, SARS-CoV-2 enters the cells via fusion of lipid viral envelope with the plasma membranes of infected cells, and so viral envelop can contain lipid components of the host neuronal membranes. Therefore, interaction of SARS-CoV-2 envelope with PM is possible in water surrounding. After drying, PM can serve as a carrier for transmission of SARS-CoV-2 immobilized at their surface. Moreover, PM and SARS-CoV-2 per se can enter human organism during nasal inhalation, and they both use the same nose-to-brain delivery pathways moving along axons directly to the brain, influencing the nervous system and exocytosis/endocytosis in nerve cells. Thus, PM can aggravate neurological symptoms of SARS-CoV-2 and vice versa, due to their identical nose-to-brain delivery mechanism and possible interference of neuronal effects. In addition, different types of PM because of their ability to interact with the plasma membranes of nerve cells can facilitate unspecific SARS-CoV-2 entrance to the cells, and can influence envelope features of SARS-CoV-2. Detailed studies are required to analyze interaction of SARS-CoV-2 with PM.

Investigating the mechanisms of BK polyomavirus egress and virus-host interactions

BK polyomavirus (BKPyV) is a small, non-enveloped dsDNA virus that infects 70-90% of the world's population and causes a lifelong, silently persistent infection. In immunocompromised individuals, BKPyV replication can result in serious pathology. Bone marrow transplant patients can develop a haemorrhagic cystitis, and in kidney transplant patients BKPyV replication can provoke a nephropathy that leads to deterioration of allograft function and eventual loss of the transplanted organ. There are currently no antiviral treatments with clinical efficacy against BKPyV associated nephropathy. While the life cycles of non-enveloped viruses are often assumed to require cell lysis to release progeny virions, we have evidence to suggest that BKPyV exits the cell via non-lytic means using an unconventional secretory pathway. We have investigated the effects of knocking out cellular genes thought to be involved in unconventional secretory pathways that bypass the Golgi apparatus on the release of BKPyV. We observe decreased BKPyV release from cells that have undergone CRISPR-mediated knockout of Golgi Reassembly Stacking Protein (GORASP) 1 or 2. Investigation of BKPyV-induced changes to the plasma membrane of infected cells demonstrated increased cell surface expression of transmembrane proteins normally resident in the endoplasmic reticulum. This appears to be inhibited by the knockout of GORASP1 or 2, suggesting that virions and ER markers are secreted via a common pathway in infected cells. These experiments are uncovering novel virus-host interactions that, when targeted, could help prevent BKPyV-associated nephropathy and allograft loss.