Human Metapneumovirus Is Capable of Entering Cells by Fusion with Endosomal Membranes.

Human metapneumovirus (HMPV), a member of the Paramyxoviridae family, is a leading cause of lower respiratory illness. Although receptor binding is thought to initiate fusion at the plasma membrane for paramyxoviruses, the entry mechanism for HMPV is largely uncharacterized. Here we sought to determine whether HMPV initiates fusion at the plasma membrane or following internalization. To study the HMPV entry process in human bronchial epithelial (BEAS-2B) cells, we used fluorescence microscopy, an R18-dequenching fusion assay, and developed a quantitative, fluorescence microscopy assay to follow virus binding, internalization, membrane fusion, and visualize the cellular site of HMPV fusion. We found that HMPV particles are internalized into human bronchial epithelial cells before fusing with endosomes. Using chemical inhibitors and RNA interference, we determined that HMPV particles are internalized via clathrin-mediated endocytosis in a dynamin-dependent manner. HMPV fusion and productive infection are promoted by RGD-binding integrin engagement, internalization, actin polymerization, and dynamin. Further, HMPV fusion is pH-independent, although infection with rare strains is modestly inhibited by RNA interference or chemical inhibition of endosomal acidification. Thus, HMPV can enter via endocytosis, but the viral fusion machinery is not triggered by low pH. Together, our results indicate that HMPV is capable of entering host cells by multiple pathways, including membrane fusion from endosomal compartments.

A broadly neutralizing human monoclonal antibody exhibits in vivo efficacy against both human metapneumovirus and respiratory syncytial virus.

BACKGROUND: Human metapneumovirus (HMPV) is a leading cause of acute respiratory tract infection, with significant morbidity and mortality. No licensed vaccines or therapeutic agents exist. Monoclonal antibodies (mAbs) are effective at preventing other infectious diseases and could be used against HMPV in high-risk hosts. METHODS: In vitro assays were performed to assess the neutralizing activity and affinity kinetics of human mAb 54G10. A new mouse model was developed to assess prophylactic and therapeutic efficacy in vivo. The epitope of 54G10 was identified by generating mAb-resistant mutants (MARMs). RESULTS: At low concentrations, 54G10 neutralized all 4 subgroups of HMPV in vitro and had subnanomolar affinity for the fusion protein. DBA/2 mice were permissive for all 4 HMPV subgroups, and 54G10 was effective both prophylactically and therapeutically against HMPV in vivo. Sequencing of HMPV MARMs identified the 54G10 epitope, which was similar to an antigenic site on respiratory syncytial virus (RSV). 54G10 also exhibited in vitro neutralizing activity and in vivo protective and therapeutic efficacy against RSV. CONCLUSIONS: Human mAb 54G10 has broad neutralizing activity against HMPV and could have prophylactic and therapeutic utility clinically. The conserved epitope could represent a structural vaccine target for HMPV and RSV.

Human metapneumovirus virus-like particles induce protective B and T cell responses in a mouse model.

UNLABELLED: Human metapneumovirus (HMPV) is a leading cause of respiratory disease in infants, children, and the elderly worldwide, yet no licensed vaccines exist. Live-attenuated vaccines present safety challenges, and protein subunit vaccines induce primarily antibody responses. Virus-like particles (VLPs) are an attractive alternative vaccine approach because of reduced safety concerns compared with live vaccines. We generated HMPV VLPs by expressing viral proteins in suspension-adapted human embryonic kidney epithelial (293-F) cells and found that the viral matrix (M) and fusion (F) proteins were sufficient to form VLPs. We previously reported that the VLPs resemble virus morphology and incorporate fusion-competent F protein (R. G. Cox, S. B. Livesay, M. Johnson, M. D. Ohi, and J. V. Williams, J. Virol. 86:12148-12160, 2012), which we hypothesized would elicit F-specific antibody and T cell responses. In this study, we tested whether VLP immunization could induce protective immunity to HMPV by using a mouse model. C57BL/6 mice were injected twice intraperitoneally with VLPs alone or with adjuvant and subsequently challenged with HMPV. Mice were euthanized 5 days postinfection, and virus titers, levels of neutralizing antibodies, and numbers of CD3(+) T cells were quantified. Mice immunized with VLPs mounted an F-specific antibody response and generated CD8(+) T cells recognizing an F protein-derived epitope. VLP immunization induced a neutralizing-antibody response that was enhanced by the addition of either TiterMax Gold or α-galactosylceramide adjuvant, though adjuvant reduced cellular immune responses. Two doses of VLPs conferred complete protection from HMPV replication in the lungs of mice and were not associated with a Th2-skewed cytokine response. These results suggest that nonreplicating VLPs are a promising vaccine candidate for HMPV. IMPORTANCE: Human metapneumovirus (HMPV) is a leading cause of acute respiratory infection in infants, children, and the elderly worldwide, yet no licensed vaccines exist. Live-attenuated vaccines present safety challenges, and protein subunit vaccines induce primarily antibody responses. Virus-like particles (VLPs) are an attractive alternative vaccine approach. We generated HMPV VLPs by expressing the viral matrix (M) and fusion (F) proteins in mammalian cells. We found that mice immunized with VLPs mounted an F-specific antibody response and generated CD8(+) T cells recognizing an F protein-derived epitope. VLP immunization induced a neutralizing-antibody response that was enhanced by the addition of either TiterMax Gold or α-galactosylceramide adjuvant. Two doses of VLPs conferred complete protection against HMPV replication in the lungs of mice and were not associated with a Th2-skewed cytokine response. These results suggest that nonreplicating VLPs are a promising vaccine candidate for HMPV.

B cell-intrinsic and -extrinsic regulation of antibody responses by PARP14, an intracellular (ADP-ribosyl)transferase.

The capacity to achieve sufficient concentrations of Ag-specific Ab of the appropriate isotypes is a critical component of immunity that requires efficient differentiation and interactions of Ag-specific B and Th cells along with dendritic cells. Numerous bacterial toxins catalyze mono(ADP-ribosyl)ation of mammalian proteins to influence cell physiology and adaptive immunity. However, little is known about biological functions of intracellular mammalian mono(ADP-ribosyl)transferases, such as any ability to regulate Ab responses. poly-(ADP-ribose) polymerase 14 (PARP14), an intracellular protein highly expressed in lymphoid cells, binds to STAT6 and encodes a catalytic domain with mammalian mono(ADP-ribosyl)transferase activity. In this article, we show that recall IgA as well as the STAT6-dependent IgE Ab responses are impaired in PARP14-deficient mice. Whereas PARP14 regulation of IgE involved a B cell-intrinsic process, the predominant impact on IgA was B cell extrinsic. Of note, PARP14 deficiency reduced the levels of Th17 cells and CD103⁺ DCs, which are implicated in IgA regulation. PARP14 enhanced the expression of RORα, Runx1, and Smad3 after T cell activation, and, importantly, its catalytic activity of PARP14 promoted Th17 differentiation. Collectively, the findings show that PARP14 influences the class distribution, affinity repertoire, and recall capacity of Ab responses in mice, as well as provide direct evidence of the requirement for protein mono-ADP-ribosylation in Th cell differentiation.

The human metapneumovirus fusion protein mediates entry via an interaction with RGD-binding integrins.

Paramyxoviruses use a specialized fusion protein to merge the viral envelope with cell membranes and initiate infection. Most paramyxoviruses require the interaction of two viral proteins to enter cells; an attachment protein binds cell surface receptors, leading to the activation of a fusion (F) protein that fuses the viral envelope and host cell plasma membrane. In contrast, human metapneumovirus (HMPV) expressing only the F protein is replication competent, suggesting a primary role for HMPV F in attachment and fusion. We previously identified an invariant arginine-glycine-aspartate (RGD) motif in the HMPV F protein and showed that the RGD-binding integrin αVß1-promoted HMPV infection. Here we show that both HMPV F-mediated binding and virus entry depend upon multiple RGD-binding integrins and that HMPV F can mediate binding and fusion in the absence of the viral attachment (G) protein. The invariant F-RGD motif is critical for infection, as an F-RAE virus was profoundly impaired. Further, F-integrin binding is required for productive viral RNA transcription, indicating that RGD-binding integrins serve as receptors for the HMPV fusion protein. Thus, HMPV F is triggered to induce virus-cell fusion by interactions with cellular receptors in a manner that is independent of the viral G protein. These results suggest a stepwise mechanism of HMPV entry mediated by the F protein through its interactions with cellular receptors, including RGD-binding integrins.

Integrin alphavbeta1 promotes infection by human metapneumovirus.

Human metapneumovirus (hMPV) is a recently described paramyxovirus that causes lower respiratory infections in children and adults worldwide. The hMPV fusion (F) protein is a membrane-anchored glycoprotein and major protective antigen. All hMPV F protein sequences determined to date contain an Arg-Gly-Asp (RGD) sequence, suggesting that F engages RGD-binding integrins to mediate cell entry. The divalent cation chelator EDTA, which disrupts heterodimeric integrin interactions, inhibits infectivity of hMPV but not the closely related respiratory syncytial virus (RSV), which lacks an RGD motif. Function-blocking antibodies specific for alphavbeta1 integrin inhibit infectivity of hMPV but not RSV. Transfection of nonpermissive cells with alphav or beta1 cDNAs confers hMPV infectivity, whereas reduction of alphav and beta1 integrin expression by siRNA inhibits hMPV infection. Recombinant hMPV F protein binds to cells, whereas Arg-Gly-Glu (RGE)-mutant F protein does not. These data suggest that alphavbeta1 integrin is a functional receptor for hMPV.

Murine hepatitis virus nonstructural protein 4 regulates virus-induced membrane modifications and replication complex function.

Positive-strand RNA viruses induce modifications of cytoplasmic membranes to form replication complexes. For coronaviruses, replicase nonstructural protein 4 (nsp4) has been proposed to function in the formation and organization of replication complexes. Murine hepatitis virus (MHV) nsp4 is glycosylated at residues Asn176 (N176) and N237 during plasmid expression of nsp4 in cells. To test if MHV nsp4 residues N176 and N237 are glycosylated during virus replication and to determine the effects of N176 and N237 on nsp4 function and MHV replication, alanine substitutions of nsp4 N176, N237, or both were engineered into the MHV-A59 genome. The N176A, N237A, and N176A/N237A mutant viruses were viable, and N176 and N237 were glycosylated during infection of wild-type (wt) and mutant viruses. The nsp4 glycosylation mutants exhibited impaired virus growth and RNA synthesis, with the N237A and N176A/N237A mutant viruses demonstrating more profound defects in virus growth and RNA synthesis. Electron microscopic analysis of ultrastructure from infected cells demonstrated that the nsp4 mutants had aberrant morphology of virus-induced double-membrane vesicles (DMVs) compared to those infected with wt virus. The degree of altered DMV morphology directly correlated with the extent of impairment in viral RNA synthesis and virus growth of the nsp4 mutant viruses. The results indicate that nsp4 plays a critical role in the organization and stability of DMVs. The results also support the conclusion that the structure of DMVs is essential for efficient RNA synthesis and optimal replication of coronaviruses.

Prenatal care utilization in Mississippi: racial disparities and implications for unfavorable birth outcomes.

The objective of the study is to identify racial disparities in prenatal care (PNC) utilization and to examine the relationship between PNC and preterm birth (PTB), low birth weight (LBW) and infant mortality in Mississippi. Retrospective cohort from 1996 to 2003 linked Mississippi birth and infant death files was used. Analysis was limited to live-born singleton infants born to non-Hispanic white and black women (n = 292,776). PNC was classified by Kotelchuck's Adequacy of Prenatal Care Utilization Index. Factors associated with PTB, LBW and infant death were identified using multiple logistic regression after controlling for maternal age, education, marital status, place of residence, tobacco use and medical risk. About one in five Mississippi women had less than adequate PNC, and racial disparities in PNC utilization were observed. Black women delayed PNC, received too few visits, and were more likely to have either "inadequate PNC" (P < 0.0001) or "no care" (P < 0.0001) compared to white women. Furthermore, among women with medical conditions, black women were twice as likely to receive inadequate PNC compared to white women. Regardless of race, "no care" and "inadequate PNC" were strong risk factors for PTB, LBW and infant death. We provide empirical evidence to support the existence of racial disparities in PNC utilization and infant birth outcomes in Mississippi. Further study is needed to explain racial differences in PNC utilization. However, this study suggests that public health interventions designed to improve PNC utilization among women might reduce unfavorable birth outcomes especially infant mortality.

Association of maternal medical conditions and unfavorable birth outcomes: findings from the 1996-2003 Mississippi linked birth and death data.

This study aimed to identify factors contributing to high rates of preterm birth (PTB), low birth weight (LBW) and infant mortality in Mississippi while considering both traditional risk factors and maternal medical conditions. The retrospective cohort study used 1996-2003 Mississippi linked birth and infant death files. Multiple logistic regression was used to investigate association between maternal medical conditions and unfavorable birth outcomes. Along with traditional risk factors, hypertension was significantly associated with PTB and LBW. Women with hypertension were about 2.2 and 3.2 times as likely to have PTB and LBW, respectively. Hydramnios/oligohydramnios increased 1.8-4.4 folds of risk for PTB, LBW and infant death and was significantly associated with the unfavorable birth outcomes. Non-Hispanic black women were about 1.5-2.0 times as likely to have an unfavorable birth outcome compared to non-Hispanic white women. Maternal education and prenatal care effect appeared to be modified by maternal race. Certain maternal medical conditions may be contributing to PTB, LBW and infant mortality rates identifying preconception and prenatal healthcare as possible strategies for reducing unfavorable outcomes. Results suggest that different risk profiles for unfavorable outcomes may exist according to maternal race highlighting the need to consider racial groups separately when further exploring the sociodemographic and/or health-related factors that contribute to unfavorable birth outcomes.

Body mass index and health related quality of life in elementary school children: a pilot study.

BACKGROUND: We investigated the relationship between Body Mass Index (BMI) and health-related quality of life (HRQOL) indicated by baseline health status in elementary school children. METHODS: Data were obtained via parents whose children enrolled in an elementary school, kindergarten to fourth grade, in southern Mississippi in spring 2004. Parents completed the SF-10 for Children, a brief 10-item questionnaire designed to measure children's HRQOL on a voluntary basis. RESULTS: A total of 279 parents completed the questionnaires for their children. On average, physical and psychosocial summary scores, major indicators for HRQOL, were significantly higher among the elementary school children in our study relative to those from U.S. children overall (p < 0.0001 and p = 0.0007, respectively). Males tended to have better physical functioning than their female classmates, whereas females had better psychosocial health. Overall, except for third graders, the physical summary scores increased as grade level increased. The means for psychosocial score fluctuated without a clear pattern over the five grade levels. High level of BMI was significantly associated with children's physical summary scores below 50, a norm used for U.S. children (p = 0.003). Gender and grade were not significant predictors of children's physical and psychosocial scores. DISCUSSION: This study can be used as baseline information to track changes over time, in BMI and health status among the elementary school children. In addition, this study can be used to investigate relationships between BMI, health status, intellectual ability, and performance in school. CONCLUSION: The findings suggest that programs designed to encourage children to lose weight in a healthy manner, thus reducing their BMI, could improve the physical and psychosocial health, and subsequently increase HRQOL.

Academic performance and substance use: findings from a state survey of public high school students.

BACKGROUND: Previous investigations have shown that low academic achievers are more likely to smoke cigarettes, drink alcohol, and use marijuana and other illicit drugs. This study investigated the relationship between academic performance and substance use among public high school students in Mississippi. METHODS: The sampling frame for the 2003 Mississippi Youth Risk Behavior Survey was obtained from the Mississippi Department of Education. A 2-stage cluster sample design produced a representative sample of Mississippi students in grades 9-12 who attended public schools. During the spring 2003, 34 of the 45 sampled schools (75.6%) participated. RESULTS: A total of 1488 from the 1672 sampled students (89.0%) completed an 87-item questionnaire (overall response rate 67.3%). Low academic performance (students with mostly Cs or below) during the 12 months preceding the survey was more prevalent among males, non-Hispanic blacks, frequent smokers, binge drinkers, and marijuana users. Logistic regression identified gender, race, frequent smoking, and marijuana use as statistically significant factors associated with increased odds for low academic performance. Adjusted odds ratios were 1.2 for binge drinkers versus other students to 2.6 for non-Hispanic black students versus non-Hispanic white students. CONCLUSIONS: The present findings highlight the extensive use of cigarettes, alcohol, and marijuana among Mississippi public high school students. Because poor academic achievers are more prevalent among students who participate in these substance-use behaviors, multifaceted approaches that encourage high levels of academic performance, while dissuading student involvement in risky/problem behaviors, may both improve low levels of academic achievement and reduce behaviors that contribute to poor health in adulthood.

The Fusion Loops of the Initial Prefusion Conformation of Herpes Simplex Virus 1 Fusion Protein Point Toward the Membrane.

All enveloped viruses, including herpesviruses, must fuse their envelope with the host membrane to deliver their genomes into target cells, making this essential step subject to interference by antibodies and drugs. Viral fusion is mediated by a viral surface protein that transits from an initial prefusion conformation to a final postfusion conformation. Strikingly, the prefusion conformation of the herpesvirus fusion protein, gB, is poorly understood. Herpes simplex virus (HSV), a model system for herpesviruses, causes diseases ranging from mild skin lesions to serious encephalitis and neonatal infections. Using cryo-electron tomography and subtomogram averaging, we have characterized the structure of the prefusion conformation and fusion intermediates of HSV-1 gB. To this end, we have set up a system that generates microvesicles displaying full-length gB on their envelope. We confirmed proper folding of gB by nondenaturing electrophoresis-Western blotting with a panel of monoclonal antibodies (MAbs) covering all gB domains. To elucidate the arrangement of gB domains, we labeled them by using (i) mutagenesis to insert fluorescent proteins at specific positions, (ii) coexpression of gB with Fabs for a neutralizing MAb with known binding sites, and (iii) incubation of gB with an antibody directed against the fusion loops. Our results show that gB starts in a compact prefusion conformation with the fusion loops pointing toward the viral membrane and suggest, for the first time, a model for gB's conformational rearrangements during fusion. These experiments further illustrate how neutralizing antibodies can interfere with the essential gB structural transitions that mediate viral entry and therefore infectivity.IMPORTANCE The herpesvirus family includes herpes simplex virus (HSV) and other human viruses that cause lifelong infections and a variety of diseases, like skin lesions, encephalitis, and cancers. As enveloped viruses, herpesviruses must fuse their envelope with the host membrane to start an infection. This process is mediated by a viral surface protein that transitions from an initial conformation (prefusion) to a final, more stable, conformation (postfusion). However, the prefusion conformation of the herpesvirus fusion protein (gB) is poorly understood. To elucidate the structure of the prefusion conformation of HSV type 1 gB, we have employed cryo-electron microscopy to study gB molecules expressed on the surface of vesicles. Using different approaches to label gB's domains allowed us to model the structures of the prefusion and intermediate conformations of gB. Overall, our findings enhance our understanding of HSV fusion and lay the groundwork for the development of new ways to prevent and block HSV infection.

Breaking in: human metapneumovirus fusion and entry.

Human metapneumovirus (HMPV) is a leading cause of respiratory infection that causes upper airway and severe lower respiratory tract infections. HMPV infection is initiated by viral surface glycoproteins that attach to cellular receptors and mediate virus membrane fusion with cellular membranes. Most paramyxoviruses use two viral glycoproteins to facilitate virus entry-an attachment protein and a fusion (F) protein. However, membrane fusion for the human paramyxoviruses in the Pneumovirus subfamily, HMPV and respiratory syncytial virus (hRSV), is unique in that the F protein drives fusion in the absence of a separate viral attachment protein. Thus, pneumovirus F proteins can perform the necessary functions for virus entry, i.e., attachment and fusion. In this review, we discuss recent advances in the understanding of how HMPV F mediates both attachment and fusion. We review the requirements for HMPV viral surface glycoproteins during entry and infection, and review the identification of cellular receptors for HMPV F. We also review our current understanding of how HMPV F mediates fusion, concentrating on structural regions of the protein that appear to be critical for membrane fusion activity. Finally, we illuminate key unanswered questions and suggest how further studies can elucidate how this clinically important paramyxovirus fusion protein may have evolved to initiate infection by a unique mechanism.

Structure of the human metapneumovirus fusion protein with neutralizing antibody identifies a pneumovirus antigenic site.

Human metapneumovirus and respiratory syncytial virus cause lower respiratory tract infections. The virus fusion (F) glycoprotein promotes membrane fusion by refolding from a metastable pre-fusion to a stable post-fusion conformation. F is also a major target of the neutralizing antibody response. Here we show that a potently neutralizing anti-human metapneumovirus antibody (DS7) binds a structurally invariant domain of F, revealing a new epitope that could be targeted in vaccine development.

A critical phenylalanine residue in the respiratory syncytial virus fusion protein cytoplasmic tail mediates assembly of internal viral proteins into viral filaments and particles.

UNLABELLED: Respiratory syncytial virus (RSV) is a single-stranded RNA virus in the Paramyxoviridae family that assembles into filamentous structures at the apical surface of polarized epithelial cells. These filaments contain viral genomic RNA and structural proteins, including the fusion (F) protein, matrix (M) protein, nucleoprotein (N), and phosphoprotein (P), while excluding F-actin. It is known that the F protein cytoplasmic tail (FCT) is necessary for filament formation, but the mechanism by which the FCT mediates assembly into filaments is not clear. We hypothesized that the FCT is necessary for interactions with other viral proteins in order to form filaments. In order to test this idea, we expressed the F protein with cytoplasmic tail (CT) truncations or specific point mutations and determined the abilities of these variant F proteins to form filaments independent of viral infection when coexpressed with M, N, and P. Deletion of the terminal three FCT residues (amino acids Phe-Ser-Asn) or mutation of the Phe residue resulted in a loss of filament formation but did not affect F-protein expression or trafficking to the cell surface. Filament formation could be restored by addition of residues Phe-Ser-Asn to an FCT deletion mutant and was unaffected by mutations to Ser or Asn residues. Second, deletion of residues Phe-Ser-Asn or mutation of the Phe residue resulted in a loss of M, N, and P incorporation into virus-like particles. These data suggest that a C-terminal Phe residue in the FCT mediates assembly through incorporation of internal virion proteins into virus filaments at the cell surface. IMPORTANCE: Respiratory syncytial virus (RSV) is a leading cause of bronchiolitis and pneumonia in infants and the elderly worldwide. There is no licensed RSV vaccine and only limited therapeutics for use in infected patients. Many aspects of the RSV life cycle have been studied, but the mechanisms that drive RSV assembly at the cell surface are not well understood. This study provides evidence that a specific residue in the RSV fusion protein cytoplasmic tail coordinates assembly into viral filaments by mediating the incorporation of internal virion proteins. Understanding the mechanisms that drive RSV assembly could lead to targeted development of novel antiviral drugs. Moreover, since RSV exits infected cells in an ESCRT (endosomal sorting complexes required for transport)-independent manner, these studies may contribute new knowledge about a general strategy by which ESCRT-independent viruses mediate outward bud formation using viral protein-mediated mechanisms during assembly and budding.

Studies of culture conditions and environmental stability of human metapneumovirus.

Human metapneumovirus (HMPV) is a paramyxovirus that is a leading cause of acute respiratory disease. HMPV is difficult to cultivate and limited published data describe the in vitro growth characteristics of the virus and its ability to replicate in different cell lines. Stability of HMPV to different temperatures or environmental conditions has not been described. Nosocomial infections due to HMPV have been reported, and thus the survival of infectious particles on environmental surfaces is important. We tested multiple cell lines for the ability to support HMPV replication both in the presence and absence of exogenous trypsin. The most permissive monkey kidney epithelial cells were LLC-MK2 and Vero, while the most permissive human airway epithelial cell line was BEAS-2B. LLC-MK2 cells were tolerant of trypsin and thus remain an ideal cell line for HMPV cultivation. Spinoculation significantly increased the infectivity of HMPV for cells in monolayer culture. Infectious virus was very stable to repeat freeze-thaw cycles, ambient room temperature, or 4 degrees C, while incubation at 37 degrees C led to degradation of virus titer. Finally, nonporous materials such as metal or plastic retained infectious virus for prolonged periods, while virus deposited on tissue and fabric rapidly lost infectivity. These findings provide guidance for laboratories attempting to culture HMPV and relevant information for infection control policies.