335 research outputs found
Roles of Non-coding RNAs in Respiratory Syncytial Virus (RSV) Infection
No full textAnalysis of host gene expression profiles following viral infections of target cells/tissues can reveal crucial insights into the host: virus interaction and enables the development of novel therapeutics and prophylactics. Regions of the host genome that do not code for protein, encode structural, and functional non-coding RNAs that are important not only in regulation of host gene expression but also may impact viral replication. This review summarizes the role of host non-coding RNAs during replication of multiple respiratory viruses with a focus on Respiratory Syncytial Virus (RSV), an important pediatric pathogen. This review highlights the current state of knowledge and understanding regarding the function(s) of ncRNAs for respiratory viral infection and host immunity in general.</p
The Structure and Replication of a Parainfluenza Virus
No full textThe simian parainfluenza virus SV5 is a member of myxovirus subgroup II, which includes parainfluenza, mumps, and Newcastle disease viruses. These viruses are 120-500 mμ in diameter, and consist of an envelope covered with surface projections and an internal ribonucleoprotein component which is a single-stranded helix, 150-180 A in diameter, A high yield of infective SV5 is produced by primary cultures of rhesus monkey kidney (MK) cells, and the virus-cell interaction is moderate: infected cells divide normally, exhibit little cyctopathic effect, and cellular macromolecular synthesis is not inhibited. An electron microscopic study of SV5 replication in MK cells was undertaken. Virus adsorbs to the cell surface and is then taken into cells by phagocytosis. Virus-induced morphological changes appear only in the cytoplasm of infected cells. The helical nucleocapsid of the virus appears to form in the cytoplasmic matrix and align under regions of the cell membrane which acquire viral surface projections. Assembly and release of virus particles at the cell surface occur by a budding process involving incorporation into the viral envelope of a unit membrane, which is continuous with and morphologically identical to that of the host cell. Both spherical and filamentous virus particles are formed. Filaments frequently contain nucleocapsid in a regular spiral which extends throughout their length. SV5 causes minimal cytopathic changes in MK cells, and there appears to be a balance between the rate of synthesis of nucleocapsid and its continuous release within mature virus particles. Under certain conditions, a gradual accumulation of nucleocapsid is seen in the cytoplasm of infected cells. This observation suggested an approach to the isolation of SV5 nucleocapsid. Equilibrium sedimentation in cesium chloride gradients was used to purify nucleocapsid released from cells by osmotic shock. The length distribution of the released nucleocapsid shows a sharp peak with a mean of 1.02 μ, and it is probable that this length contains one SV5 genome. Chemical determinations indicate that the nucleocapsid is a ribonucleoprotein with an RNA content of 4.1%. The length distribution of the nucleocapsid of Newcastle disease virus closely resembles that obtained for SV5. On the basis of these and other results, it seems likely that ~ 1μ, is the unit length of the nucleocapsid of all subgroup II myxoviruses. SV5 RNA was isolated from purified nucleocapsid or from virus purified equilibrium zonal centrifugation in a potassium tartrate gradient, in which the virus bands at a density of 1.22-1.23. The RNA was dissociated from protein by treatment with sodium dodecyl sulfate, and purified by sedimentation in a sucrose density gradient. RNA\u27s isolated from virions and from purified nucleocapsid are indistinguishable in sedimentation behavior. The sedimentation coefficient of SV5 RNA was estimated to be 50 S in sucrose gradients containing 0.05 M NaCl. On the basis of its ribonuclease sensitivity, base composition, and sedimentation behavior, SV5 RNA appears to be single-stranded. The methods used in the studies of SV5 were applied to pneumonia virus of mice (PVM), an unclassified virus whose structure had not been previously determined. PVM virions are spheres 80-120 mμ in diameter, or filaments of similar diameter with lengths up to 3 μ. The particles possess an outer, spike-covered envelope and helical internal component 120-150 A in diameter. Virus particles acquire their envelope by a budding process at the cell membrane; mature particles are seen only extracellularly. Dense inclusions are prominent in the cytoplasm of PVM-infected BHK21 cells and appear to consist of aggregates of the PVM internal component. The helical component was isolated in a cesium chloride gradient from extracts of osmotically shocked cells. Murine erythrocytes, which are agglutinated by PVM, adsorb to the surfaces of infected cells and to budding and extracellular PVM particles. On the basis of its structure and morphogenesis, PVM appears to be a myxovirus; however, the details of its structure and replication differ frora either of the two established subgroups of myxoviruses and suggests that a third subgroup of these viruses exists
Palmitoylation of the Murine Leukemia Virus Envelope Glycoprotein Transmembrane Subunits
No full textAbstractThe envelope protein of Friend murine leukemia virus is modified by fatty acylation of the transmembrane (TM) protein subunit. The labeling by [3H]palmitic acid was found to be sensitive to treatment with the reducing reagents 2-mercaptoethanol and hydroxylamine, indicating the presence of a thioester linkage. Pulse-chase experiments showed that the precursor protein can be labeled by [3H]palmitic acid prior to its cleavage into the surface and TM subunits. By using site-directed mutagenesis, we determined that palmitoylation occurs on a cysteine residue, Cys 606, located in the transmembrane domain. A thin-layer chromatography assay after acid hydrolysis showed that incorporated label comigrated with palmitic acid. When another cysteine residue was introduced into the cytoplasmic tail 22 amino acids from the transmembrane domain, no palmitoylation was observed to occur on this cysteine residue, demonstrating the importance of the position of the cysteine residue for palmitoylation. Sequence comparison revealed that most retrovirus envelope proteins have one or two conserved cysteine residues in their transmembrane domain. Mutations that change the palmitoylation state of the murine leukemia virus envelope protein did not affect its transport, processing, surface expression, or cell fusion activity. The palmitate-deficient viral envelope proteins were incorporated into virus particles, and replication of the virusin vitrowas not affected significantly by the mutation of the palmitoylation site
Oligomerization, Secretion, and Biological Function of an Anchor-Free Parainfluenza Virus Type 2 (PI2) Fusion Protein
No full textAbstractA number of studies indicate that the transmembrane domain, the cytoplasmic domain, or both regions of viral surface glycoproteins are involved in quaternary structure formation. In this report, the transmembrane domain and cytoplasmic tail coding sequence of the fusion (F) glycoprotein gene from parainfluenza type 2 virus was truncated by PCR and the resulting gene (PI2F′) was expressed in HeLa-T4 cells by using the vaccinia virus-T7 transient expression system. Pulse–chase experiments indicated that the anchor-free PI2F′ was expressed and processed into F1 and F2 subunits. Both the processed and the unprocessed anchor-free PI2F′ proteins were found to be efficiently secreted into the culture medium. Examination of the oligomeric form of the anchor-free PI2F′ by chemical cross-linking demonstrated that it assembles posttranslationally into dimers and trimers with a pattern similar to that of the wild-type PI2F protein. In an effort to better understand the biological properties of the truncated form of PI2F′, we anchored PI2F′ by a glycosyl-phosphatidylinositol (GPI) linkage. The GPI-anchored PI2F′ protein, when coexpressed with PI2HN, did not induce cell fusion seen as syncytium formation, but was found to initiate lipid mixing (hemifusion) as observed by transfer of R-18 rhodamine from red blood cells to the GPI-PI2F′/PI2HN cotransfected cells. The results therefore indicate that the extracellular domain of the PI2 fusion protein contains not only the structural information sufficient to direct assembly into higher oligomers, but also is competent to initiate membrane fusion, suggesting that the anchor-free PI2F′ may be useful for further structural studies
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