Low temperature and pressure stability of picornaviruses: implications for virus uncoating.
(1/876)
The family Picornaviridae includes several viruses of great economic and medical importance. Poliovirus replicates in the human digestive tract, causing disease that may range in severity from a mild infection to a fatal paralysis. The human rhinovirus is the most important etiologic agent of the common cold in adults and children. Foot-and-mouth disease virus (FMDV) causes one of the most economically important diseases in cattle. These viruses have in common a capsid structure composed of 60 copies of four different proteins, VP1 to VP4, and their 3D structures show similar general features. In this study we describe the differences in stability against high pressure and cold denaturation of these viruses. Both poliovirus and rhinovirus are stable to high pressure at room temperature, because pressures up to 2.4 kbar are not enough to promote viral disassembly and inactivation. Within the same pressure range, FMDV particles are dramatically affected by pressure, with a loss of infectivity of more than 4 log units observed. The dissociation of polio and rhino viruses can be observed only under pressure (2.4 kbar) at low temperatures in the presence of subdenaturing concentrations of urea (1-2 M). The pressure and low temperature data reveal clear differences in stability among the three picornaviruses, FMDV being the most sensitive, polio being the most resistant, and rhino having intermediate stability. Whereas rhino and poliovirus differ little in stability (less than 10 kcal/mol at 0 degrees C), the difference in free energy between these two viruses and FMDV was remarkable (more than 200 kcal/mol of particle). These differences are crucial to understanding the different factors that control the assembly and disassembly of the virus particles during their life cycle. The inactivation of these viruses by pressure (combined or not with low temperature) has potential as a method for producing vaccines. (+info)
unr, a cellular cytoplasmic RNA-binding protein with five cold-shock domains, is required for internal initiation of translation of human rhinovirus RNA.
(2/876)
Initiation of translation of the animal picornavirus RNAs occurs via a mechanism of direct ribosome entry, which requires a segment of the 5' UTR of the RNA, known as the internal ribosome entry site (IRES). In addition, translation of the enterovirus and rhinovirus (HRV) subgroups requires cellular trans-acting factors that are absent from, or limiting in rabbit reticulocytes, but are more abundant in HeLa cell extracts. It has been shown previously that HeLa cells contain two separable activities, each of which independently stimulates HRV IRES-dependent translation when used to supplement reticulocyte lysate; one of these activities was identified as polypyrimidine tract-binding protein (PTB). Here, the purification of the second activity is achieved by use of an RNA-affinity column based on the HRV 5' UTR. It comprises two components: a 38-kD protein (p38), which is a novel member of the GH-WD repeat protein family and has no intrinsic RNA-binding activity; and a 96- to 97-kD protein doublet, which was identified as unr, an RNA-binding protein with five cold-shock domains. Coimmunoprecipitation with antibodies against either protein shows that the two proteins interact with each other, and thus p38 is named unrip (unr-interacting protein). Recombinant unr acts synergistically with recombinant PTB to stimulate translation dependent on the rhinovirus IRES. In contrast, unr did not significantly augment the PTB-dependent stimulation of poliovirus IRES activity. (+info)
Eukaryotic initiation factor 4GII (eIF4GII), but not eIF4GI, cleavage correlates with inhibition of host cell protein synthesis after human rhinovirus infection.
(3/876)
For many members of the Picornaviridae family, infection of cells results in a shutoff of host protein synthesis. For rhinoviruses and enteroviruses, the shutoff has been explained in part by the cleavage of eukaryotic initiation factor 4GI (eIF4GI), a component of the cap-binding protein complex eIF4F. The cleavage of eIF4GI is mediated by the virus-specific proteinase 2Apro and results in inhibition of cap-dependent, but not cap-independent, translation. The inhibition of host protein synthesis after infection with human rhinovirus 14 (HRV-14) lags behind the cleavage of eIF4GI. Recently, we discovered a functional homolog of eIF4GI, termed eIF4GII, and showed that cleavage of eIF4GII coincides with the shutoff of host cell protein synthesis after poliovirus infection (Gradi et al., Proc. Natl. Acad. Sci. USA 95:11089-11094, 1998). We wished to determine whether eIF4GII cleavage kinetics could also explain the lack of correlation between the kinetics of eIF4GI cleavage and the shutoff of host protein synthesis after rhinovirus infection. In this study, we examined the correlation between human rhinovirus-induced shutoff of host protein synthesis and cleavage of eIF4GI and eIF4GII. In HRV-14-infected HeLa cells, almost no intact eIF4GI could be detected by 4 h postinfection, while only 4% of eIF4GII was cleaved at this time. By 6 h, however, 67% of eIF4GII was cleaved, and this cleavage coincided with a significant (60%) decline of host translation. These results suggest that cleavage of both eIF4GI and eIF4GII is required for HRV-mediated inhibition of host cell protein synthesis and that the cleavage of eIF4GII is the rate-limiting step in the shutoff of host cell protein synthesis after rhinovirus infection. (+info)
Rhinovirus infection induces expression of its own receptor intercellular adhesion molecule 1 (ICAM-1) via increased NF-kappaB-mediated transcription.
(4/876)
Virus infections, the majority of which are rhinovirus infections, are the major cause of asthma exacerbations. Treatment is unsatisfactory, and the pathogenesis unclear. Lower airway lymphocyte and eosinophil recruitment and activation are strongly implicated, but the mechanisms regulating these processes are unknown. Intercellular adhesion molecule-1 (ICAM-1) has a central role in inflammatory cell recruitment to the airways in asthma and is the cellular receptor for 90% of rhinoviruses. We hypothesized that rhinovirus infection of lower airway epithelium might induce ICAM-1 expression, promoting both inflammatory cell infiltration and rhinovirus infection. We therefore investigated the effect of rhinovirus infection on respiratory epithelial cell ICAM-1 expression and regulation to identify new targets for treatment of virus-induced asthma exacerbations. We observed that rhinovirus infection of primary bronchial epithelial cells and the A549 respiratory epithelial cell line increased ICAM-1 cell surface expression over 12- and 3-fold, respectively. We then investigated the mechanisms of this induction in A549 cells and observed rhinovirus-induction of ICAM-1 promoter activity and ICAM-1 mRNA transcription. Rhinovirus induction of ICAM-1 promoter activity was critically dependent upon up-regulation of NF-kappaB proteins binding to the -187/-178 NF-kappaB binding site on the ICAM-1 promoter. The principal components of the rhinovirus-induced binding proteins were NF-kappaB p65 homo- or heterodimers. These studies identify ICAM-1 and NF-kappaB as new targets for the development of therapeutic interventions for virus-induced asthma exacerbations. (+info)
Polypyrimidine-tract binding protein (PTB) is necessary, but not sufficient, for efficient internal initiation of translation of human rhinovirus-2 RNA.
(5/876)
Initiation of translation of the animal picornavirus RNAs is via a mechanism of direct internal ribosome entry, which requires a substantial segment of the viral 5'-untranslated region, generally known as the IRES (for "internal ribosome entry site"). Because, however, translation of the RNAs of members of the enterovirus, and more especially, the rhinovirus subgroups of the Picornaviridae is restricted in the reticulocyte lysate system, but is greatly stimulated by the addition of HeLa cell extracts, the implication is that, in these cases, internal initiation also requires cellular trans-acting factors that are more abundant in HeLa cell extracts than in rabbit reticulocytes. This was used as the basis of a functional assay for the purification of the HeLa cell factors required for translation dependent on the human rhinovirus-2 (HRV) IRES. There are two such HeLa cell factors separable by ion-exchange chromatography, each of which is individually active in the assay, although their combined effect is synergistic. One of these activities is shown to be polypyrimidine-tract binding protein (PTB) on the grounds that (1) the activity copurifies to homogeneity with PTB and (2) recombinant PTB expressed in Escherichia coli stimulates HRV IRES-dependent translation with a specific activity similar to that of the purified HeLa cell factor. Furthermore, it is shown that recombinant PTB also stimulates the translation of RNAs bearing the poliovirus type 1 (Mahoney) IRES. (+info)
Antiviral effect of hyperthermic treatment in rhinovirus infection.
(6/876)
Human rhinoviruses (HRV) are recognized as the major etiologic agents for the common cold. Starting from the observation that local hyperthermic treatment is beneficial in patients with natural and experimental common colds, we have studied the effect of brief hyperthermic treatment (HT) on HRV replication in HeLa cells. We report that a 20-min HT at 45 degrees C is effective in suppressing HRV multiplication by more than 90% when applied at specific stages of the virus replication cycle. Synthesis of virus proteins is not affected by HT, indicating that the target for treatment is a posttranslational event. The antiviral effect is a transient cell-mediated event and is associated with the synthesis of the 70-kDa heat shock protein hsp70. Unlike poliovirus, rhinovirus infection does not inhibit the expression of hsp70 induced by heat. The possibility that hsp70 could play a role in the control of rhinovirus replication is suggested by the fact that a different class of HSP inducers, the cyclopentenone prostaglandins PGA1 and delta 12-PGJ2, were also effective in inhibiting HRV replication in HeLa cells. Inhibition of hsp70 expression by actinomycin D prevented the antiviral activity of prostaglandins in HRV-infected cells. These results indicate that the beneficial effect of respiratory hyperthermia may be mediated by the induction of a cytoprotective heat shock response in rhinovirus-infected cells. (+info)
Rhinovirus regulation of IL-1 receptor antagonist in vivo and in vitro: a potential mechanism of symptom resolution.
(7/876)
Rhinovirus (RV) upper respiratory tract infections are prototypic transient inflammatory responses. To address the mechanism of disease resolution in these infections, we determined if RV stimulated the production of the IL-1 receptor antagonist (IL-1ra) in vivo and in vitro. In contrast to IL-1alpha and IL-1beta, immunoreactive IL-1ra was readily detected in the nasal washings of normal human volunteers. Symptomatic RV infection caused a small increase in IL-1alpha, a modest increase in IL-1beta, and an impressive increase in IL-1ra. Maximal induction of IL-1alpha and IL-1beta was transiently noted 48 h after RV infection. In contrast, maximal induction of IL-1ra was prolonged appearing 48-72 h after RV infection. These time points corresponded to the periods of peak symptomatology and the onset of symptom resolution, respectively. Western analysis of nasal washings demonstrated that RV stimulated the accumulation of intracellular IL-1ra type I in all and secreted IL-1ra in a subset of volunteers. Unstimulated normal respiratory epithelial cells contained intracellular IL-1ra type I mRNA and protein. RV infection increased the intracellular levels and extracellular transport of this IL-1ra moiety without causing significant changes in the levels of IL-1ra mRNA. IL-1ra may play an important role in the resolution of RV respiratory infections. RV stimulates epithelial cell IL-1ra elaboration, at least in part, via a novel translational and/or posttranslational mechanism. (+info)
Rhinovirus-mediated changes in airway smooth muscle responsiveness: induced autocrine role of interleukin-1beta.
(8/876)
An important interplay exists between specific viral respiratory pathogens, most commonly rhinovirus (RV), and altered airway responsiveness in the development and exacerbations of asthma. Given that RV infection reportedly induces the release of various cytokines in different cell types and that the reported effects of RV on airway smooth muscle (ASM) responsiveness are highly comparable to those obtained in ASM exposed to the proinflammatory cytokine interleukin (IL)-1beta, this study examined whether RV (serotype 16)-mediated pertubations in ASM responsiveness are mechanistically coupled to altered induced expression and action of IL-1beta in RV-exposed isolated rabbit and human ASM tissue and cultured cells. Relative to control tissues, ASM inoculated with RV exhibited significantly increased maximal isometric contractility to ACh (P < 0.01) and attenuated relaxation to isoproterenol (P < 0. 005). In extended studies, we found that 1) the RV-induced changes in ASM responsiveness were ablated by pretreating the tissues with the IL-1 recombinant human receptor antagonist; 2) in contrast to their respective controls, RV-inoculated ASM tissue and cultured cells exhibited progressively induced expression of IL-1beta mRNA and elaboration of IL-1beta protein at 6 and 24 h after viral exposure; and 3) the latter effect of RV was inhibited in the presence of a monoclonal antibody to intercellular adhesion molecule-1, the endogenous receptor for most RV. Collectively, these observations provide new evidence demonstrating that "pro-asthmatic-like" pertubations in agonist responsiveness elicited in RV-exposed ASM are largely attributed to the induced autologous expression and autocrine action of IL-1beta in the virus-infected ASM. (+info)