Incorporation Of Glycoproteins Research Articles

Further Characterization of the Antiviral Transmembrane Protein MARCH8.

The cellular transmembrane protein MARCH8 impedes the incorporation of various viral envelope glycoproteins, such as the HIV-1 envelope glycoprotein (Env) and vesicular stomatitis virus G-glycoprotein (VSV-G), into virions by downregulating them from the surface of virus-producing cells. This downregulation significantly reduces the efficiency of virus infection. In this study, we aimed to further characterize this host protein by investigating its species specificity and the domains responsible for its antiviral activity, as well as its ability to inhibit cell-to-cell HIV-1 infection. We found that the antiviral function of MARCH8 is well conserved in the rhesus macaque, mouse, and bovine versions. The RING-CH domains of these versions are functionally important for inhibiting HIV-1 Env and VSV-G-pseudovirus infection, whereas tyrosine motifs are crucial for the former only, consistent with findings in human MARCH8. Through analysis of chimeric proteins between MARCH8 and non-antiviral MARCH3, we determined that both the N-terminal and C-terminal cytoplasmic tails, as well as presumably the N-terminal transmembrane domain, of MARCH8 are critical for its antiviral activity. Notably, we found that MARCH8 is unable to block cell-to-cell HIV-1 infection, likely due to its insufficient downregulation of Env. These findings offer further insights into understanding the biology of this antiviral transmembrane protein.

MicroRNA-25/93 induction by Vpu as a mechanism for counteracting MARCH1-restriction on HIV-1 infectivity in macrophages.

In order to efficiently produce infectious viral particles, HIV must counter several restrictions exerted by host cell antiviral proteins. MARCH1 is a member of the MARCH protein family that restricts HIV infection by limiting the incorporation of viral envelope glycoproteins into nascent virions. Here, we identified two regulatory RNAs, microRNAs-25 and -93, induced by the HIV-1 accessory protein Vpu, that downregulate MARCH1 mRNA. We also show that Vpu induces these cellular microRNAs in macrophages by hijacking the cellular β-catenin pathway. The notion that HIV-1 has evolved a mechanism to counteract MARCH1 restriction on viral infectivity underlines the importance of MARCH1 in the host antiviral response.

Deleting UL49.5 in duck plague virus causes attachment, entry and spread defects

Duck plague is a disease with high morbidity and mortality rates, and it causes great losses for the duck breeding industry. Duck plague virus (DPV) is the causative agent of duck plague, and DPV UL49.5 protein (pUL49.5) is homologue of glycoprotein N (gN), which is conserved in herpesviruses. UL49.5 homologues are known to be involved in processes such as immune escape, virus assembly, viral fusion, transporter associated with antigen processing (TAP) inhibition and degradation, and maturation and incorporation of glycoprotein M. However, few studies have focused on the role of gN in the early stage of virus infection cells. In this study, we determined that DPV pUL49.5 was distributed in the cytoplasm and colocalized with the endoplasmic reticulum (ER). Moreover, we found that DPV pUL49.5 was a virion component and nonglycosylated protein. To better explore its function, BAC-DPV-ΔUL49.5 was constructed, and its attachment was only approximately 25 % of the revertant virus. Additionally, the penetration ability of BAC-DPV-ΔUL49.5 has only reached 73 % of the revertant virus. The plaque sizes produced by the UL49.5-deleted virus were approximately 58 % smaller than those produced by the revertant virus. Deleting UL49.5 mainly resulted in attachment and cell-to-cell-spread defects. Taken together, these findings suggest important roles for DPV pUL49.5 in viral attachment, penetration and spread.

Endocytosed HIV-1 Envelope Glycoprotein Traffics to Rab14+ Late Endosomes and Lysosomes to Regulate Surface Levels in T-Cell Lines.

ABSTRACTProduction of infectious HIV-1 particles requires incorporation of the viral envelope glycoprotein (Env) at the plasma membrane (PM) of infected CD4+ T cells. Env trafficking to the PM exposes viral epitopes that can be exploited by the host immune system; however, HIV-1 can evade this response by endocytosis of excess Env from the PM. The fate of Env after internalization remains unclear, with evidence suggesting several different vesicular trafficking steps may be involved, including recycling pathways. To date, there have been very few studies documenting the trafficking pathways of native Env in infected T cells. Furthermore, it remains unclear whether there are T-cell-specific endosomal pathways regulating the fate of endocytic Env. Here, we use a pulse-labeling approach with a monovalent anti-Env Fab probe to characterize the trafficking of internalized Env within infected CD4+ T-cell lines, together with CRISPR/Cas9-mediated endogenous protein tagging, to assess the role of host cell Rab GTPases in Env trafficking. We show that endocytosed Env traffics to Rab14+ compartments that possess hallmarks of late endosomes and lysosomes. We also demonstrate that Env can recycle back to the PM, although we find that recycling does not occur at high rates when compared to the model recycling protein transferrin. These results help to resolve open questions about the fate and relevance of endocytosed Env in HIV-infected cells and suggest a novel role for Rab14 in a cell-type-specific late-endosomal/lysosomal trafficking pathway in T cells.IMPORTANCE HIV-1 envelope glycoprotein (Env) evades immune neutralization through many mechanisms. One immune evasion strategy may result from the internalization of excess surface-exposed Env to prevent antibody-dependent cellular cytotoxicity or neutralization. Characterization of the fate of endocytosed Env is critical to understand which vesicular pathways could be targeted to promote display of Env epitopes to the immune system. In this study, we characterize the endocytic fate of native Env, expressed from infected human T-cell lines. We demonstrate that Env is rapidly trafficked to a late-endosome/lysosome-like compartment and can be recycled to the cell surface for incorporation into virus assembly sites. This study implicates a novel intracellular compartment, marked by host-cell Rab14 GTPases, for the sequestration of Env. Therapeutic approaches aimed at mobilizing this intracellular pool of Env could lead to stronger immune control of HIV-1 infection via antibody-dependent cell-mediated cytotoxicity.

An increase in glycoprotein concentration on extracellular virions dramatically alters vaccinia virus infectivity and pathogenesis without impacting immunogenicity.

The extracellular virion (EV) form of Orthopoxviruses is required for cell-to-cell spread and pathogenesis, and is the target of neutralizing antibodies in the protective immune response. EV have a double envelope that contains several unique proteins that are involved in its intracellular envelopment and/or subsequent infectivity. One of these, F13, is involved in both EV formation and infectivity. Here, we report that replacement of vaccinia virus F13L with the molluscum contagiosum virus homolog, MC021L, results in the production of EV particles with significantly increased levels of EV glycoproteins, which correlate with a small plaque phenotype. Using a novel fluorescence-activated virion sorting assay to isolate EV populations based on glycoprotein content we determine that EV containing either higher or lower levels of glycoproteins are less infectious, suggesting that there is an optimal concentration of glycoproteins in the outer envelope that is required for maximal infectivity of EV. This optimal glycoprotein concentration was required for lethality and induction of pathology in a cutaneous model of animal infection, but was not required for induction of a protective immune response. Therefore, our results demonstrate that there is a sensitive balance between glycoprotein incorporation, infectivity, and pathogenesis, and that manipulation of EV glycoprotein levels can produce vaccine vectors in which pathologic side effects are attenuated without a marked diminution in induction of protective immunity.

Mechanism of Viral Glycoprotein Targeting by Membrane-Associated RING-CH Proteins.

An emerging class of cellular inhibitory proteins has been identified that targets viral glycoproteins. These include the membrane-associated RING-CH (MARCH) family of E3 ubiquitin ligases that, among other functions, downregulate cell surface proteins involved in adaptive immunity. The RING-CH domain of MARCH proteins is thought to function by catalyzing the ubiquitination of the cytoplasmic tails (CTs) of target proteins, leading to their degradation. MARCH proteins have recently been reported to target retroviral envelope glycoproteins (Env) and vesicular stomatitis virus G glycoprotein (VSV-G). However, the mechanism of antiviral activity remains poorly defined. Here we show that MARCH8 antagonizes the full-length forms of HIV-1 Env, VSV-G, Ebola virus glycoprotein (EboV-GP), and the spike (S) protein of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), thereby impairing the infectivity of virions pseudotyped with these viral glycoproteins. This MARCH8-mediated targeting of viral glycoproteins requires the E3 ubiquitin ligase activity of the RING-CH domain. We observe that MARCH8 protein antagonism of VSV-G is CT dependent. In contrast, MARCH8-mediated targeting of HIV-1 Env, EboV-GP, and SARS-CoV-2 S protein by MARCH8 does not require the CT, suggesting a novel mechanism of MARCH-mediated antagonism of these viral glycoproteins. Confocal microscopy data demonstrate that MARCH8 traps the viral glycoproteins in an intracellular compartment. We observe that the endogenous expression of MARCH8 in several relevant human cell types is rapidly inducible by type I interferon. These results help to inform the mechanism by which MARCH proteins exert their antiviral activity and provide insights into the role of cellular inhibitory factors in antagonizing the biogenesis, trafficking, and virion incorporation of viral glycoproteins.IMPORTANCE Viral envelope glycoproteins are an important structural component on the surfaces of enveloped viruses that direct virus binding and entry and also serve as targets for the host adaptive immune response. In this study, we investigate the mechanism of action of the MARCH family of cellular proteins that disrupt the trafficking and virion incorporation of viral glycoproteins across several virus families. This research provides novel insights into how host cell factors antagonize viral replication, perhaps opening new avenues for therapeutic intervention in the replication of a diverse group of highly pathogenic enveloped viruses.

Elucidating the Antiviral Mechanism of Different MARCH Factors.

The membrane-associated RING-CH (MARCH) proteins belong to a family of E3 ubiquitin ligases, whose main function is to remove transmembrane proteins from the plasma membrane. Recent work has shown that the human MARCH1, 2, and 8 are antiretroviral factors that target the human immunodeficiency virus type 1 (HIV-1) envelope glycoproteins by reducing their incorporation in the budding virions. Nevertheless, the dearth of information regarding the antiviral mechanism of this family of proteins necessitates further examination. In this study, using both the human MARCH proteins and their mouse homologues, we provide a comprehensive analysis of the antiretroviral mechanism of this family of proteins. Moreover, we show that human MARCH proteins restrict to various degrees the envelope glycoproteins of a diverse number of viruses. This report sheds light on the important antiviral function of MARCH proteins and their significance in cell intrinsic immunity.IMPORTANCE This study examines the mechanism utilized by different MARCH proteins to restrict retrovirus infection. MARCH proteins block the incorporation of envelope glycoproteins to the budding virions. In this report, by comparing the human and mouse MARCH genes and using murine leukemia virus (MLV) and HIV-1, we identify differences in the mechanism of restriction among MARCH proteins. Furthermore, we perform a comprehensive analysis on a number of envelope glycoproteins and show that MARCH proteins have broad antiviral functions.

The interplays between Crimean-Congo hemorrhagic fever virus (CCHFV) M segment-encoded accessory proteins and structural proteins promote virus assembly and infectivity.

Crimean-Congo hemorrhagic fever virus (CCHFV) is a tick-borne orthonairovirus that has become a serious threat to the public health. CCHFV has a single-stranded, tripartite RNA genome composed of L, M, and S segments. Cleavage of the M polyprotein precursor generates the two envelope glycoproteins (GPs) as well as three secreted nonstructural proteins GP38 and GP85 or GP160, representing GP38 only or GP38 linked to a mucin-like protein (MLD), and a double-membrane-spanning protein called NSm. Here, we examined the relevance of each M-segment non-structural proteins in virus assembly, egress and infectivity using a well-established CCHFV virus-like-particle system (tc-VLP). Deletion of MLD protein had no impact on infectivity although it reduced by 60% incorporation of GPs into particles. Additional deletion of GP38 abolished production of infectious tc-VLPs. The loss of infectivity was associated with impaired Gc maturation and exclusion from the Golgi, showing that Gn is not sufficient to target CCHFV GPs to the site of assembly. Consistent with this, efficient complementation was achieved in cells expressing MLD-GP38 in trans with increased levels of preGc to Gc conversion, co-targeting to the Golgi, resulting in particle incorporation and restored infectivity. Contrastingly, a MLD-GP38 variant retained in the ER allowed preGc cleavage but failed to rescue miss-localization or infectivity. NSm deletion, conversely, did not affect trafficking of Gc but interfered with Gc processing, particle formation and secretion. NSm expression affected N-glycosylation of different viral proteins most likely due to increased speed of trafficking through the secretory pathway. This highlights a potential role of NSm in overcoming Golgi retention and facilitating CCHFV egress. Thus, deletions of GP38 or NSm demonstrate their important role on CCHFV particle production and infectivity. GP85 is an essential viral factor for preGc cleavage, trafficking and Gc incorporation into particles, whereas NSm protein is involved in CCHFV assembly and virion secretion.

MARCH8 inhibits viral infection by two different mechanisms.

Membrane-associated RING-CH 8 (MARCH8) inhibits infection with both HIV-1 and vesicular stomatitis virus G-glycoprotein (VSV-G)-pseudotyped viruses by reducing virion incorporation of envelope glycoproteins. The molecular mechanisms by which MARCH8 targets envelope glycoproteins remain unknown. Here, we show two different mechanisms by which MARCH8 inhibits viral infection. Viruses pseudotyped with the VSV-G mutant, in which cytoplasmic lysine residues were mutated, were insensitive to the inhibitory effect of MARCH8, whereas those with a similar lysine mutant of HIV-1 Env remained sensitive to it. Indeed, the wild-type VSV-G, but not its lysine mutant, was ubiquitinated by MARCH8. Furthermore, the MARCH8 mutant, which had a disrupted cytoplasmic tyrosine motif that is critical for intracellular protein sorting, did not inhibit HIV-1 Env-mediated infection, while it still impaired infection by VSV-G-pseudotyped viruses. Overall, we conclude that MARCH8 reduces viral infectivity by downregulating envelope glycoproteins through two different mechanisms mediated by a ubiquitination-dependent or tyrosine motif-dependent pathway.

Plasma membrane vesicles of human umbilical cord mesenchymal stem cells ameliorate acetaminophen-induced damage in HepG2 cells: a novel stem cell therapy

BackgroundAcetaminophen (APAP) overdose is the common cause of acute liver failure (ALF) due to the oxidative damage of multiple cellular components. This study aimed to investigate whether plasma membrane vesicles (PMVs) from human umbilical cord mesenchymal stem cells (hUCMSCs) could be exploited as a novel stem cell therapy for APAP-induced liver injury.MethodsPMVs from hUCMSCs were prepared with an improved procedure including a chemical enucleation step followed by a mechanical extrusion. PMVs of hUCMSCs were characterized and supplemented to hepatocyte cultures. Rescue of APAP-induced hepatocyte damage was evaluated.ResultsThe hUCMSCs displayed typical fibroblastic morphology and multipotency when cultivated under adipogenic, osteogenic, or chondrogenic conditions. PMVs of hUCMSCs maintained the stem cell phenotype, including the presence of CD13, CD29, CD44, CD73, and HLA-ABC, but the absence of CD45, CD117, CD31, CD34, and HLA-DR on the plasma membrane surface. RT-PCR and transcriptomic analyses showed that PMVs were similar to hUCMSCs in terms of mRNA profile, including the expression of stemness genes GATA4/5/6, Nanog, and Oct1/2/4. GO term analysis showed that the most prominent reduced transcripts in PMVs belong to integral membrane components, extracellular vesicular exosome, and extracellular matrix. Immunofluorescence labeling/staining and confocal microscopy assays showed that PMVs enclosed cellular organelles, including mitochondria, lysosomes, proteasomes, and endoplasmic reticula. Incorporation of the fusogenic VSV-G viral membrane glycoprotein stimulated the endosomal release of PMV contents into the cytoplasm. Further, the addition of PMVs and a mitochondrial-targeted antioxidant Mito-Tempo into cultures of APAP-treated HepG2 cells resulted in reduced cell death, enhanced viability, and increased mitochondrial membrane potential. Lastly, this study demonstrated that the redox state and activities of aminotransferases were restored in APAP-treated HepG2 cells.ConclusionsThe results suggest that PMVs from hUCMSCs could be used as a novel stem cell therapy for the treatment of APAP-induced liver injury.

The Interplay between HIV-1 Gag Binding to the Plasma Membrane and Env Incorporation.

Advancement in drug therapies and patient care have drastically improved the mortality rates of HIV-1 infected individuals. Many of these therapies were developed or improved upon by using structure-based techniques, which underscore the importance of understanding essential mechanisms in the replication cycle of HIV-1 at the structural level. One such process which remains poorly understood is the incorporation of the envelope glycoprotein (Env) into budding virus particles. Assembly of HIV particles is initiated by targeting of the Gag polyproteins to the inner leaflet of the plasma membrane (PM), a process mediated by the N-terminally myristoylated matrix (MA) domain and phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2). There is strong evidence that formation of the Gag lattice on the PM is a prerequisite for the incorporation of Env into budding particles. It is also suggested that Env incorporation is mediated by an interaction between its cytoplasmic tail (gp41CT) and the MA domain of Gag. In this review, we highlight the latest developments and current efforts to understand the interplay between gp41CT, MA, and the membrane during assembly. Elucidation of the molecular determinants of Gag–Env–membrane interactions may help in the development of new antiviral therapeutic agents that inhibit particle assembly, Env incorporation and ultimately virus production.

The Genetics and Biochemistry of Cell Wall Structure and Synthesis in Neurospora crassa, a Model Filamentous Fungus.

This review discusses the wealth of information available for the N. crassa cell wall. The basic organization and structure of the cell wall is presented and how the wall changes during the N. crassa life cycle is discussed. Over forty cell wall glycoproteins have been identified by proteomic analyses. Genetic and biochemical studies have identified many of the key enzymes needed for cell wall biogenesis, and the roles these enzymes play in cell wall biogenesis are discussed. The review includes a discussion of how the major cell wall components (chitin, β-1,3-glucan, mixed β-1,3-/ β-1,4- glucans, glycoproteins, and melanin) are synthesized and incorporated into the cell wall. We present a four-step model for how cell wall glycoproteins are covalently incorporated into the cell wall. In N. crassa, the covalent incorporation of cell wall glycoproteins into the wall occurs through a glycosidic linkage between lichenin (a mixed β-1,3-/β-1,4- glucan) and a “processed” galactomannan that has been attached to the glycoprotein N-linked oligosaccharides. The first step is the addition of the galactomannan to the N-linked oligosaccharide. Mutants affected in galactomannan formation are unable to incorporate glycoproteins into their cell walls. The second step is carried out by the enzymes from the GH76 family of α-1,6-mannanases, which cleave the galactomannan to generate a processed galactomannan. The model suggests that the third and fourth steps are carried out by members of the GH72 family of glucanosyltransferases. In the third step the glucanosyltransferases cleave lichenin and generate enzyme/substrate intermediates in which the lichenin is covalently attached to the active site of the glucanosyltransferases. In the final step, the glucanosyltransferases attach the lichenin onto the processed galactomannans, which creates new glycosidic bonds and effectively incorporates the glycoproteins into the cross-linked cell wall glucan/chitin matrix.

Improving Baculovirus Transduction of Mammalian Cells by Incorporation of Thogotovirus Glycoproteins.

Baculovirus can transduce a wide range of mammalian cells and is considered a promising gene therapy vector. However, the low transduction efficiency of baculovirus into many mammalian cells limits its practical application. Co-expressing heterologous viral glycoproteins (GPs), such as vesicular stomatitis virus G protein (VSV G), with baculovirus native envelope protein GP64 is one of the feasible strategies for improving virus transduction. Tick-borne thogotoviruses infect mammals and their GPs share sequence/structure homology and common evolutionary origins with baculovirus GP64. Herein, we tested whether thogotovirus GPs could facilitate the entry of the prototype baculovirus Autographa californica multiple multiple nucleopolyhedrovirus (AcMNPV) into mammalian cells. The gp genes of two thogotoviruses, Thogoto virus and Dhori virus, were inserted into the AcMNPV genome. Both GPs were properly expressed and incorporated into the envelope of the recombinant AcMNPVs. The transduction rates of recombinant AcMNPVs expressing the two thogotovirus GPs increased for approximately 4-12 fold compared to the wild type AcMNPV in six of the 12 tested mammalian cell lines. It seemed that thogotovirus GPs provide the recombinant AcMNPVs with different cell tropisms and showed better performance in several mammalian cells compared to VSV G incorporated AcMNPV. Further studies showed that the improved transduction was a result of augmented virus-endosome fusion and endosome escaping, rather than increased cell binding or internalization. We found the AcMNPV envelope protein GP64-mediated fusion was enhanced by the thogotovirus GPs at relatively higher pH conditions. Therefore, the thogotovirus GPs represent novel candidates to improve baculovirus-based gene delivery vectors.

Incorporation of Ebola glycoprotein into HIV particles facilitates dendritic cell and macrophage targeting and enhances HIV-specific immune responses.

The development of an effective vaccine against HIV infection remains a global priority. Dendritic cell (DC)-based HIV immunotherapeutic vaccine is a promising approach which aims at optimizing the HIV-specific immune response using primed DCs to promote and enhance both the cellular and humoral arms of immunity. Since the Ebola virus envelope glycoprotein (EboGP) has strong DC-targeting ability, we investigated whether EboGP is able to direct HIV particles towards DCs efficiently and promote potent HIV-specific immune responses. Our results indicate that the incorporation of EboGP into non-replicating virus-like particles (VLPs) enhances their ability to target human monocyte-derived dendritic cells (MDDCs) and monocyte-derived macrophages (MDMs). Also, a mucin-like domain deleted EboGP (EboGPΔM) can further enhanced the MDDCs and MDMs-targeting ability. Furthermore, we investigated the effect of EboGP on HIV immunogenicity in mice, and the results revealed a significantly stronger HIV-specific humoral immune response when immunized with EboGP-pseudotyped HIV VLPs compared with those immunized with HIV VLPs. Splenocytes harvested from mice immunized with EboGP-pseudotyped HIV VLPs secreted increased levels of macrophage inflammatory proteins-1α (MIP-1α) and IL-4 upon stimulation with HIV Env and/or Gag peptides compared with those harvested from mice immunized with HIV VLPs. Collectively, this study provides evidence for the first time that the incorporation of EboGP in HIV VLPs can facilitate DC and macrophage targeting and induce more potent immune responses against HIV.

Deficient Incorporation of Rabies Virus Glycoprotein into Virions Enhances Virus-Induced Immune Evasion and Viral Pathogenicity.

Previous studies have shown that wild-type (wt) rabies virus (RABV) evades the host immune response by restricting expression of glycoprotein (G), which blocks activation of dendritic cells (DCs) and induces production of virus-neutralizing antibodies (VNAs). In the present study, wt RABVs not only restricted G expression but also reduced incorporation of G into mature virions compared with laboratory-adapted viruses. A recombinant RABV expressing triple G was used to further determine whether G expression relates to incorporation. The recombinant virus showed higher expression and incorporation of G and activated more DCs than the virus that expressed a single copy of G. Removal of G from viruses using subtilisin or Dithiothreitol (DTT)/ Nonidet P-40 (NP40) almost completely abolishes DC activation and VNA production. Consequently, these G-depleted viruses cause lethal infection in mice. Thus, wt RABVs can subvert DC-induced antiviral immune response and maintain pathogenicity by decreasing G expression in infected cells and G incorporation into virions.

Membrane-associated RING-CH (MARCH) 1 and 2 are MARCH family members that inhibit HIV-1 infection

Membrane-associated RING-CH 8 (MARCH8) is one of 11 members of the MARCH family of RING finger E3 ubiquitin ligases and down-regulates several membrane proteins (e.g. major histocompatibility complex II [MHC-II], CD86, and transferrin receptor). We recently reported that MARCH8 also targets HIV-1 envelope glycoproteins and acts as an antiviral factor. However, it remains unclear whether other family members might have antiviral functions similar to those of MARCH8. Here we show that MARCH1 and MARCH2 are MARCH family members that reduce virion incorporation of envelope glycoproteins. Infectivity assays revealed that MARCH1 and MARCH2 dose-dependently suppress viral infection. Treatment with type I interferon enhanced endogenous expression levels of MARCH1 and MARCH2 in monocyte-derived macrophages. Expression of these proteins in virus-producing cells decreased the efficiency of viral entry and down-regulated HIV-1 envelope glycoproteins from the cell surface, resulting in reduced incorporation of envelope glycoproteins into virions, as observed in MARCH8 expression. With the demonstration that MARCH1 and MARCH2 are antiviral MARCH family members as presented here, these two proteins join a growing list of host factors that inhibit HIV-1 infection.

The Botanical Glycoside Oleandrin Inhibits Human T-cell Leukemia Virus Type-1 Infectivity and Env-Dependent Virological Synapse Formation.

At present, there are no antiretroviral drugs that inhibit incorporation of the envelope glycoprotein into newly-synthesized virus particles. The botanical glycoside, oleandrin, derived from extracts of Nerium oleander, has previously been shown to reduce the levels of the gp120 envelope glycoprotein on human immunodeficiency virus type-1 (HIV-1) particles and inhibit HIV-1 infectivity in vitro. We therefore tested whether oleandrin or an extract from N. oleander could also inhibit the infectivity of the human T-cell leukemia virus type-1 (HTLV-1): A related enveloped retrovirus and emerging tropical infectious agent. The treatment of HTLV-1+ lymphoma T-cells with either oleandrin or a N. oleander extract did not significantly inhibit viral replication or the release of p19Gag-containing particles into the culture supernatants. However, the collected virus particles from treated cells exhibited reduced infectivity on primary human peripheral blood mononuclear cells (huPBMCs). Unlike HIV-1, extracellular HTLV-1 particles are poorly infectious and viral transmission typically occurs via direct intercellular interactions across a virological synapse. We therefore investigated whether oleandrin or a N. oleander extract could inhibit virus transmission from a GFP-expressing HTLV-1+ lymphoma T-cell-line to huPBMCs in co-culture assays. These results demonstrated that both oleandrin and the crude phytoextract inhibited the formation of virological synapses and the transmission of HTLV-1 in vitro. Importantly, these findings suggest oleandrin may have broad antiviral activity against enveloped viruses by reducing the incorporation of the envelope glycoprotein into mature particles, a stage of the infection cycle not targeted by modern HAART.

The Antiviral Activity of the Cellular Glycoprotein LGALS3BP/90K Is Species Specific.

ABSTRACTCellular antiviral proteins interfere with distinct steps of replication cycles of viruses. The galectin 3 binding protein (LGALS3BP, also known as 90K) was previously shown to lower the infectivity of nascent human immunodeficiency virus type 1 (HIV-1) virions when expressed in virus-producing cells. This antiviral effect was accompanied by impaired gp160Env processing and reduced viral incorporation of mature Env glycoproteins. Here, we examined the ability of 90K orthologs from primate species to reduce the particle infectivity of distinct lentiviruses. We show that 90K's ability to diminish the infectivity of lentiviral particles is conserved within primate species, with the notable exception of 90K from rhesus macaque. Comparison of active and inactive 90K orthologs and variants uncovered the fact that inhibition of processing of the HIV-1 Env precursor and reduction of cell surface expression of HIV-1 Env gp120 are required, but not sufficient, for 90K-mediated antiviral activity. Rather, 90K-mediated reduction of virion-associated gp120 coincided with antiviral activity, suggesting that 90K impairs the incorporation of HIV-1 Env into budding virions. We show that a single “humanizing” amino acid exchange in the BTB (broad-complex, tramtrack, and bric-à-brac)/POZ (poxvirus and zinc finger) domain is sufficient to fully rescue the antiviral activity of a shortened version of rhesus macaque 90K, but not that of the full-length protein. Comparison of the X-ray structures of the BTB/POZ domains of 90K from rhesus macaques and humans point toward a slightly larger hydrophobic patch at the surface of the rhesus macaque BTB domain that may modulate a direct interaction with either a second 90K domain or a different protein.IMPORTANCE The cellular 90K protein has been shown to diminish the infectivity of nascent HIV-1 particles. When produced in 90K-expressing cells, particles bear smaller amounts of the HIV-1 Env glycoprotein, which is essential for attaching to and entering new target cells in the subsequent infection round. However, whether the antiviral function of 90K is conserved across primates is unknown. Here, we found that 90K orthologs from most primate species, but, surprisingly, not from rhesus macaques, inhibit HIV-1. The introduction of a single amino acid exchange into a short version of the rhesus macaque 90K protein, consisting of the two intermediate domains of 90K, resulted in full restoration of antiviral activity. Structural elucidation of the respective domain suggests that the absence of antiviral activity in the rhesus macaque factor may be linked to a subtle change in protein-protein interaction.

Statins Suppress Ebola Virus Infectivity by Interfering with Glycoprotein Processing.

ABSTRACTEbola virus (EBOV) infection is a major public health concern due to high fatality rates and limited effective treatments. Statins, widely used cholesterol-lowering drugs, have pleiotropic mechanisms of action and were suggested as potential adjunct therapy for Ebola virus disease (EVD) during the 2013–2016 outbreak in West Africa. Here, we evaluated the antiviral effects of statin (lovastatin) on EBOV infection in vitro. Statin treatment decreased infectious EBOV production in primary human monocyte-derived macrophages and in the hepatic cell line Huh7. Statin treatment did not interfere with viral entry, but the viral particles released from treated cells showed reduced infectivity due to inhibition of viral glycoprotein processing, as evidenced by decreased ratios of the mature glycoprotein form to precursor form. Statin-induced inhibition of infectious virus production and glycoprotein processing was reversed by exogenous mevalonate, the rate-limiting product of the cholesterol biosynthesis pathway, but not by low-density lipoprotein. Finally, statin-treated cells produced EBOV particles devoid of the surface glycoproteins required for virus infectivity. Our findings demonstrate that statin treatment inhibits EBOV infection and suggest that the efficacy of statin treatment should be evaluated in appropriate animal models of EVD.

Promotion of virus assembly and organization by the measles virus matrix protein

Measles virus (MeV) remains a major human pathogen, but there are presently no licensed antivirals to treat MeV or other paramyxoviruses. Here, we use cryo-electron tomography (cryo-ET) to elucidate the principles governing paramyxovirus assembly in MeV-infected human cells. The three-dimensional (3D) arrangement of the MeV structural proteins including the surface glycoproteins (F and H), matrix protein (M), and the ribonucleoprotein complex (RNP) are characterized at stages of virus assembly and budding, and in released virus particles. The M protein is observed as an organized two-dimensional (2D) paracrystalline array associated with the membrane. A two-layered F–M lattice is revealed suggesting that interactions between F and M may coordinate processes essential for MeV assembly. The RNP complex remains associated with and in close proximity to the M lattice. In this model, the M lattice facilitates the well-ordered incorporation and concentration of the surface glycoproteins and the RNP at sites of virus assembly.