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Mammalian orthoreovirus attachment to target cells is mediated by the outer capsid protein σ1, which projects from the virion surface. The σ1 protein is a homotrimer consisting of a filamentous tail, which is partly inserted into the virion; a body domain constructed from β-spiral repeats; and a globular head with receptor-binding properties. The σ1 tail is predicted to form an α-helical coiled coil. Although σ1 undergoes a conformational change during cell entry, the nature of this change and its contributions to viral replication are unknown. Electron micrographs of σ1 molecules released from virions identified three regions of flexibility, including one at the midpoint of the molecule, that may be involved in its structural rearrangement. To enable a detailed understanding of essential σ1 tail organization and properties, we determined high-resolution structures of the reovirus type 1 Lang (T1L) and type 3 Dearing (T3D) σ1 tail domains. Both molecules feature extended α-helical coiled coils, with T1L σ1 harboring central chloride ions. Each molecule displays a discontinuity (stutter) within the coiled coil and an unexpectedly seamless transition to the body domain. The transition region features conserved interdomain interactions and appears rigid rather than highly flexible. Functional analyses of reoviruses containing engineered σ1 mutations suggest that conserved residues predicted to stabilize the coiled-coil-to-body junction are essential for σ1 folding and encapsidation, whereas central chloride ion coordination and the stutter are dispensable for efficient replication. Together, these findings enable modeling of full-length reovirus σ1 and provide insight into the stabilization of a multidomain virus attachment protein. While it is established that different conformational states of attachment proteins of enveloped viruses mediate receptor binding and membrane fusion, less is understood about how such proteins mediate attachment and entry of nonenveloped viruses. The filamentous reovirus attachment protein σ1 binds cellular receptors; contains regions of predicted flexibility, including one at the fiber midpoint; and undergoes a conformational change during cell entry. Neither the nature of the structural change nor its contribution to viral infection is understood. We determined crystal structures of large σ1 fragments for two different reovirus serotypes. We observed an unexpectedly tight transition between two domains spanning the fiber midpoint, which allows for little flexibility. Studies of reoviruses with engineered changes near the σ1 midpoint suggest that the stabilization of this region is critical for function. Together with a previously determined structure, we now have a complete model of the full-length, elongated reovirus σ1 attachment protein.
Copyright © 2018 American Society for Microbiology.
BACKGROUND - Mitochondria are abundant organelles critical for energy metabolism and brain function. Mitochondrial DNA (mtDNA), released during cellular injury and as part of the innate immune response to viral pathogens, contains CpG motifs that act as TLR-9 ligands. We investigated relationships between cerebrospinal fluid (CSF) cell-free mtDNA levels and HIV viral load (VL), biomarkers of inflammation and iron transport, and neurocognitive (NC) function in the CNS HIV Antiretroviral Therapy Effects Research (CHARTER) cohort.
METHODS - We quantified cell-free mtDNA in CSF by droplet digital PCR in 332 CHARTER participants who underwent comprehensive neuropsychiatric evaluation. NC performance was assessed using the global deficit score (GDS) as either a continuous or a binary measure (GDS ≥ 0.5, impaired vs. GDS < 0.5, unimpaired). CSF, clinical, and biomarker data from the earliest available time point were analyzed. Cell-free mtDNA associations with CSF inflammation and iron-related biomarkers [CXCL10, IL-6, IL-8, TNF-a, transferrin (TF), ceruloplasmin (CP), and vascular endothelial growth factor (VEGF)], VL, and GDS were evaluated by multivariable regression.
RESULTS - CSF cell-free mtDNA levels were significantly lower in participants with undetectable (vs. detectable) VL in either plasma (p < 0.001) or CSF (p < 0.001) and in those on antiretroviral therapy (ART; p < 0.001). Participants on ART with undetectable VL in both CSF and plasma had lower mtDNA levels than those with detectable VL in both compartments (p = 0.001). Higher mtDNA levels were observed in participants in the highest vs. lowest tertile (T3 vs. T1) of CSF CXCL10 (T3 vs. T1, p < 0.001) and TNF-a (T3 vs. T1, p < 0.05) in unadjusted analyses. MtDNA levels also correlated with CSF leukocyte count. After adjusting for CSF leukocyte count and VL, mtDNA levels were also associated with other inflammation- and iron-related biomarkers in CSF, including TF (T3 vs. T1, p < 0.05) and CP (T3 vs. T1, p < 0.05). With additional correction for ART use, mtDNA was also negatively associated with CSF VEGF (p < 0.05) and IL-6 (p = 0.05). We observed no associations of CSF mtDNA levels with age or GDS-defined NC impairment.
CONCLUSIONS - CSF cell-free mtDNA levels were associated with HIV RNA and ART status, as well as with biomarkers of iron transport and VEGF, a growth factor with known effects on mitochondrial integrity and autophagy. CSF mtDNA may be a biomarker of iron dysregulation and/or neuroinflammation during HIV infection.
Reovirus attachment protein σ1 engages glycan receptors and junctional adhesion molecule-A (JAM-A) and is thought to undergo a conformational change during the proteolytic disassembly of virions to infectious subvirion particles (ISVPs) that accompanies cell entry. The σ1 protein is also the primary target of neutralizing antibodies. Here, we present a structural and functional characterization of two neutralizing antibodies that target σ1 of serotype 1 (T1) and serotype 3 (T3) reoviruses. The crystal structures revealed that each antibody engages its cognate σ1 protein within the head domain via epitopes distinct from the JAM-A-binding site. Surface plasmon resonance and cell-binding assays indicated that both antibodies likely interfere with JAM-A engagement by steric hindrance. To define the interplay between the carbohydrate receptor and antibody binding, we conducted hemagglutination inhibition assays using virions and ISVPs. The glycan-binding site of T1 σ1 is located in the head domain and is partly occluded by the bound Fab in the crystal structure. The T1-specific antibody inhibited hemagglutination by virions and ISVPs, probably via direct interference with glycan engagement. In contrast to T1 σ1, the carbohydrate-binding site of T3 σ1 is located in the tail domain, distal to the antibody epitope. The T3-specific antibody inhibited hemagglutination by T3 virions but not ISVPs, indicating that the antibody- and glycan-binding sites in σ1 are in closer spatial proximity on virions than on ISVPs. Our results provide direct evidence for a structural rearrangement of σ1 during virion-to-ISVP conversion and contribute new information about the mechanisms of antibody-mediated neutralization of reovirus.
IMPORTANCE - Virus attachment proteins mediate binding to host cell receptors, serve critical functions in cell and tissue tropism, and are often targeted by the neutralizing antibody response. The structural investigation of antibody-antigen complexes can provide valuable information for understanding the molecular basis of virus neutralization. Studies with enveloped viruses, such as HIV and influenza virus, have helped to define sites of vulnerability and guide vaccination strategies. By comparison, less is known about antibody binding to nonenveloped viruses. Here, we structurally investigated two neutralizing antibodies that bind the attachment protein σ1 of reovirus. Furthermore, we characterized the neutralization efficiency, the binding affinity for σ1, and the effect of the antibodies on reovirus receptor engagement. Our analysis defines reovirus interactions with two neutralizing antibodies, allows us to propose a mechanism by which they block virus infection, and provides evidence for a conformational change in the σ1 protein during viral cell entry.
Copyright © 2017 American Society for Microbiology.
Zika virus (ZIKV) is an emerging mosquito-transmitted flavivirus that can cause severe disease, including congenital birth defects during pregnancy. To develop candidate therapeutic agents against ZIKV, we isolated a panel of human monoclonal antibodies from subjects that were previously infected with ZIKV. We show that a subset of antibodies recognize diverse epitopes on the envelope (E) protein and exhibit potent neutralizing activity. One of the most inhibitory antibodies, ZIKV-117, broadly neutralized infection of ZIKV strains corresponding to African and Asian-American lineages. Epitope mapping studies revealed that ZIKV-117 recognized a unique quaternary epitope on the E protein dimer-dimer interface. We evaluated the therapeutic efficacy of ZIKV-117 in pregnant and non-pregnant mice. Monoclonal antibody treatment markedly reduced tissue pathology, placental and fetal infection, and mortality in mice. Thus, neutralizing human antibodies can protect against maternal-fetal transmission, infection and disease, and reveal important determinants for structure-based rational vaccine design efforts.
Megakaryocyte-erythrocyte progenitor (MEP) cells are potential in vivo targets of dengue virus (DENV); the virus has been found associated with megakaryocytes ex vivo and platelets during DENV-induced thrombocytopenia. We report here that DENV serotype 2 (DENV2) propagates well in human nondifferentiated MEP cell lines (Meg01 and K562). In comparison to virus propagated in Vero cells, viruses from MEP cell lines had similar structure and buoyant density. However, differences in MEP-DENV2 stability and composition were suggested by distinct protein patterns in western blot analysis. Also, antibody neutralization of envelope domain I/II on MEP-DENV2 was reduced relative to that on Vero-DENV2. Infectious DENV2 was produced at comparable kinetics and magnitude in MEP and Vero cells. However, fewer virion structures appeared in electron micrographs of MEP cells. We propose that DENV2 infects and produces virus efficiently in megakaryocytes and that megakaryocyte impairment might contribute to dengue disease pathogenesis.
Copyright © 2016 Elsevier Inc. All rights reserved.
UNLABELLED - The proposed antibody-dependent enhancement (ADE) mechanism for severe dengue virus (DENV) disease suggests that non-neutralizing serotype cross-reactive antibodies generated during a primary infection facilitate entry into Fc receptor bearing cells during secondary infection, resulting in enhanced viral replication and severe disease. One group of cross-reactive antibodies that contributes considerably to this serum profile target the premembrane (prM) protein. We report here the isolation of a large panel of naturally occurring human monoclonal antibodies (MAbs) obtained from subjects following primary DENV serotype 1, 2, or 3 or secondary natural DENV infections or following primary DENV serotype 1 live attenuated virus vaccination to determine the antigenic landscape on the prM protein that is recognized by human antibodies. We isolated 25 prM-reactive human MAbs, encoded by diverse antibody-variable genes. Competition-binding studies revealed that all of the antibodies bound to a single major antigenic site on prM. Alanine scanning-based shotgun mutagenesis epitope mapping studies revealed diverse patterns of fine specificity of various clones, suggesting that different antibodies use varied binding poses to recognize several overlapping epitopes within the immunodominant site. Several of the antibodies interacted with epitopes on both prM and E protein residues. Despite the diverse genetic origins of the antibodies and differences in the fine specificity of their epitopes, each of these prM-reactive antibodies was capable of enhancing the DENV infection of Fc receptor-bearing cells.
IMPORTANCE - Antibodies may play a critical role in the pathogenesis of enhanced DENV infection and disease during secondary infections. A substantial proportion of enhancing antibodies generated in response to natural dengue infection are directed toward the prM protein. The fine specificity of human prM antibodies is not understood. Here, we isolated a panel of dengue prM-specific human monoclonal antibodies from individuals after infection in order to define the mode of molecular recognition by enhancing antibodies. We found that only a single antibody molecule can be bound to each prM protein at any given time. Distinct overlapping epitopes were mapped, but all of the epitopes lie within a single major antigenic site, suggesting that this antigenic domain forms an immunodominant region of the protein. Neutralization and antibody-dependent enhanced replication experiments showed that recognition of any of the epitopes within the major antigenic site on prM was sufficient to cause enhanced infection of target cells.
Copyright © 2015, American Society for Microbiology. All Rights Reserved.
Dengue virus is a major global health threat and can lead to life-threatening hemorrhagic complications due to immune activation and cytokine production. Cross-reactive antibodies to an earlier dengue virus infection are a recognized risk factor for severe disease. These antibodies bind heterologous dengue serotypes and enhance infection into Fc-receptor-bearing cells, a process known as antibody-dependent enhancement of infection. One crucial cytokine seen elevated in severe dengue patients is IL-1β, a potent inflammatory cytokine matured by the inflammasome. We used a highly-physiologic system by studying antibody-dependent enhancement of IL-1β in primary human monocytes with anti-dengue human monoclonal antibodies isolated from patients. Antibody-enhancement increased viral replication in primary human monocytes inoculated with supernatant harvested from Vero cells infected with dengue virus serotype 2 (DENV-2) 16681. Surprisingly, IL-1β secretion induced by infectious supernatant harvested from two independent Vero cell lines was not enhanced by antibody. Secretion of multiple other inflammatory cytokines was also independent of antibody signaling. However, IL-1β secretion did require NLRP3 and caspase-1 activity. Immunodepletion of dengue virions from the infectious supernatant confirmed that virus was not the main IL-1β-inducing agent, suggesting that a supernatant component(s) not associated with the virion induced IL-1β production. We excluded RNA, DNA, contaminating LPS, viral NS1 protein, complement, and cytokines. In contrast, purified Vero-derived DENV-2 16681 exhibited antibody-enhancement of both infection and IL-1β induction. Furthermore, C6/36 mosquito cells did not produce such an inflammatory component, as crude supernatant harvested from insect cells infected with DENV-2 16681 induced antibody-dependent IL-1β secretion. This study indicates that Vero cells infected with DENV-2 16681 may produce inflammatory components during dengue virus propagation that mask the virus-specific immune response. Thus, the choice of host cell and viral purity should be carefully considered, while insect-derived virus represents a system that elicits antibody-dependent cytokine responses to dengue virus with fewer confounding issues.
UNLABELLED - During HIV-1 infection of cells, the viral capsid plays critical roles in reverse transcription and nuclear entry of the virus. The capsid-targeting small molecule PF74 inhibits HIV-1 at early stages of infection. HIV-1 resistance to PF74 is complex, requiring multiple amino acid substitutions in the viral CA protein. Here we report the identification and analysis of a novel PF74-resistant mutant encoding amino acid changes in both domains of CA, three of which are near the pocket where PF74 binds. Interestingly, the mutant virus retained partial PF74 binding, and its replication was stimulated by the compound. The mutant capsid structure was not significantly perturbed by binding of PF74; rather, the mutations inhibited capsid interactions with CPSF6 and Nup153 and altered HIV-1 dependence on these host factors and on TNPO3. Moreover, the replication of the mutant virus was markedly impaired in activated primary CD4(+) T cells and macrophages. Our results suggest that HIV-1 escapes a capsid-targeting small molecule inhibitor by altering the virus's dependence on host factors normally required for entry into the nucleus. They further imply that clinical resistance to inhibitors targeting the PF74 binding pocket is likely to be strongly limited by functional constraints on HIV-1 evolution.
IMPORTANCE - The HIV-1 capsid plays critical roles in early steps of infection and is an attractive target for therapy. Here we show that selection for resistance to a capsid-targeting small molecule inhibitor can result in viral dependence on the compound. The mutant virus was debilitated in primary T cells and macrophages--cellular targets of infection in vivo. The mutations also altered the virus's dependence on cellular factors that are normally required for HIV-1 entry into the nucleus. This work provides new information regarding mechanisms of HIV-1 resistance that should be useful in efforts to develop clinically useful drugs targeting the HIV-1 capsid.
Copyright © 2015, American Society for Microbiology. All Rights Reserved.
Despite the availability of >30 effective drugs for managing HIV-1 infection, no current therapy is curative, and long-term management is challenging owing to the emergence and spread of drug-resistant mutants. Identification of drugs against novel HIV-1 targets would expand the current treatment options and help to control resistance. The highly conserved HIV-1 capsid protein represents an attractive target because of its multiple roles in replication of the virus. However, the low antiviral potencies of the reported HIV-1 capsid-targeting inhibitors render them unattractive for therapeutic development. To facilitate the identification of more-potent HIV-1 capsid inhibitors, we developed a scintillation proximity assay to screen for small molecules that target a biologically active and specific intersubunit interface in the HIV-1 capsid. The assay, which is based on competitive displacement of a known capsid-binding small-molecule inhibitor, exhibited a signal-to-noise ratio of >9 and a Z factor of >0.8. In a pilot screen of a chemical library containing 2,400 druglike compounds, we obtained a hit rate of 1.8%. This assay has properties that are suitable for screening large compound libraries to identify novel HIV-1 capsid ligands with antiviral activity.
Copyright © 2015, American Society for Microbiology. All Rights Reserved.
UNLABELLED - Human metapneumovirus (HMPV) is a major cause of respiratory disease in infants, the elderly, and immunocompromised individuals worldwide. There is currently no licensed HMPV vaccine. Virus-like particles (VLPs) are an attractive vaccine candidate because they are noninfectious and elicit a neutralizing antibody response. However, studies show that serum neutralizing antibodies are insufficient for complete protection against reinfection and that adaptive T cell immunity is important for viral clearance. HMPV and other respiratory viruses induce lung CD8(+) T cell (TCD8) impairment, mediated by programmed death 1 (PD-1). In this study, we generated HMPV VLPs by expressing the fusion and matrix proteins in mammalian cells and tested whether VLP immunization induces functional HMPV-specific TCD8 responses in mice. C57BL/6 mice vaccinated twice with VLPs and subsequently challenged with HMPV were protected from lung viral replication for at least 20 weeks postimmunization. A single VLP dose elicited F- and M-specific lung TCD8s with higher function and lower expression of PD-1 and other inhibitory receptors than TCD8s from HMPV-infected mice. However, after HMPV challenge, lung TCD8s from VLP-vaccinated mice exhibited inhibitory receptor expression and functional impairment similar to those of mice experiencing secondary infection. HMPV challenge of VLP-immunized μMT mice also elicited a large percentage of impaired lung TCD8s, similar to mice experiencing secondary infection. Together, these results indicate that VLPs are a promising vaccine candidate but do not prevent lung TCD8 impairment upon HMPV challenge.
IMPORTANCE - Human metapneumovirus (HMPV) is a leading cause of acute respiratory disease for which there is no licensed vaccine. Virus-like particles (VLPs) are an attractive vaccine candidate and induce antibodies, but T cell responses are less defined. Moreover, HMPV and other respiratory viruses induce lung CD8(+) T cell (TCD8) impairment mediated by programmed death 1 (PD-1). In this study, HMPV VLPs containing viral fusion and matrix proteins elicited epitope-specific TCD8s that were functional with low PD-1 expression. Two VLP doses conferred sterilizing immunity in C57BL/6 mice and facilitated HMPV clearance in antibody-deficient μMT mice without enhancing lung pathology. However, regardless of whether responding lung TCD8s had previously encountered HMPV antigens in the context of VLPs or virus, similar proportions were impaired and expressed comparable levels of PD-1 upon viral challenge. These results suggest that VLPs are a promising vaccine candidate but do not prevent lung TCD8 impairment upon HMPV challenge.
Copyright © 2015, American Society for Microbiology. All Rights Reserved.