Gag proteins direct the process of retroviral particle assembly and form the major protein constituents of the viral core. The matrix region of the HIV-1 Gag polyprotein plays a critical role in the transport of Gag to the plasma membrane assembly site. Recent evidence indicates that Gag trafficking to late endosomal compartments, including multivesicular bodies, occurs prior to viral particle budding from the plasma membrane. Here we demonstrate that the matrix region of HIV-1 Gag interacts directly with the delta subunit of the AP-3 complex, and that this interaction plays an important functional role in particle assembly. Disruption of this interaction eliminated Gag trafficking to multivesicular bodies and diminished HIV particle formation. These studies illuminate an early step in retroviral particle assembly and provide evidence that the trafficking of Gag to late endosomes is part of a productive particle assembly pathway.

HCPs within 1.829 m of patients with influenza could be exposed to infectious doses of influenza virus, primarily in small-particle aerosols. This finding questions the current paradigm of localized droplet transmission during non-aerosol-generating procedures.

Reovirus replication occurs in the cytoplasm of infected cells and culminates in the formation of crystalline arrays of progeny virions within viral inclusions. Two viral nonstructural proteins, NS and NS, and structural protein 3 form protein-RNA complexes early in reovirus infection. To better understand the minimal requirements of viral inclusion formation, we expressed NS, NS, and 3 alone and in combination in the absence of viral infection. In contrast to its concentration in inclusion structures during reovirus replication, NS expressed in cells in the absence of infection is distributed diffusely throughout the cytoplasm and does not form structures that resemble viral inclusions. Expressed NS is functional as it complements the defect in temperature-sensitive, NS-mutant virus tsE320. In both transfected and infected cells, NS is found in punctate cytoplasmic structures and 3 is distributed diffusely in the cytoplasm and the nucleus. The subcellular localization of NS and 3 is not altered when the proteins are expressed together or with NS. However, when expressed with NS, NS colocalizes with NS to punctate structures similar in morphology to inclusion structures observed early in viral replication. During reovirus infection, both NS and NS are detectable 4 h after adsorption and colocalize to punctate structures throughout the viral life cycle. In concordance with these results, NS interacts with NS in a yeast two-hybrid assay and by coimmunoprecipitation analysis. These data suggest that NS and NS are the minimal viral components required to form inclusions, which then recruit other reovirus proteins and RNA to initiate viral genome replication.

BackgroundGroup A Streptococcus (GAS) causes acute tonsillopharyngitis in children, and approximately 20% of this population are chronic carriers of GAS. Antibacterial therapy has previously been shown to be insufficient at clearing GAS carriage. Bacterial biofilms are a surface-attached bacterial community that is encased in a matrix of extracellular polymeric substances. Biofilms have been shown to provide a protective niche against the immune response and antibiotic treatments, and are often associated with recurrent or chronic bacterial infections. The objective of this study was to test the hypothesis that GAS is present within tonsil tissue at the time of tonsillectomy.MethodsBlinded immunofluorescent and histological methods were employed to evaluate palatine tonsils from children undergoing routine tonsillectomy for adenotonsillar hypertrophy or recurrent GAS tonsillopharyngitis.ResultsImmunofluorescence analysis using anti-GAS antibody was positive in 11/30 (37%) children who had tonsillectomy for adenotonsillar hypertrophy and in 10/30 (33%) children who had tonsillectomy for recurrent GAS pharyngitis. Fluorescent microscopy with anti-GAS and anti-cytokeratin 8 & 18 antibodies revealed GAS was localized to the tonsillar reticulated crypts. Scanning electron microscopy identified 3-dimensional communities of cocci similar in size and morphology to GAS. The characteristics of these communities are similar to GAS biofilms from in vivo animal models.ConclusionOur study revealed the presence of GAS within the tonsillar reticulated crypts of approximately one-third of children who underwent tonsillectomy for either adenotonsillar hypertrophy or recurrent GAS tonsillopharyngitis at the Wake Forest School of Medicine.Trial RegistrationThe tissue collected was normally discarded tissue and no patient identifiers were collected. Thus, no subjects were formally enrolled.

Transocular transmission of LAIV occured in most participants suggesting the necessity of eye protection. An N95 respirator provided the best guard further enhanced by eye protection.

Coronaviruses encode the largest replicase polyprotein of any known positive-strand RNA virus. Replicase protein precursors and mature products are thought to mediate the formation and function of viral replication complexes on the surfaces of intracellular double-membrane vesicles. However, the functions of only a few of these proteins are known. For the coronavirus mouse hepatitis virus (MHV), the first proteolytic processing event of the replicase polyprotein liberates an amino-terminal 28-kDa product (p28). While previous biochemical studies have suggested that p28 is associated with viral replication complexes, the intracellular localization and interactions of p28 with other proteins during the course of MHV replication have not been defined. We used immunofluorescence confocal microscopy to show that p28 localizes to viral replication complexes in the cytoplasm during early times postinfection. However, at late times postinfection, p28 localizes to sites of M accumulation distinct from the replication complex. Furthermore, by yeast two-hybrid and coimmunoprecipitation analyses, we demonstrate that p28 specifically binds to p10 and p15, two coronavirus replicase proteins of unknown function. Deletion mutagenesis experiments determined that the carboxy terminus of p28 is not required for its interactions with p10 and p15. These results suggest that p28 may play a part at the replication complex by interacting with p10 and p15. Moreover, our findings highlight a potential role for p28 at virion assembly sites.Coronaviruses are enveloped, positive-strand RNA viruses that cause a wide spectrum of disease in both humans and animals. The identification of a newly discovered human coronavirus as the causative agent of severe acute respiratory syndrome (SARS) underscores the pathogenic potential of this diverse virus family. However, the mechanisms by which coronaviruses replicate are not fully understood. Based on genome organization and phylogenetic analysis of gene 1 (replicase gene), coronaviruses are categorized into three distinct groups (1, 2, and 3), with the SARS coronavirus (SARS-CoV) being most closely related to group 2 (25, 34). Mouse hepatitis virus (MHV) is a prototype group 2 coronavirus and a model for studies of viral replication and pathogenesis. MHV and SARSCoV share orthologous protein domains encoded by the 5Ј end of the replicase gene that are absent in coronaviruses belonging to groups 1 and 3 (25, 34). Although the functions of these amino-terminal replicase proteins are not known, the similarity between MHV and SARS-CoV suggests that the proteins may have important roles during group 2-like coronavirus replication. Thus, determination of the intracellular localization and interactions of these unique replicase proteins during MHV replication may provide insight into processes by which the SARS-CoV replicates and causes disease.Following viral attachment and cell entry, the first event in the MHV life cycle is translation of the 22-kb replicase gene to yield two large polyproteins from two...

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Human metapneumovirus (HMPV) is a recently discovered paramyxovirus of the subfamily Pneumovirinae, which also includes avian pneumovirus and human respiratory syncytial virus (HRSV). HMPV is an important cause of respiratory disease worldwide. To understand early events in HMPV replication, cDNAs encoding the HMPV nucleoprotein (N), phosphoprotein (P), matrix protein (M), M2-1 protein and M2-2 protein were cloned from cells infected with the genotype A1 HMPV wild-type strain TN/96-12. HMPV N and P were shown to interact using a variety of techniques: yeast two-hybrid assays, co-immunoprecipitation and fluorescence resonance energy transfer (FRET). Confocal microscopy studies showed that, when expressed individually, fluorescently tagged HMPV N and P exhibited a diffuse expression pattern in the hostcell cytoplasm of uninfected cells but were recruited to cytoplasmic viral inclusion bodies in HMPV-infected cells. Furthermore, when HMPV N and P were expressed together, they also formed cytoplasmic inclusion-like complexes, even in the absence of viral infection. FRET microscopy revealed that HMPV N and P interacted directly within cytoplasmic inclusion-like complexes. Moreover, it was shown by yeast two-hybrid analysis that the N-terminal 28 aa are required for the recruitment to and formation of cytoplasmic inclusions, but are dispensable for binding to HMPV P. This work showed that HMPV N and P proteins provide the minimal viral requirements for HMPV inclusion body formation, which may be a distinguishing characteristic of members of the subfamily Pneumovirinae.