Human Parainfluenza Virus Type 3 HN-Receptor Interaction: Effect of 4-Guanidino-Neu5Ac2en on a Neuraminidase-Deficient Variant

The envelope of human parainfluenza virus type 3 (HPF3) contains two viral glycoproteins, the hemagglutinin-neuraminidase (HN) and the fusion protein (F). HN, which is responsible for receptor attachment and for promoting F-mediated fusion, also possesses neuraminidase (receptor-destroying) activity. We reported previously that 4-guanidino-neu5Ac2en (4-GU-DANA) and related sialic acid-based inhibitors of HPF3 neuraminidase activity also inhibit HN-mediated receptor binding and fusion processes not involving neuraminidase activity. We have now examined this mechanism, as well as neuraminidase's role in the viral life cycle, using a neuraminidase-deficient HPF3 variant (C28a) and stable cell lines expressing C28a or wild-type (wt) HN. C28a, which has a wt F sequence and two point mutations in the HN gene corresponding to two amino acid changes in the HN protein, is the first HPF3 variant with insignificant neuraminidase activity. Cells expressing C28a HN did not bind erythrocytes at 4 degrees C unless pretreated with neuraminidase, but no such pretreatment was required for hemadsorption activity (HAD) at 22 or 37 degrees C. HAD was blocked by 4-GU-DANA, attesting to the ability of this compound to inhibit HN's receptor-binding activity. C28a or wt plaque enlargement, a process that involves cell-cell fusion and does not depend on virion release, is diminished by the presence of 4-GU-DANA, confirming the inhibitory effect of 4-GU-DANA on the fusogenic function of C28a HN. In C28a-infected cell monolayers, virion release and thus multicycle replication are severely restricted. This defect was corrected by supplementation of exogenous neuraminidase and also by the addition of 4-GU-DANA; neuraminidase destroys the receptors whereby newly formed C28a virions would remain attached to the cell surface, whereas 4-GU-DANA prevents the attachment itself, obviating the need for receptor cleavage. In accord with the ability of 4-GU-DANA to prevent attachment, the neuraminidase inhibitory effect of 4-GU-DANA on wt HPF3 did not diminish virion release into the medium. Thus, it is by inhibition of viral entry and syncytium formation that sialic acid analogs like 4-GU-DANA may counteract wt HPF3 infection.     

Mutations in human parainfluenza virus type 3 hemagglutinin-neuraminidase causing increased receptor binding activity and resistance to the transition state sialic acid analog 4-GU-DANA (Zanamivir)

Entry and fusion of human parainfluenza virus type 3 (HPF3) require the interaction of the viral hemagglutinin-neuraminidase (HN) glycoprotein with its sialic acid receptor. 4-GU-DANA, a potent inhibitor of influenza virus neuraminidase, inhibits not only HPF3 neuraminidase but also the receptor binding activity of HPF3 HN and thus its ability to promote attachment and fusion. We previously generated a 4-GU-DANA-resistant HPF3 virus variant (ZM1) with a markedly fusogenic plaque morphology that harbored two HN gene mutations resulting in amino acid alterations. The present study using cells that express the individual mutations of ZM1 HN shows that one of these mutations is responsible for the increases in receptor binding and neuraminidase activities as well as the diminished sensitivity of both activities to the inhibitory effect of 4-GU-DANA. To examine the hypothesis that increased receptor binding avidity underlies 4-GU-DANA resistance, parallel studies were carried out on the high-affinity HN variant virus C22 and cells expressing the C22 variant HN. This variant also exhibited reduced sensitivity to 4-GU-DANA in terms of receptor binding and infectivity but without concomitant changes in the neuraminidase activity of HN. Another high-affinity HN variant, C0, was not resistant in terms of infectivity; however, a small increase in the receptor binding activity of C0 HN and a partial resistance of this activity to 4-GU-DANA were revealed by sensitive methods that we developed. In each virus variant, one mutation in HN accounted for both increased receptor binding avidity and 4-GU-DANA resistance; the higher affinity for the receptor overcomes the inhibitory effect of 4-GU-DANA. Thus, in contrast to influenza viruses for which 4-GU-DANA escape variants include hemagglutinin mutants with decreased receptor binding avidity that promotes virion release, for HPF3, HN mutants with increased receptor binding avidity are those that can escape the growth inhibitory effect of 4-GU-DANA.     

The anti-influenza virus agent 4-GU-DANA (zanamivir) inhibits cell fusion mediated by human parainfluenza virus and influenza virus HA

4-GU-DANA (zanamivir) (as well as DANA and 4-AM-DANA) was found to inhibit the neuraminidase activity of human parainfluenza virus type 3 (HPF3). The viral neuraminidase activity is attributable to hemagglutinin-neuraminidase (HN), an envelope protein essential for viral attachment and for fusion mediated by the other envelope protein, F. While there is no evidence that HN's neuraminidase activity is essential for receptor binding and syncytium formation, we found that 4-GU-DANA prevented hemadsorption and fusion of persistently infected cells with uninfected cells. In plaque assays, 4-GU-DANA reduced the number (but not the area) of plaques if present only during the adsorption period and reduced plaque area (but not number) if added only after the 90-min adsorption period. 4-GU-DANA also reduced the area of plaques formed by a neuraminidase-deficient variant, confirming that its interference with cell-cell fusion is unrelated to inhibition of neuraminidase activity. The order-of-magnitude lower 50% inhibitory concentrations of 4-GU-DANA (and also DANA and 4-AM-DANA) for plaque area reduction and for inhibition in the fusion assay than for reducing plaque number or blocking hemadsorption indicate the particular efficacy of these sialic acid analogs in interfering with cell-cell fusion. In cell lines expressing influenza virus hemagglutinin (HA) as the only viral protein, we found that 4-GU-DANA had no effect on hemadsorption but did inhibit HA2b-red blood cell fusion, as judged by both lipid mixing and content mixing. Thus, 4-GU-DANA can interfere with both influenza virus- and HPF3-mediated fusion. The results indicate that (i) in HPF3, 4-GU-DANA and its analogs have an affinity not only for the neuraminidase active site of HN but also for sites important for receptor binding and cell fusion and (ii) sialic acid-based inhibitors of influenza virus neuraminidase can also exert a direct, negative effect on the fusogenic function of the other envelope protein, HA.     

A Single Amino Acid Alteration in the Human Parainfluenza Virus Type 3 Hemagglutinin-Neuraminidase Glycoprotein Confers Resistance to the Inhibitory Effects of Zanamivir on Receptor Binding and Neuraminidase Activity

Entry and fusion of human parainfluenza virus type 3 (HPF3) requires interaction of the viral hemagglutinin-neuraminidase (HN) glycoprotein with its sialic acid receptor. 4-Guanidino-2,4-dideoxy-2,3-dehydro-N-acetylneuraminic acid (4-GU-DANA; zanamivir), a sialic acid transition-state analog designed to fit the influenza virus neuraminidase catalytic site, possesses antiviral activity at nanomolar concentrations in vitro. We have shown previously that 4-GU-DANA also inhibits both HN-mediated binding of HPF3 to host cell receptors and HN's neuraminidase activity. In the present study, a 4-GU-DANA-resistant HPF3 virus variant (ZM1) was generated by serial passage in the presence of 4-GU-DANA. ZM1 exhibited a markedly fusogenic plaque morphology and harbored two HN gene mutations resulting in two amino acid alterations, T193I and I567V. Another HPF3 variant studied in parallel, C-0, shared an alteration at T193 and exhibited similar plaque morphology but was not resistant to 4-GU-DANA. Neuraminidase assays revealed a 15-fold reduction in 4-GU-DANA sensitivity for ZM1 relative to the wild type (WT) and C-0. The ability of ZM1 to bind sialic acid receptors was inhibited 10-fold less than for both WT and C-0 in the presence of 1 mM 4-GU-DANA. ZM1 also retained infectivity at 15-fold-higher concentrations of 4-GU-DANA than WT and C-0. A single amino acid alteration at HN residue 567 confers these 4-GU-DANA-resistant properties. An understanding of ZM1 and other escape variants provides insight into the effects of this small molecule on HN function as well as the role of the HN glycoprotein in HPF3 pathogenesis.     

Contribution of the Human Parainfluenza Virus Type 3 HN-Receptor Interaction to Pathogenesis In Vivo

The envelope of human parainfluenza virus type 3 (HPF3) contains two viral glycoproteins, the hemagglutinin-neuraminidase (HN) protein and the fusion (F) protein. In a previous study, highly fusogenic variant HPF3 viruses were isolated, including two, C-0 and C-22, that exhibit increased avidity for sialic acid receptors due to single amino acid changes in the HN protein and one, C-28, that has decreased neuraminidase activity relative to that of the wild type (wt) and is delayed in the release of virus particles into the supernatant fluid. These variants form very large plaques and destroy a cell monolayer more rapidly than does wt HPF3 in cell culture. These variant viruses allowed us to formulate hypotheses about the roles of HN in pathogenesis. We investigated the behavior of wt HPF3 and the three variant viruses in the cotton rat model. In the cotton rat, there was no delayed clearance of any of the variant viruses compared to that of the wt. The variant plaque morphology was preserved in vivo, and there was no reversion to the wt phenotype in the infected animals. In spite of a slight advantage of wt virus in viral titer, there were no differences in the severities of peribronchiolitis between wt viruses and the variants. However, there were marked differences in severities in alveolitis and interstitial pneumonitis when each of the three variants was compared to the wt, with the variants causing enhanced disease. Thus, despite similar or lower viral titers and similar clearance rates, the variants caused more extensive disease in the lung. The results show that mutations in HN conferring altered fusion properties in cell culture also confer striking differences in the ability of HPF3 to cause extensive disease in the cotton rat lung and that this effect is dissociated from any effect on viral replication.     

Fusion properties of cells infected with human parainfluenza virus type 3: receptor requirements for viral spread and virus-mediated membrane fusion.

Cells can be persistently infected with human parainfluenza virus type 3 (HPF3) by using a high multiplicity of infection (MOI) (> or = 5 PFU per cell). The persistently infected cells exhibit no cytopathic effects and do not fuse with each other, yet they readily fuse with uninfected cells. We have previously shown that the failure of the persistently infected cells to fuse with each other is due to the lack of a receptor on these cells for the viral hemagglutinin-neuraminidase glycoprotein, and we have established that both fusion and hemagglutinin-neuraminidase proteins are needed for cell fusion mediated by HPF3. We then postulated that the generation of persistent infection and the failure of cells infected with HPF3 at high MOI to form syncytia are both due to the action of viral neuraminidase in the high-MOI inoculum. In this report, we describe experiments to test this hypothesis and further investigate the receptor requirements for HPF3 infection and cell fusion. A normally cytopathic low-MOI HPF3 infection can be converted into a noncytopathic infection by the addition of exogenous neuraminidase, either in the form of a purified enzyme or as UV-inactivated HPF3 virions. Evidence is presented that the receptor requirements for an HPF3 virus particle to infect a cell are different from those for fusion between cells. By treating infected cells in culture with various doses of neuraminidase, we demonstrate that virus spreads from cell to cell in the complete absence of cell-cell fusion. We compare the outcome of HPF3 infection in the presence of excess neuraminidase with that of another paramyxovirus (simian virus 5) and provide evidence that these two viruses differ in their receptor requirements for mediating fusion.     

Fusion properties of cells persistently infected with human parainfluenza virus type 3: participation of hemagglutinin-neuraminidase in membrane fusion.

Cells persistently infected with human parainfluenza virus type 3 (HPF3) exhibit a novel phenotype. They are completely resistant to fusion with each other but readily fuse with uninfected cells. We demonstrate that the inability of these cells to fuse with each other is due to a lack of cell surface neuraminic acid. Neuraminic acid is the receptor for the HPF3 hemagglutinin-neuraminidase (HN) glycoprotein, the molecule responsible for binding of the virus to cell surfaces. Uninfected CV-1 cells were treated with neuraminidase and then tested for their ability to fuse with the persistently infected (pi) cells. Neuraminidase treatment totally abolished cell fusion. To extend this result, we used a cell line deficient in sialic acid and demonstrated that these cells, like the neuraminidase-treated CV-1 cells, were unable to fuse with pi cells. We then tested whether mimicking the agglutinating function of the HN molecule with lectins would result in cell fusion. We added a panel of five lectins to the neuraminic acid-deficient cells and showed that binding of these cells to the pi cells did not result in fusion; the lectins could not substitute for interaction of neuraminic acid with the HN molecule in promoting membrane fusion. These results provide compelling evidence that the HN molecule of HPF3 and its interaction with neuraminic acid participate in membrane fusion and that cell fusion is mediated by an interaction more complex than mere juxtaposition of the cell membranes.     

Role of reticuloendotheliosis virus envelope glycoprotein in superinfection interference.

Cells expressing specific proviruses are resistant to superinfection by viruses of the same subgroup. To investigate the role of the reticuloendotheliosis virus (REV) envelope glycoprotein (env-gp) in the establishment of resistance to superinfection, we constructed plasmids that express either the wild-type env-gp or an env-gp derivative that lacks part of the transmembrane (TM) protein. After transfection, transient expression of the wild-type env gene resulted in syncytium formation in a mammalian cell line permissive for virus replication, whereas synthesis of the TM-defective env-gp did not result in syncytium formation. Several stable cell lines expressing either the normal or TM-defective env-gp were isolated. Expression of the normal env-gp in the absence of expression of other viral genes induced resistance to infection by REV. Immunofluorescence analysis of cells expressing the TM-defective env derivative and an examination of the glycosylation pattern of this peptide indicated that it is not translocated to the cell surface but resides primarily in the rough endoplasmic reticulum. However, these cells were also resistant to REV infection. Thus, interaction between the env derivative and the cellular component that functions as a receptor for the virus can occur in the endoplasmic reticulum and renders the cell immune to superinfection.     

Relative affinity of the human parainfluenza virus type 3 hemagglutinin-neuraminidase for sialic acid correlates with virus-induced fusion activity.

The ability of enveloped viruses to cause disease depends on their ability to enter the host cell via membrane fusion events. An understanding of these early events in infection, crucial for the design of methods of blocking infection, is needed for viruses that mediate membrane fusion at neutral pH, such as paramyxoviruses and human immunodeficiency virus. Sialic acid is the receptor for the human parainfluenza virus type 3 (HPF3) hemagglutinin-neuraminidase (HN) glycoprotein, the molecule responsible for binding of the virus to cell surfaces. In order for the fusion protein (F) of HPF3 to promote membrane fusion, the HN must interact with its receptor. In the present report, two variants of HPF3 with increased fusion-promoting phenotypes were selected and used to study the function of the HN glycoprotein in membrane fusion. Increased fusogenicity correlated with single amino acid changes in the HN protein that resulted in increased binding of the variant viruses to the sialic acid receptor. These results suggest that the avidity of binding of the HN protein to its receptor regulates the level of F protein-mediated fusion and begin to define one role of the receptor-binding protein of a paramyxovirus in the membrane fusion process.     

Fusion promotion by a paramyxovirus hemagglutinin-neuraminidase protein: pH modulation of receptor avidity of binding sites I and II

Paramyxoviruses, including the childhood respiratory pathogen human parainfluenza virus type 3 (HPIV3), possess an envelope protein hemagglutinin-neuraminidase (HN) that has receptor-cleaving (neuraminidase), as well as receptor-binding, activity. HN is a type II transmembrane glycoprotein, present on the surface of the virus as a tetramer composed of two dimers. HN is also essential for activating the fusion protein (F) to mediate merger of the viral envelope with the host cell membrane. This initial step of viral entry occurs at the host cell surface at neutral pH. The HN molecule carries out these three different critical activities at specific points in the process of viral entry, and understanding the regulation of these activities is key for the design of strategies that block infection. One bifunctional site (site I) on the HN of HPIV3 possesses both receptor binding and neuraminidase activities, and we recently obtained experimental evidence for a second receptor binding site (site II) on HPIV3 HN. Mutation of HN at specific residues at this site, which is next to the HN dimer interface, confers enhanced fusion properties, without affecting neuraminidase activity or receptor binding at neutral pH. We now demonstrate that mutations at this site II, as well as at site I, confer pH dependence on HN's receptor avidity. These mutations permit pH to modulate the binding and fusion processes of the virus, potentially providing regulation at specific stages of the viral life cycle.     

Influence of the human parainfluenza virus 3 attachment protein's neuraminidase activity on its capacity to activate the fusion protein

In order to examine functions of the hemagglutinin-neuraminidase (HN) protein that quantitatively influence fusion promotion, human parainfluenza virus 3 (HPIV3) variants with alterations in HN were studied. The variant HNs have mutations that affect either receptor binding avidity, neuraminidase activity, or fusion protein (F) activation. Neuraminidase activity was regulated by manipulation of temperature and pH. F activation was assessed by quantitating the irreversible binding of target erythrocytes (RBC) to HN/F-coexpressing cells in the presence of 4-GU-DANA (zanamivir) to release target cells bound only by HN-receptor interactions; the remaining, irreversibly bound target cells are retained via the fusion protein. In cells coexpressing wild-type (wt) or variant HNs with wt F, the fusion promotion capacity of HN was distinguished from target cell binding by measuring changes with time in the amounts of target RBC that were (i) reversibly bound by HN-receptor interaction (released only upon the addition of 4-GU-DANA), (ii) released by HN's neuraminidase, and (iii) irreversibly bound by F-insertion or fusion (F triggered). For wt HN, lowering the pH (to approach the optimum for HPIV3 neuraminidase) decreased F triggering via release of HN from its receptor. An HN variant with increased receptor binding avidity had F-triggering efficiency like that of wt HN at pH 8.0, but this efficiency was not decreased by lowering the pH to 5.7, which suggested that the variant HN's higher receptor binding activity counterbalanced the receptor dissociation promoted by increased neuraminidase activity. To dissect the specific contribution of neuraminidase to triggering, two variant HNs that are triggering-defective due to a mutation in the HN stalk were evaluated. One of these variants has, in addition, a mutation in the globular head that renders it neuraminidase dead, while the HN with the stalk mutation alone has 30% of wt neuraminidase. While the variant without neuraminidase activity triggered F effectively at 37 degrees C irrespective of pH, the variant possessing effective neuraminidase activity completely failed to activate F at pH 5.7 and was capable of only minimal triggering activity even at pH 8.0. These results demonstrate that neuraminidase activity impacts the extent of HPIV3-mediated fusion by releasing HN from contact with receptor. Any particular HN's competence to promote F-mediated fusion depends on the balance between its inherent F-triggering efficacy and its receptor-attachment regulatory functions (binding and receptor cleavage).     

Receptor-mediated interference mechanism responsible for resistance to polytropic leukemia viruses in Mus castaneus

The Asian mouse Mus castaneus is resistant to infection by the polytropic mink cell focus-inducing (MCF) subgroup of murine leukemia viruses (MuLVs). Genetic crosses showed this recessive resistance to be governed by a single gene that maps at or near the gene encoding the polytropic viral receptor, Rmc1. To investigate this resistance, we mated M. castaneus with mice carrying the wild mouse Sxv variant of the Rmc1 receptor that allows infection by xenotropic as well as polytropic virus. Unlike other F1 hybrids of M. castaneus, these F1 mice were resistant to both xenotropic and polytropic classes of MuLVs. Analysis of backcrossed progeny of the F1 hybrids mated to Sxv mice indicates that resistance is due to inheritance of two M. castaneus genes. Cells from individual backcross mice were also examined for cell surface antigen by fluorescence-activated cell sorter analysis with monoclonal antibodies reactive with xenotropic or MCF virus env glycoproteins. A correlation was observed between virus resistance and antigen, suggesting that virus resistance is due to expression of endogenous viral envelope genes that interfere with infection by exogenous virus. Since the inbred strain Rmc1 receptor remains functional in the presence of these M. castaneus genes, and since M. castaneus contains multiple copies of xenotropic MuLV env genes, we suggest that these resistance genes control expression of xenotropic env glycoprotein that interferes with exogenous virus in cells containing the Sxv variant of Rmc1.     

Influenza A virus neuraminidase limits viral superinfection

Enveloped viruses use multiple mechanisms to inhibit infection of a target cell by more than one virion. These mechanisms may be of particular importance for the evolution of segmented viruses, because superinfection exclusion may limit the frequency of reassortment of viral genes. Here, we show that cellular expression of influenza A virus neuraminidase (NA), but not hemagglutinin (HA) or the M2 proton pump, inhibits entry of HA-pseudotyped retroviruses. Cells infected with H1N1 or H3N2 influenza A virus were similarly refractory to HA-mediated infection and to superinfection with a second influenza A virus. Both HA-mediated entry and viral superinfection were rescued by the neuraminidase inhibitors oseltamivir carboxylate and zanamivir. These inhibitors also prevented the removal of alpha-2,3- and alpha-2,6-linked sialic acid observed in cells expressing NA or infected with influenza A viruses. Our data indicate that NA alone among viral proteins limits influenza A virus superinfection.     

Inhibition of parainfluenza virus type 3 and Newcastle disease virus hemagglutinin-neuraminidase receptor binding: effect of receptor avidity and steric hindrance at the inhibitor binding sites

Zanamivir (4-guanidino-Neu5Ac2en [4-GU-DANA]) inhibits not only the neuraminidase activity but also the receptor interaction of the human parainfluenza virus type 3 (HPIV3) hemagglutinin-neuraminidase (HN), blocking receptor binding and subsequent fusion promotion. All activities of the HPIV3 variant ZM1 HN (T193I/I567V) are less sensitive to 4-GU-DANA's effects. The T193I mutation in HN confers both increased receptor binding and increased neuraminidase activity, as well as reduced sensitivities of both activities to 4-GU-DANA inhibition, consistent with a single site on the HN molecule carrying out both catalysis and binding. We now provide evidence that the HPIV3 variant's resistance to receptor-binding inhibition by 4-GU-DANA is related to a reduced affinity of the HN receptor-binding site for this compound as well as to an increase in the avidity of HN for the receptor. Newcastle disease virus (NDV) HN and HPIV3 HN respond differently to inhibition in ways that suggest a fundamental distinction between them. NDV HN-receptor binding is less sensitive than HPIV3 HN-receptor binding to 4-GU-DANA, while its neuraminidase activity is highly sensitive. Both HPIV3 and NDV HNs are sensitive to receptor-binding inhibition by the smaller molecule DANA. However, for NDV HN, some receptor binding cannot be inhibited. These data are consistent with the presence in NDV HN of a second receptor-binding site that is devoid of enzyme activity and has a negligible, if any, affinity for 4-GU-DANA. Avidity for the receptor contributes to resistance by allowing the receptor to compete effectively with inhibitors for interaction with HN, while the further determinant of resistance is the reduced binding of the inhibitor molecule to the binding pocket on HN. Based upon our data and recent three-dimensional structural information on the HPIV3 and NDV HNs, we propose mechanisms for the observed sensitivity and resistance of HN to receptor-binding inhibition and discuss the implications of these mechanisms for the distribution of HN functions.     

Expression of Pokeweed Antiviral Protein in Transgenic Plants Induces Virus Resistance in Grafted Wild-Type Plants Independently of Salicylic Acid Accumulation and Pathogenesis-Related Protein Synthesis

Pokeweed antiviral protein (PAP), a 29-kD protein isolated from Phytolacca americana, inhibits translation by catalytically removing a specific adenine residue from the large rRNA of the 60S subunit of eukaryotic ribosomes. Transgenic tobacco (Nicotiana tabacum) plants expressing PAP or a variant (PAP-v) were shown to be resistant to a broad spectrum of plant viruses. Expression of PAP-v in transgenic plants induces synthesis of pathogenesis-related proteins and a very weak (<2-fold) increase in salicylic acid levels. Using reciprocal grafting experiments, we demonstrate here that transgenic tobacco rootstocks expressing PAP-v induce resistance to tobacco mosaic virus infection in both N. tabacum NN and nn scions. Increased resistance to potato virus X was also observed in N. tabacum nn scions grafted on transgenic rootstocks. PAP expression was not detected in the wild-type scions or rootstocks that showed virus resistance, nor was there any increase in salicylic acid levels or pathogenesis-related protein synthesis. Grafting experiments with transgenic plants expressing an inactive PAP mutant demonstrated that an intact active site of PAP is necessary for induction of virus resistance in wild-type scions. These results indicate that enzymatic activity of PAP is responsible for generating a signal that renders wild-type scions resistant to virus infection in the absence of increased salicylic acid levels and pathogenesis-related protein synthesis.     

Molecular anatomy of mouse hepatitis virus persistence: coevolution of increased host cell resistance and virus virulence.

Persistent infection of murine astrocytoma (DBT) cells with mouse hepatitis virus (MHV) has been established. From this in vitro virus-host system, persistence is mediated at the level of cellular MHV receptor (MHVR) expression and increased virus virulence. MHV persistence selects for resistant host cell populations which abate virus replication. Reductions in MHVR expression were significantly associated with increased host resistance, and transfection of MHVR into resistant host cells completely restored the capacity of cells to support efficient replication of MHV strain A59. The emergence of resistant host cells coselected for variant viruses that had increased avidity for MHVR and also recognized different receptors for entry into resistant cells. These data illustrate that MHV persistence in vitro provides a model to identify critical sites of virus-host interaction at the cellular level which are altered during the evolution of host cell resistance to viral infection and the coevolution of virus virulence.     

Analysis of hepatitis C virus superinfection exclusion by using novel fluorochrome gene-tagged viral genomes

Studies of the complete hepatitis C virus (HCV) life cycle have become possible with the development of an infectious cell culture system using the genotype 2a isolate JFH-1. Taking advantage of this system in the present study, we investigated whether HCV infection leads to superinfection exclusion, a state in which HCV-infected cells are resistant to secondary HCV infection. To discriminate between viral genomes, we inserted genes encoding fluorescent proteins in frame into the 3'-terminal NS5A coding region. These genomes replicated to wild-type levels and supported the production of infectious virus particles. Upon simultaneous infection of Huh-7 cells, co-replication of both viral genomes in the same cell was detected. However, when infections were performed sequentially, secondary infection was severely impaired. This superinfection exclusion was neither due to a reduction of cell surface expression of CD81 and scavenger receptor BI, two molecules implicated in HCV entry, nor due to a functional block at the level of virus entry. Instead, superinfection exclusion was mediated primarily by interference at the level of HCV RNA translation and, presumably, also replication. In summary, our results describe the construction and characterization of viable monocistronic HCV reporter genomes allowing detection of viral replication in infected living cells. By using these genomes, we found that HCV induces superinfection exclusion, which is primarily due to interference at a post-entry step.     

Human cytomegalovirus UL38 protein blocks apoptosis

Apoptosis is an innate cellular defense response to viral infection. The slow-replicating human cytomegalovirus (HCMV) blocks premature death of host cells prior to completion of the infection cycle. In this study, we report that the HCMV UL38 gene encodes a cell death inhibitory protein. A mutant virus lacking the pUL38 coding sequence, ADdlUL38, grew poorly in human fibroblasts, failed to accumulate viral DNA to wild-type levels, and induced excessive death of infected cells. Cells expressing pUL38 were resistant to cell death upon infection and effectively supported the growth of ADdlUL38. Cells infected with the pUL38-deficient virus showed morphological changes characteristic of apoptosis, including cell shrinkage, membrane blebbing, vesicle release, and chromatin condensation and fragmentation. The proteolytic cleavage of two key enzymes involved in apoptosis, namely, caspase 3 and poly(ADP-ribose) polymerase, was activated upon ADdlUL38 infection, and the cleavage was blocked in cells expressing pUL38. The pan-caspase inhibitor Z-VAD-FMK largely restored the growth of ADdlUL38 in normal fibroblasts, indicating that the defective growth of the mutant virus mainly resulted from premature death of host cells. Furthermore, cells expressing pUL38 were resistant to cell death induced by a mutant adenovirus lacking the antiapoptotic E1B-19K protein or by thapsigargin, which disrupts calcium homeostasis in the endoplasmic reticulum. Taken together, these results indicate that the HCMV protein pUL38 suppresses apoptosis, blocking premature death of host cells to facilitate efficient virus replication.     

Purification of the Porcine rubulavirus attachment protein by liquid isoelectric focusing

Porcine rubulavirus (PoRV) is an emerging virus responsible for meningoencephalitis, respiratory distress, and reproductive alterations in pigs. The hemagglutinin-neuraminidase (HN) glycoprotein is the most exposed and antigenic of the virus proteins. HN plays central roles in PoRV infection; i.e., it recognizes sialic acid-containing cell receptors that mediate virus attachment and penetration; in addition, its neuraminidase (sialic acid hydrolysis) activity has been proposed to be a virulence factor. So, HN is an ideal target for therapeutic treatment and prevention of this viral infection. This work describes a simple, fast, and sensitive method to purify the active form of HN protein based on its isoelectric point. HN was purified at a pH of 4.4, at which a single protein band of 66 kDa was observed on SDS-PAGE. Pure HN showed a maximal enzymatic activity at pH 3.5 and 37 degrees C using bovine fetuin as substrate. However, it retains circa 80% of its activity at a wide temperature range from 30 to 55 degrees C. We also describe improvements of neuraminidase determination method, which permits analysis in a microplate spectrophotometer, thereby increasing the sensitivity and reducing the costs of valuable reagents and biological samples.