Human parainfluenza virus fusion complex glycoproteins imaged in action on authentic viral surfaces.

Infection by human parainfluenza viruses (HPIVs) causes widespread lower respiratory diseases, including croup, bronchiolitis, and pneumonia, and there are no vaccines or effective treatments for these viruses. HPIV3 is a member of the Respirovirus species of the Paramyxoviridae family. These viruses are pleomorphic, enveloped viruses with genomes composed of single-stranded negative-sense RNA. During viral entry, the first step of infection, the viral fusion complex, comprised of the receptor-binding glycoprotein hemagglutinin-neuraminidase (HN) and the fusion glycoprotein (F), mediates fusion upon receptor binding. The HPIV3 transmembrane protein HN, like the receptor-binding proteins of other related viruses that enter host cells using membrane fusion, binds to a receptor molecule on the host cell plasma membrane, which triggers the F glycoprotein to undergo major conformational rearrangements, promoting viral entry. Subsequent fusion of the viral and host membranes allows delivery of the viral genetic material into the host cell. The intermediate states in viral entry are transient and thermodynamically unstable, making it impossible to understand these transitions using standard methods, yet understanding these transition states is important for expanding our knowledge of the viral entry process. In this study, we use cryo-electron tomography (cryo-ET) to dissect the stepwise process by which the receptor-binding protein triggers F-mediated fusion, when forming a complex with receptor-bearing membranes. Using an on-grid antibody capture method that facilitates examination of fresh, biologically active strains of virus directly from supernatant fluids and a series of biological tools that permit the capture of intermediate states in the fusion process, we visualize the series of events that occur when a pristine, authentic viral particle interacts with target receptors and proceeds from the viral entry steps of receptor engagement to membrane fusion.

Electron tomography imaging of surface glycoproteins on human parainfluenza virus 3: association of receptor binding and fusion proteins before receptor engagement.

UNLABELLED: In order to deliver their genetic material to host cells during infection, enveloped viruses use specialized proteins on their surfaces that bind cellular receptors and induce fusion of the viral and host membranes. In paramyxoviruses, a diverse family of single-stranded RNA (ssRNA) viruses, including several important respiratory pathogens, such as parainfluenza viruses, the attachment and fusion machinery is composed of two separate proteins: a receptor binding protein (hemagglutinin-neuraminidase [HN]) and a fusion (F) protein that interact to effect membrane fusion. Here we used negative-stain and cryo-electron tomography to image the 3-dimensional ultrastructure of human parainfluenza virus 3 (HPIV3) virions in the absence of receptor engagement. We observed that HN exists in at least two organizations. The first were arrays of tetrameric HN that lacked closely associated F proteins: in these purely HN arrays, HN adopted a "heads-down" configuration. In addition, we observed regions of complex surface density that contained HN in an apparently extended "heads-up" form, colocalized with prefusion F trimers. This colocalization with prefusion F prior to receptor engagement supports a model for fusion in which HN in its heads-up state and F may interact prior to receptor engagement without activating F, and that interaction with HN in this configuration is not sufficient to activate F. Only upon receptor engagement by HN's globular head does HN transmit its activating signal to F. IMPORTANCE: Human parainfluenza virus 3 (HPIV3) is an enveloped, ssRNA virus that can cause serious respiratory illness, especially in children. HPIV3, like most other paramyxoviruses, uses two specialized proteins to mediate cell entry: the fusion protein (F) and the receptor binding protein, hemagglutinin-neuraminidase (HN). F becomes activated to mediate fusion during entry when it is triggered by a signal from HN. Here we used electron tomography to reconstruct the 3-dimensional ultrastructure of HPIV3. From these structures, we could discern the distribution and, in some cases, conformation of HN and F proteins, which provided an understanding of their interrelationship on virions. HN is found in arrays alone in one conformation and interspersed with prefusion F trimers in another. The data support a model of paramyxovirus membrane fusion in which HN associates with F before receptor engagement, and receptor engagement by the globular head of HN switches the HN-F interaction into one of fusion activation.

Characterization of a novel parainfluenza virus, caviid parainfluenza virus 3, from laboratory guinea pigs (Cavia porcellus).

A novel Respirovirus was isolated from nasopharyngeal swab specimens from clinically normal laboratory guinea pigs, and was characterized and named caviid parainfluenza virus 3 (CavPIV-3). The CavPIV-3 is enveloped, is 100 to 300 nm in diameter, and has a characteristic 15-nm-diameter chevron-shaped virus ribonucleocapsid protein. Sequence analysis of the fusion glycoprotein of CavPIV-3 revealed it to be 94% identical to human and guinea pig parainfluenza 3 (PIV-3) viruses and 80% identical to bovine PIV-3. To determine whether CavPIV-3 causes clinical disease in laboratory guinea pigs and to compare the serologic response of guinea pigs to CavPIV-3 and to other paramyxoviruses, an infection study was performed, in which groups of guinea pigs were inoculated with CavPIV-3, Sendai virus, simian virus 5 (SV-5), murine pneumonia virus (PVM), or bovine PIV-3 virus. During the course of the study, guinea pigs were maintained in an infectious disease suite, housed in Micro-Isolator cages, and were only manipulated under a laminar flow hood. Clinical signs of disease were not observed in any of the paramyxovirus-inoculated guinea pigs during the eight-week course of the study, and histologic signs of disease were not evident at necropsy eight weeks after inoculation. Guinea pigs inoculated with CavPIV-3, Sendai virus, PVM, and bovine PIV-3 developed robust homologous or heterologous serologic responses. In contrast, guinea pigs inoculated with SV-5 developed modest or equivocal serologic responses, as assessed by use of an enzyme-linked immunosorbent assay. Further, use of the SV-5 enzyme-linked immunosorbent assay resulted in the highest degree of non-specific reactivity among all of the paramyxovirus assays. In summary, CavPIV-3 is a novel guinea pig Respirovirus that subclinically infects laboratory guinea pigs, resulting in a robust serologic response, but no observed clinical or histologic disease. The CavPIV-3 fusion glycoprotein gene sequence is available from GenBank as accession No. AF394241, and the CavPIV-3 virus is available from the American Type Culture Collection as accession No. DR-1547.

Replication of parainfluenza type-3 virus and bovine respiratory syncytial virus in isolated bovine type-II alveolar epithelial cells.

The objectives of our research were to determine whether bovine pulmonary type-II alveolar epithelial cells could be isolated from bovine lung and maintained in tissue culture and to determine whether isolated bovine type-II alveolar epithelial cells would support productive viral replication of bovine parainfluenza type-3 virus and bovine respiratory syncytial virus. Type-II alveolar epithelial cells were isolated from lungs of 4- to 7-day-old male Holstein calves by enzymatic dissociation of pulmonary tissue with trypsin and by separation of cells with the use of filtration and centrifugation on continuous Percoll gradients. Cells were further separated by panning on IgG-coated plastic plates and by lectin binding. Isolated type-II alveolar cells were maintained on basement membrane-coated tissue cultured plates. In culture, type-II cells formed alveolar structures and maintained other cytologic features of type-II cells, including osmiophilic lamellar inclusions. Cell cultures were inoculated with and supported productive replication of bovine parainfluenza type-3 virus and bovine respiratory syncytial virus. This was determined by recovery of infectious viruses from inoculated cell cultures and by identification of viral structures in type-II alveolar epithelial cells by transmission electron microscopy.

Analysis of bovine parainfluenza-3 virus replication in bovine embryonic lung cells by indirect fluorescent antibody and hemadsorption assays.

The temporal manifestation of bovine parainfluenza-3 virus (BPI3V) proteins in the cytoplasm and on the surface of bovine embryonic lung (BEL) cells was characterized by indirect fluorescent antibody (IFA) and hemadsorption (HAd) assays. Intracellular proteins appeared earliest at 0.5 h post inoculation (p.i.) and the infection spread to virtually all cells by 48 h p.i. Viral proteins on the surface of cells were seen first at 16 h p.i., and by 48 h p.i. the entire cell monolayer was IFA positive. Hundred-fold less virus in the inoculum delayed appearance of the intracellular as well as cell-surface viral proteins by 24 h and allowed the infection of only about 1/3 of the cells by 48 h p.i. Kinetics of the development in the proportion of HAd-positive cells correlated with those of the surface fluorescence-positive cells. Morphology of the manifestation of BPI3V proteins is characterized by microphotography.

[Mechanism of penetration of human type 3 parainfluenza virus into monkey kidney cells]. / Mekhanizm proniknoveniia paragrippoznogo virusa cheloveka tipa 3 v kletki pochek obez'iany.

The mechanism of human parainfluenza type 3 virus penetration into monkey kidney cells was studied by morphological and biochemical methods. The results of electron microscopic studies permit a conclusion that the virus penetrates into the cells by the mechanism of receptor endocytosis. Analysis of subvirus structures in cytosole revealed two types of particles: nucleocapsids and structures of a larger size and lower buoyant density containing, in addition to NP protein, matrix (M) protein. It is presumed that nucleocapsid is released from the endocytic vacuole into the cytosole in association with M protein which is gradually eliminated from the nucleocapsid surface.

[Structural organization and proteins of the human type-3 para-influenza virus]. / Strukturnaia organizatsiia i belki virusa paragrippa cheloveka tipa 3.

The structure of human parainfluenza type 3 virus was studied by electron microscopy and virion fractionation by treatment with a detergent and high ionic strength. The protein spectrum of the virus was studied. The presence of 6 structural proteins was revealed of which two, HN and F, are glycoproteins. Intracellular cleavage of F0 protein into F1+2 proteins was demonstrated in a pulse-chase experiment. A tighter binding of HN protein than of F protein with the virus lipoprotein membrane was observed which may be useful for obtaining purified F protein preparations.

Application of computer-assisted image analysis for studying viral structures.

The application of CAIE has been shown to be useful for analyzing the structures of RNA viruses. Critical assessment of this method is essential for the selection of the micrographs of viruses. In our experience this procedure was helpful for resolving some questions concerning virus morphology.

Structural characterization of virion proteins and genomic RNA of human parainfluenza virus 3.

The virion proteins and genomic RNA of human parainfluenza virus 3 have been characterized. The virion contains seven major and two minor proteins. Three proteins of 195 X 10(3) molecular weight (195K), 87K, and 67K are associated with the nucleocapsid of the virion and have been designated L, P, and NP, respectively. Three proteins can be labeled with [14C]glucosamine and have molecular weights of 69K, 60K, and 46K. We have designated these proteins as HN, F0, and F1, respectively. HN protein has interchain disulfide bonds, but does not participate in disulfide bonding to form homomultimeric forms. F1 appears to be derived from a complex, F1,2, that has an electrophoretic mobility similar to that of F0 under nonreducing conditions. A protein of 35K is associated with the envelope components of the virion and aggregates under low-salt conditions; this protein has been designated M. The genome of human parainfluenza virus 3 is a linear RNA molecule with a molecular weight of approximately 4.6 X 10(6).

Electron microscopic evidence for parainfluenza type III/retrovirus phenotypically mixed particles.

Sublines of malignant permanent human fibroblast cell lines were shown to be infected by a type-D retrovirus (PMFV) and by a parainfluenza virus type III in an earlier study (2, 6). Careful electron microscopical investigations of these sublines have proved "viral structural elements" of both viruses in the same cell. We obtained electron microscopic evidence--though rarely--for parainfluenza type III/PMFV mixed-particles.

Some characteristics of a natural infection by parainfluenza-3 virus in a group of calves.

Parainfluenza-3 (Pi3) virus infection in a group of 25 calves is described. The virus was isolated from the lungs of four calve at days 6, 7, 13 and 55 after they were housed together at birth. Intracytoplasmic inclusion bodies were seen by light microscopy in bronchial and bronchiolar epithelial cells of two of these calves. Virus infected cells were detected by electron microscopy in three of the four calves. Haemagglutination inhibition antibodies to Pi3 virus were found in the sera of the calves. Despite the virus being present in the group from one week, a significant increase in antibody titre was found in only two animals although all the calves were in contact with each other during the study period. The pulmonary lesions in the four infected calves consisted of a bronchitis and bronchiolitis with infiltration of the walls and lumena of these structures by neutrophils and an adjacent neutrophil infiltration of alveoli some of which were collapsed.

Replication of parainfluenza type 3 virus in alveolar macrophages: evidence of in vivo infection and of in vitro temperature sensitivity in virus maturation.

Approximately 2% of cultured alveolar macrophages (AM), originally lavaged from the lungs of parainfluenza type 3 virus (PI-3V)-infected calves, were observed to contain viral antigen (by fluorescent antibody method) or viral nucleocapsids (by electron microscopy). Plaque assays, however, indicated that virus titers were generally low when cultures were incubated at 37 degrees C for 10 days. AM, obtained from "in vivo infected" and "noninfected" calves, were found to be equally susceptible to further in vitro PI-3V infection when cultures were incubated at 37 degrees C. AM that were obtained from the lungs of normal calves, cultured at 37 degrees C, and inoculated with PI-3V were observed to produce relatively high virus titers when the incubation temperature was shifted down to 32 degrees C. Results from hemagglutinin assays showed that considerable amounts of hemagglutinin were detected when AM cultures were incubated at 32 degrees C, but only limited amounts were detected at 37 degrees C. Results from electron microscopic examinations at both temperatures substantiated the results of plaque and hemagglutinin assays. The PI-3V, isolated from AM cultures incubated at 32 degrees C, grew well in Madin-Darby bovine kidney cells at 32 degrees C, but little virus was produced at 37 degrees C. In contrast, parent PI-3V grew equally well at both temperatures. The results are discussed in terms of host susceptibility, temperature-sensitivity and virus maturation, and surface viral antigens and persistent viral infection.

Effect of prostaglandins on cell function and multiplication of parainfluenza 3 viruses in WISH cells.

Cytotoxic effect of prostaglandins E2 and F2alpha on cells grown in vitro and the influence of these compounds on multiplication of myxovirus parainfluenza 3 were investigated. The prostaglandins were added to culture medium (0-01-10 mug/ml) 24 hr before virus infection, or for 2 and 48 hr after inoculation with viruses. WISH cells and monkey kidney cell cultures were used. No direct cytotoxic effect of prostaglandins at concentrations 0-01-1 mug/ml was found (viability, supravital staining, phase-contrast system, Nitro-BT reduction and succinic dehydrogenase tests), whereas the concentration of 10 mug/ml within 48 hr led to reduction and succinic dehydrogenase tests), whereas the concentration of 10 mug/ml within 48 hr led to partial injury of the cell population with symptoms of damage to mitochondria. Prostaglandins E2 and F2alpha inhibited multiplication of parainfluenza 3 virus at concentrations 0-1-10 mug/ml. The inhibitory effect was most pronounced if prostaglandins were added to medium for the whole period of virus multiplication i.e. for 48 hr but little or no effect was found if they were added prior to inoculation or for 2 hr after it. Inhibitory effect of prostaglandins on replication phase of viruses is suggested.