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.

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 ␣v␤1 integrin inhibit infectivity of hMPV but not RSV. Transfection of nonpermissive cells with ␣v or ␤1 cDNAs confers hMPV infectivity, whereas reduction of ␣v and ␤1 integrin expression by siRNA inhibits hMPV infection. Recombinant hMPV F protein binds to cells, whereas ArgGly-Glu (RGE)-mutant F protein does not. These data suggest that ␣v␤1 integrin is a functional receptor for hMPV.receptor ͉ paramyxovirus ͉ fusion protein ͉ viral entry

ABSTRACTRespiratory syncytial virus (RSV) is a single-stranded RNA virus in theParamyxoviridaefamily 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.IMPORTANCERespiratory 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.

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.

SUMMARY Human metapneumovirus (HMPV) and respiratory syncytial virus (RSV) cause lower respiratory 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-HMPV antibody (DS7) binds a structurally invariant domain of F, identifying a new epitope that could be targeted in vaccine development.