Sphingomyelin-Sequestered Cholesterol Domain Recruits Formin-Binding Protein 17 for Constricting Clathrin-Coated Pits in Influenza Virus Entry

Influenza A virus (IAV) is a global health threat. The cellular endocytic machineries harnessed by IAV remain elusive. Here, by tracking single IAV particles and quantifying the internalized IAV, we found that the sphingomyelin (SM)-sequestered cholesterol, but not the accessible cholesterol, is essential for the clathrin-mediated endocytosis (CME) of IAV. The clathrin-independent endocytosis of IAV is cholesterol-independent. Whereas, the CME of transferrin depends on SM-sequestered cholesterol and accessible cholesterol. Furthermore, three-color single-virus tracking and electron microscopy showed that the SM-cholesterol complex nanodomain is recruited to the IAV-containing clathrin-coated structure (CCS) and facilitates neck constriction of the IAV-containing CCS. Meanwhile, formin-binding protein 17 (FBP17), a membrane-bending protein which activates actin nucleation, is recruited to IAV-CCS complex in a manner dependent on the SM-cholesterol complex. We propose that the SM-cholesterol nanodomain at the neck of CCS recruits FBP17 to induce neck constriction by activating actin assembly. These results unequivocally show the physiological importance of the SM-cholesterol complex in IAV entry. Importance: IAV infects the cells by harnessing cellular endocytic machineries. Better understanding of the cellular machineries used for its entry might lead to the development of antiviral strategies, and would also provide important insights into physiological endocytic processes. This work demonstrated that a special pool of cholesterol in plasma membrane, SM-sequestered cholesterol, recruits FBP17 for the constriction of clathrin-coated pits in IAV entry. Meanwhile, the clathrin-independent cell entry of IAV is cholesterol-independent. The internalization of transferrin, the gold-standard cargo endocytosed solely via CME, is much less dependent on the SM-cholesterol complex. These results would provide new insights into IAV infection and pathway/cargo-specific involvement of cholesterol pool(s).

Human Cytomegalovirus Hijacks WD Repeat Domain 11 for Virion Assembly Compartment Formation and Virion Morphogenesis

Human cytomegalovirus (HCMV) has a large (∼235-kb) genome with over 200 predicted open reading frames and exploits numerous cellular factors to facilitate its replication. A key feature of HCMV-infected cells is the emergence of a distinctive membranous cytoplasmic compartment termed the virion assembly compartment (vAC). Here we report that host protein WD repeat domain 11 (WDR11) plays a key role in vAC formation and virion morphogenesis. We found that WDR11 was up-regulated at both mRNA and protein levels during HCMV infection. At the late stage of HCMV replication, WDR11 relocated to the vAC and co-localized with markers of the trans-Golgi network (TGN) and vAC. Depletion of WDR11 hindered HCMV-induced membrane reorganization of the Golgi and TGN, altered vAC formation, and impaired HCMV secondary envelopment and virion morphogenesis. Further, motifs critical for the localization of WDR11 in TGN were identified by alanine-scanning mutagenesis. Mutation of these motifs led to WDR11 mislocation outside of the TGN and loss of vAC formation. Taken together, these data indicate that host protein WDR11 is required for efficient viral replication at the stage of virion assembly, possibly by facilitating the remodeling of the endomembrane system for vAC formation and virion morphogenesis. Importance During the late phase of human cytomegalovirus (HCMV) infection, the endomembrane system is dramatically reorganized, resulting in the formation of a unique structure termed the virion assembly compartment (vAC), which is critical for the assembly of infectious virions. The mechanism of HCMV-induced vAC formation is still not fully understood. In this report, we identified a host factor, WDR11, that plays an important role in vAC formation. Our findings argue that WDR11 contributes to the relocation of the Golgi and trans-Golgi network to the vAC, a membrane reorganization process that appears to be required for efficient virion maturation. The present work provides new insights into the vAC formation and HCMV virion morphogenesis and a potential novel target for anti-viral treatment.

Expression of a phage-encoded Gp21 protein protects Pseudomonas aeruginosa against phage infection

There is a continuously expanding gap between predicted phage gene sequences and their corresponding functions, which largely hampered the development of phage therapy. Previous studies reported several phage proteins that could interfere with the intracellular processes of the host to obtain efficient infection. But few phage proteins that protect host against phage infection has been identified and characterized in detail. Here, we isolate a phage vB_Pae_QDWS capable of infecting Pseudomonas aeruginosa PAO1, and report its encoded Gp21 protein protects PAO1 against phage infection. Expressing of Gp21 regulate bacterial quorum sensing with an inhibitory effect in low cell density and activation effect in high cell density. By testing the TFPs-mediated twitching motility and transmission electron microscopy analysis, Gp21 was found decreased the pilus synthesis. Further constructing the TFPs synthesis gene pilB mutant and performing adsorption and phage resistance assay, we demonstrated Gp21 protein could block phage infection via decreasing the TFPs-mediated phage adsorption. Gp21 is a novel protein that inhibit phage efficacy against bacteria. The study deepens our understanding of phage-host interactions. Importance The majority of the annotated phage genes are currently deposited as “hypothetical protein” with unknown function. Researches revealed that some phage proteins serve to inhibit or redirect the host intracellular processes for phage infection. Differently, we report a phage encoded protein Gp21 that protect the host against phage infection. The pathways that Gp21 involved in anti-phage defense in Pseudomonas aeruginosa PAO1 are interfering with quorum sensing and decreasing the type IV pilus-mediated phage adsorption. Gp21 is a novel protein with a low sequence homology with other reported twitching inhibitory proteins. As a lytic phage derived protein, Gp21 expression protects P. aeruginosa PAO1 from reinfection by phage vB_Pae_QDWS, which may explain the well-known pseudolysogeny caused by virulent phages. Our discoveries provide valuable new insight into the phage-host evolutionary dynamics.

Antiviral CD19 + CD27 + Memory B Cells Are Associated with Protection from Recurrent Asymptomatic Ocular Herpes Infection

Reactivation of herpes simplex virus 1 (HSV-1) from latently infected neurons of the trigeminal ganglia (TG) leads to blinding recurrent herpetic disease in symptomatic (SYMP) individuals. Although the role of T cells in herpes immunity seen in asymptomatic (ASYMP) individuals is heavily explored, the role of B cells is less investigated. In the present study, we evaluated whether B cells are associated with protective immunity against recurrent ocular herpes. The frequencies of circulating HSV-specific memory B cells and of memory follicular helper T cells (CD4 + T fh cells), that help B cells produce antibodies, were compared between HSV-1 infected SYMP and ASYMP individuals. The levels of IgG/IgA and neutralizing antibodies were compared in SYMP and ASYMP individuals. We found that: ( i ) the ASYMP individuals had increased frequencies of HSV-specific CD19 + CD27 + memory B cells; and ( ii ) high frequencies of HSV-specific switched IgG + CD19 + CD27 + memory B cells detected in ASYMP individuals were directly proportional to high frequencies of CD45R0 + CXCR5 + CD4 + memory T fh cells. However, no differences were detected in the level of HSV-specific IgG/IgA antibodies in SYMP and ASYMP individuals. Using the UV-B-induced HSV-1 reactivation mouse model, we found increased frequencies of HSV-specific antibody-secreting plasma HSV-1 gD + CD138 + B cells within the TG and circulation of ASYMP mice compared to SYMP mice. In contrast, no significant differences in the frequencies of B cells were found in the cornea, spleen, and bone-marrow. Our findings suggest that circulating antibody-producing HSV-specific memory B cells recruited locally to the TG may contribute to protection from symptomatic recurrent ocular herpes. IMPORTANCE Reactivation of herpes simplex virus 1 (HSV-1) from latently infected neurons of the trigeminal ganglia (TG) leads to blinding recurrent herpetic disease in symptomatic (SYMP) individuals. Although the role of T cells in herpes immunity against blinding recurrent herpetic disease is heavily explored, the role of B cells is less investigated. In the present study, we found that in both asymptomatic (ASYMP) individuals and ASYMP mice there was increased frequencies of HSV-specific memory B cells that were directly proportional to high frequencies of memory T fh cells. Moreover, following UV-B induce reactivation, we found increased frequencies of HSV-specific antibody-secreting plasma B cells within the TG and circulation of ASYMP mice, compared to SYMP mice. Our findings suggest that circulating antibody-producing HSV-specific memory B cells recruited locally to the TG may contribute to protection from recurrent ocular herpes.

Establishment of a monoclonal antibody against human NTCP that blocks HBV infection

Hepatitis B virus (HBV) infects 240 million people worldwide. Current therapy profoundly suppresses HBV replication but requires long-term maintenance therapy. Therefore there is still a medical need for an efficient HBV cure. HBV enters host cells by binding via the preS1-domain of the viral L protein to the Na + /Taurocholate Cotransporting Polypeptide (NTCP). Thus, NTCP should be a key target for the development of anti-HBV therapeutics. Indeed, Myrcludex B, a synthetic form of the myristoylated preS1 peptide, effectively reduces HBV/HDV infection and has been approved as Hepcludex® in Europe for the treatment of patients with chronic hepatitis D virus (HDV) infection. We established a monoclonal antibody (mAb) N6HB426-20 that recognizes the extracellular domain of human NTCP and blocks HBV entry in vitro into human liver cells but has much less of an inhibitory effect on bile acid uptake. In vivo , administration of the N6HB426-20 mAb prevented HBV viremia for an extended period of time after HBV inoculation in a mouse model system without strongly inhibiting bile acid absorption. Among the extracellular loops (ECLs) of NTCP, regions of amino acids (aa) 84-87 in ECL1 and aa 157-165 near ECL2 of transmembrane domain 5 are critically important for HBV/HDV infection. Epitope-mapping and the 3D model of the NTCP structure suggested that the N6HB426-20 mAb may recognize aa 276/277 at the tip of ECL4 and interfere with an binding of HBV to the aa 84-87 region. In summary, we identified an in vivo neutralizing NTCP-targeting antibody capable of preventing HBV infection. Further improvements in efficacy of this drug will pave the way for its clinical applications. IMPORTANCE A number of entry inhibitors are being developed to enhance the treatment of HBV patients with oral nucleos(t)ide analogues (NA). To amplify the effectiveness of NA therapy, several efforts have been made to develop therapeutic mAbs with neutralizing activity against HBs antigens. However, the neutralizing effect of these mAbs may be muted by a large excess of HBsAg-positive noninfectious particles in the blood of infected patients. The advantage of NTCP-targeted HBV entry inhibitors is that they remain effective regardless of viral genotype, viral mutations and the presence of subviral particles. Although N6HB426-20 requires a higher dose than Myrcludex to obtain equivalent suppression of HBV in a model mouse system, it maintained the inhibitory effect for a long time post administration in proportion to the half-life of an IgG mAb. We believe that further improvements will make this antibody a promising treatment option for patients with chronic hepatitis B.

Vaccine-associated enhanced respiratory disease following influenza virus infection in ferrets recapitulates the model in pigs

Influenza A virus (IAV) causes respiratory disease in swine and humans. Vaccines are used to prevent influenza illness in both populations but must be frequently updated due to rapidly evolving strains. Mismatch between the circulating strains and strains contained in vaccines may cause loss in efficacy. Whole inactivated virus (WIV) vaccines with adjuvant utilized by the swine industry are effective against antigenically similar viruses; however, vaccine-associated enhanced respiratory disease (VAERD) may happen when the WIV is antigenically mismatched with the infecting virus. VAERD is a repeatable model in pigs, but had yet to be experimentally demonstrated in other mammalian species. We recapitulated VAERD in ferrets, a standard benchmark animal model for studying human influenza infection, in a direct comparison to VAERD in pigs. Both species were vaccinated with WIV with oil in water adjuvant containing a δ-1 H1N2 (1B.2.2) derived from the pre-2009 human seasonal lineage, then challenged with a 2009 pandemic H1N1 (H1N1pdm09, 1A.3.3.2) five weeks after vaccination. Nonvaccinated and challenged groups showed typical signs of influenza disease, but the mismatched vaccinated and challenged pigs and ferrets showed elevated clinical signs, despite similar viral loads. VAERD affected pigs exhibited a 2-fold increase in lung lesions, while VAERD affected ferrets showed a 4-fold increase. Similar to pigs, antibodies from VAERD affected ferrets preferentially bound to the HA2 domain of the H1N1pdm09 challenge strain. These results indicate VAERD is not limited to pigs, as demonstrated here in ferrets, and the need to consider VAERD when evaluating new vaccine platforms and strategies. Importance We demonstrated the susceptibility of ferrets, a laboratory model species for human influenza A virus research, to vaccine associated enhanced respiratory disease (VAERD) using an experimental model previously demonstrated in pigs. Ferrets developed clinical characteristics of VAERD very similar to that in pigs. The hemagglutinin (HA) stalk is a potential vaccine target to develop more efficacious, broadly reactive influenza vaccine platforms and strategies. However, non-neutralizing antibodies directed towards a conserved epitope on the HA stalk induced by an oil-in-water adjuvanted whole influenza virus vaccine were previously shown in VAERD-affected pigs and were also identified here in VAERD-affected ferrets. The induction of VAERD in ferrets highlights the potential risk of mismatched influenza vaccines to humans and the need to consider VAERD when designing and evaluating vaccine strategies.

Activation of the MKK3-p38-MK2-ZFP36 axis by coronavirus infection restricts the upregulation of AU-rich element-containing transcripts in proinflammatory response

Coronavirus infections induce the expression of multiple proinflammatory cytokines and chemokines. We have previously shown that in cells infected with gammacoronavirus infectious bronchitis virus (IBV), interleukin 6 (IL-6) and IL-8 were drastically upregulated, and the MAP kinase p38 and the integrated stress response pathways were implicated in this process. In this study, we report that coronavirus infection activates a negative regulatory loop that restricts the upregulation of a number of proinflammatory genes. As revealed by the initial transcriptomic and subsequent validation analyses, the anti-inflammatory adenine-uridine (AU)-rich element (ARE)-binding protein, Zinc finger protein 36 (ZFP36) and its related family members were upregulated in cells infected with IBV and three other coronaviruses, alphacoronaviruses porcine epidemic diarrhea virus (PEDV) and human coronavirus 229E (HCoV-229E), and betacoronavirus HCoV-OC43, respectively. Characterization of the functional roles of ZFP36 during IBV infection demonstrated that ZFP36 promoted the degradation of transcripts coding for IL-6, IL-8, dual-specificity phosphatase 1 (DUSP1), prostaglandin-endoperoxide synthase 2 (PTGS2) and TNF-α-induced protein 3 (TNFAIP3), through binding to AREs in these transcripts. Consistently, knockdown and inhibition of JNK and p38 kinase activities reduced the expression of ZFP36, as well as the expression of IL-6 and IL-8. On the contrary, overexpression of mitogen-activated protein kinase kinase 3 (MKK3) and MAPKAP kinase-2 (MK2), the upstream and downstream kinases of p38, respectively, increased the expression of ZFP36 and decreased the expression of IL-8. Taken together, this study reveals an important regulatory role of the MKK3-p38-MK2-ZFP36 axis in coronavirus infection-induced proinflammatory response. Importance Excessive and uncontrolled induction and release of proinflammatory cytokines and chemokines, the so-called cytokine release syndrome (CRS), would cause life-threatening complications and multiple organ failure in severe coronavirus infections, including severe acute respiratory syndrome (SARS), Middle East respiratory syndrome (MERS) and COVID-19. This study reveals that coronavirus infection also induces the expression of ZFP36, an anti-inflammatory ARE-binding protein, promoting the degradation of ARE-containing transcripts coding for IL-6 and IL-8 as well as a number of other proteins related to inflammatory response. Furthermore, the p38 MAP kinase, its upstream kinase MKK3 and downstream kinase MK2 were shown to play a regulatory role in upregulation of ZFP36 during coronavirus infection cycles. This MKK3-p38-MK2-ZFP36 axis would constitute a potential therapeutic target for severe coronavirus infections.

A Pseudorabies Virus Serine/Threonine Kinase, US3, Promotes Retrograde Transport in Axons via Akt/mToRC1

Infection of peripheral axons by alpha herpesviruses (AHVs) is a critical stage in establishing a life-long infection in the host. Upon entering the cytoplasm of axons, AHV nucleocapsids and associated inner-tegument proteins must engage the cellular retrograde transport machinery to promote the long-distance movement of virion components to the nucleus. The current model outlining this process is incomplete and further investigation is required to discover all viral and cellular determinants involved as well as the temporality of the events. Using a modified tri-chamber system, we have discovered a novel role of the pseudorabies virus (PRV) serine/threonine kinase, US3, in promoting efficient retrograde transport of nucleocapsids. We discovered that transporting nucleocapsids move at similar velocities both in the presence and absence of a functional US3 kinase; however fewer nucleocapsids are moving when US3 is absent and move for shorter periods of time before stopping, suggesting US3 is required for efficient nucleocapsid engagement with the retrograde transport machinery. This led to fewer nucleocapsids reaching the cell bodies to produce a productive infection 12hr later. Furthermore, US3 was responsible for the induction of local translation in axons as early as 1hpi through the stimulation of a PI3K/Akt-mToRC1 pathway. These data describe a novel role for US3 in the induction of local translation in axons during AHV infection, a critical step in transport of nucleocapsids to the cell body. Importance Neurons are highly polarized cells with axons that can reach centimeters in length. Communication between axons at the periphery and the distant cell body is a relatively slow process involving the active transport of chemical messengers. There’s a need for axons to respond rapidly to extracellular stimuli. Translation of repressed mRNAs present within the axon occurs to enable rapid, localized responses independently of the cell body. AHVs have evolved a way to hijack local translation in the axons to promote their transport to the nucleus. We have determined the cellular mechanism and viral components involved in the induction of axonal translation. The US3 serine/threonine kinase of PRV activates Akt-mToRC1 signaling pathways early during infection to promote axonal translation. When US3 is not present, the number of moving nucleocapsids and their processivity are reduced, suggesting that US3 activity is required for efficient engagement of nucleocapsids with the retrograde transport machinery.

Attenuation of Getah virus by a single amino acid substitution at residue 253 of the E2 protein that might be part of a new heparan sulfate binding site on alphaviruses

The emergence of new epidemic variants of alphaviruses poses a public health risk. It is associated with adaptive mutations that often cause increased pathogenicity. Getah virus (GETV), a neglected and re-emerging mosquito-borne alphavirus, poses threat to many domestic animals and probably even humans. At present, the underlying mechanisms of GETV pathogenesis are not well defined. We identified a residue in the E2 glycoprotein that is critical for viral adsorption to cultured cells and pathogenesis in vivo . Viruses containing an arginine instead of a lysine at residue 253 displayed enhanced infectivity in mammalian cells and diminished virulence in a mouse model of GETV disease. Experiments in cell culture show that heparan sulfate (HS) is a new attachment factor for GETV, and the exchange Lys253Arg improves virus attachment by enhancing binding to HS. The mutation also results in more effective binding to glycosaminoglycan (GAG), linked to low virulence due to rapid virus clearance from the circulation. Localization of residue 253 in the 3D structure of the spike revealed several other basic residues in E2 and E1 in close vicinity that might constitute an HS-binding site different from sites previously identified in other alphaviruses. Overall, our study reveals that HS acts as the attachment factor of GETV and provides convincing evidence for an HS-binding determinant at residue 253 in the E2 glycoprotein of GETV, which contributes to infectivity and virulence. IMPORTANCE Due to decades of inadequate monitoring and lack of vaccines and specific treatment, a large number of people have been infected with alphaviruses. GETV is a re-emerging alphavirus that has the potential to infect humans. This specificity of the GETV disease, particularly its propensity for chronic musculoskeletal manifestations, underscores the need to identify the genetic determinants that govern GETV virulence in the host. Using a mouse model, we show that a single amino acid substitution at residue 253 in the E2 glycoprotein causes attenuation of the virus. Residue 253 might be part of a binding site for HS, a ubiquitous attachment factor on the cell surface. The substitution of Lys by Arg improves cell attachment of the virus in vitro and virus clearance from the blood in vivo by enhancing binding to HS. In summary, we have identified HS as a new attachment factor for GETV and the corresponding binding site in the E2 protein for the first time. Our research potentially improved understanding of the pathogenic mechanism of GETV and provided a potential target for the development of new attenuated vaccines and antiviral drugs.

Role of Sw5 gene cluster in the fight against plant viruses

The Sw5 gene cluster furnishes robust resistance to Tomato spotted wilt virus in tomato, which has led to its widespread applicability in agriculture. Among the five orthologs, Sw5b functions as a resistance gene against a broad-spectrum Tospovirus and is linked with Tospovirus resistance. However, its paralog, Sw5a, has been recently implicated in providing resistance against Tomato leaf curl New Delhi virus , broadening the relevance of the Sw5 gene cluster in promoting defense against plant viruses. We propose that plants have established modifications within the homologs of R genes that permit identification of different effector proteins and provide broad and robust resistance against different pathogens through activation of hypersensitive response and cell death.