Svetlana P. Amineva

Similar Frequency of Rhinovirus-Infectible Cells in Upper and Lower Airway Epithelium

Rhinovirus (RV) infections can alter lower airway physiology and inflammation, yet the characteristics of RV replication in lower airway cells are incompletely understood. An RV serotype 16 (RV16)-specific monoclonal antibody was identified. Immunohistochemistry and an infectious center assay were used to quantitate the infectivity of RV16 in primary bronchial and adenoidal epithelial cells. The proportion of infectible epithelial cells increased with the inoculum but did not exceed 10%. Analysis of bronchial tissue samples infected ex vivo demonstrated a small subset of RV-infected cells in the epithelial layer. These data confirm previous reports that RV infects only a small subset of epithelial cells in upper airway tissues and indicate that lower airway epithelial cells have a similar susceptibility to RV infection. In confirming that RV can infect cells in the lower airway, these results suggest that lower airway dysfunction occurs through this mechanism in susceptible persons.

Molecular modeling, organ culture and reverse genetics for a newly identified human rhinovirus C

A recently recognized human rhinovirus species C (HRV-C) is associated with up to half of HRV infections in young children. Here we propagated two HRV-C isolates ex vivo in organ culture of nasal epithelial cells, sequenced a new C15 isolate and developed the first, to our knowledge, reverse genetics system for HRV-C. Using contact points for the known HRV receptors, intercellular adhesion molecule-1 (ICAM-1) and low-density lipoprotein receptor (LDLR), inter- and intraspecies footprint analyses predicted a unique cell attachment site for HRV-Cs. Antibodies directed to binding sites for HRV-A and -B failed to inhibit HRV-C attachment, consistent with the alternative receptor footprint. HRV-A and HRV-B infected HeLa and WisL cells but HRV-C did not. However, HRV-C RNA synthesized in vitro and transfected into both cell types resulted in cytopathic effect and recovery of functional virus, indicating that the viral attachment mechanism is a primary distinguishing feature of HRV-C.

Encephalomyocarditis virus (EMCV) proteins 2A and 3BCD localize to nuclei and inhibit cellular mRNA transcription but not rRNA transcription.

We have followed the viral processing cascade and polyprotein precursor fates during encephalomyocarditis virus (EMCV) infection of HeLa cells using a panel of monoclonal antibodies (mAbs). Within the first 2-4 h of infection, signals of antibodies specific for the 2A, 3B(VPg), 3C(pro) and 3D(pol) proteins were found to co-localize in nucleoli at the rRNA synthesis and cellular protein B23 (nucleophosmin) sites. Cellular fractionation identified viral protein precursor 3BCD as the common source of the P3-region antibody signals. Previously thought to be a minor product of the polymerase region cleavage pathways, the nuclear targeting of this precursor was localized with engineered mutations to five P2 and P3 region polyprotein processing sites. A nuclear localization motif (NLS), similar to that in many yeast ribosomal proteins, was identified near the N-terminus of the 3D(pol) sequence. Point mutations within this motif prevented nuclear and nucleolar localization by all forms of 3B(VPg), 3C(pro) and 3D(pol), and were lethal to the virus because they also prevented genome replication. However, viral RNA synthesis was not required for nucleolar transport and 3BCD was found in nuclei, even when the 3D(pol) was inactivated. Co-immunoprecipitation experiments showed a tight association between 3BCD and B23 (nucleophosmin), suggesting a possible ribosomal protein-like mechanism for nuclear transport. Infected cell extracts analyzed with microarrays, quantitative slot-blots and pulse-labeling experiments confirmed a nearly complete shutoff of host pol-II-dependent mRNA synthesis during EMCV infection, in reactions that depended on wild-type 2A protein. In contrast to human rhinovirus-16 infection, rRNA synthesis by pol-I and pol-III were not turned off by EMCV, although the cellular concentration of rRNA decreased during infection, relative to control samples. The data suggest that nuclear targeting by 2A and 3BCD may be responsible for regulating cellular mRNA and rRNA transcription during infection, perhaps via a proteolytic mechanism catalyzed by the endogenous 3C(pro) sequence.

Encephalomyocarditis viral protein 2A localizes to nucleoli and inhibits cap-dependent mRNA translation.

Panels of monoclonal antibodies were raised against viral non-structural proteins of encephalomyocarditis virus (EMCV) and used to probe infected cells in laser confocal microscopy experiments and Western analyses. Surprisingly, all Mengovirus and EMCV-infected cells showed strong targeting of protein 2A, 3B(VPg), 3C(pro), and 3D(pol) signals to cellular nuclei, in particular to nucleoli, from the earliest times of infection. Viral capsid proteins (1AB, 1C, and 1D) and other non-structural proteins (2B, 2C, and 3A) did not target nuclei and remained cytoplasmic throughout the infection. The cardioviral 2A protein (subject of this article) has a novel 143 amino acid sequence, terminating in a 19 amino acid COOH-terminal processing cassette (PCC) that participates in autocatalytic, co-translational primary cleavage of the viral polyprotein. The remainder of the 2A protein shares only limited similarity with other viral or cellular sequences, except for a short motif (KRvRPFRLP) near PCC resembling the nuclear localization signals (NLS) common to many yeast ribosomal proteins. Deletions within the EMCV 2A protein that impinge on this region have been reported to diminish the ability of virus to inhibit cap-dependent translation of cellular mRNAs. We have now observed that these same deletions prevented nuclear localization. Cellular expression of 2A protein from RNA transcripts or cDNAs confirmed that it does not require other viral proteins or activities for nuclear transport; even when expressed as a single protein, 2A protein effectively shuts off translation from capped reporter mRNAs. Within infected, transfected, or DNA vector-transformed cells, the 2A protein was always found in close association with the nucleolar ribosomal chaperone protein B23, which may help the traffic 2A into nucleoli like a surrogate ribosomal protein, by virtue of the putative nucleolar localization signal (NoLS). The data are consistent with a novel mechanism for virus-induced host protein shut off in cardioviruses, whereby 2A helps to upregulate the synthesis of new and modified ribosomes that have an inherent preference for internal ribosomal entry site (IRES)-dependent viral genome translation over cap-dependent host mRNA translation.

A rat model of picornavirus-induced airway infection and inflammation.

BackgroundInfection of the lower airways by rhinovirus, a member of the picornavirus family, is an important cause of wheezing illnesses in infants, and plays an important role in the pathogenesis of rhinovirus-induced asthma exacerbations. Given the absence of natural rhinovirus infections in rodents, we investigated whether an attenuated form of mengovirus, a picornavirus whose wild-type form causes systemic rather than respiratory infections in its natural rodent hosts, could induce airway infections in rats with inflammatory responses similar to those in human rhinovirus infections.ResultsAfter inoculation with 107 plaque-forming units of attenuated mengovirus through an inhalation route, infectious mengovirus was consistently recovered on days 1 and 3 postinoculation from left lung homogenates (median Log10 plaque-forming units = 6.0 and 4.8, respectively) and right lung bronchoalveolar lavage fluid (median Log10 plaque-forming units = 5.8 and 4.0, respectively). Insufflation of attenuated mengovirus, but not vehicle or UV-inactivated virus, into the lungs of BN rats caused significant increases (P < 0.05) in lower airway neutrophils and lymphocytes in the bronchoalveolar lavage fluid and patchy peribronchiolar, perivascular, and alveolar cellular infiltrates in lung tissue sections. In addition, infection with attenuated mengovirus significantly increased (P < 0.05) lower airway levels of neutrophil chemoattractant CXCR2 ligands [cytokine-induced neutrophil chemoattractant-1 (CINC-1; CXCL1) and macrophage inflammatory protein-2 (MIP-2; CXCL2)] and monocyte chemoattractant protein-1 (MCP-1; CCL2) in comparison to inoculation with vehicle or UV-inactivated virus.ConclusionAttenuated mengovirus caused a respiratory infection in rats with several days of viral shedding accompanied by a lower airway inflammatory response consisting of neutrophils and lymphocytes. These features suggest that mengovirus-induced airway infection in rodents could be a useful model to define mechanisms of rhinovirus-induced airway inflammation in humans.

Serial culture of murine primary airway epithelial cells and ex vivo replication of human rhinoviruses.

Human rhinoviruses (HRV) are the primary etiological agents in cold infections, and represent a serious risk to individuals with chronic respiratory disease such as asthma. In order to develop treatment options for HRV infections, murine models are a crucial component in the study of infection mechanisms due to the wide array of reagents and techniques available to study murine immunology. We present here a cell culture system for studying isolated murine epithelial cell responses to HRV. Monolayers of primary mouse airway epithelial cells were maintained in a serial culture system, and the identity and purity of the cell population was confirmed via immunostaining (positive for cytokeratin, negative for vimentin). Infection of these cells with a minor group rhinovirus (HRV-1A) was evidenced by increases in viral RNA, de novo synthesis of viral proteins, and production of infectious virus. This model will be useful in experiments to define mechanisms of viral replication and host/virus interactions within airway epithelial cells.

Comparison of rhinovirus A infection in human primary epithelial and HeLa cells.

HeLa cells are used to study the life cycles of many different viruses, including the human rhinoviruses (HRV) in the family Picornaviridae. Although the natural targets of HRV are human bronchial epithelial cells (hBE), it is generally more difficult to obtain and maintain the relevant primary cell cultures, relative to HeLa cells. Given that the HRV are now identified as a major cause of human asthma exacerbations, it becomes important to document how much of the virus biology learned from HeLa cells is common also to natural primary cells. When compared directly in matched infections using A01a virus, the kinetics of RNA replication, the synthesis and processing of viral proteins and the general subcellular localization of key non-structural proteins were resembled in hBE and HeLa cells. Viral-induced shutoff of host cell processes (e.g. nucleo-cytoplasmic trafficking) was also comparable.

Lower Respiratory Tract Infection Induced by a Genetically Modified Picornavirus in Its Natural Murine Host

Infections with the picornavirus, human rhinovirus (HRV), are a major cause of wheezing illnesses and asthma exacerbations. In developing a murine model of picornaviral airway infection, we noted the absence of murine rhinoviruses and that mice are not natural hosts for HRV. The picornavirus, mengovirus, induces lethal systemic infections in its natural murine hosts, but small genetic differences can profoundly affect picornaviral tropism and virulence. We demonstrate that inhalation of a genetically attenuated mengovirus, vMC0, induces lower respiratory tract infections in mice. After intranasal vMC0 inoculation, lung viral titers increased, peaking at 24 h postinoculation with viral shedding persisting for 5 days, whereas HRV-A01a lung viral titers decreased and were undetectable 24 h after intranasal inoculation. Inhalation of vMC0, but not vehicle or UV-inactivated vMC0, induced an acute respiratory illness, with body weight loss and lower airway inflammation, characterized by increased numbers of airway neutrophils and lymphocytes and elevated pulmonary expression of neutrophil chemoattractant CXCR2 ligands (CXCL1, CXCL2, CXCL5) and interleukin-17A. Mice inoculated with vMC0, compared with those inoculated with vehicle or UV-inactivated vMC0, exhibited increased pulmonary expression of interferon (IFN-α, IFN-β, IFN-λ), viral RNA sensors [toll-like receptor (TLR)3, TLR7, nucleotide-binding oligomerization domain containing 2 (NOD2)], and chemokines associated with HRV infection in humans (CXCL10, CCL2). Inhalation of vMC0, but not vehicle or UV-inactivated vMC0, was accompanied by increased airway fluid myeloperoxidase levels, an indicator of neutrophil activation, increased MUC5B gene expression, and lung edema, a sign of infection-related lung injury. Consistent with experimental HRV inoculations of nonallergic, nonasthmatic human subjects, there were no effects on airway hyperresponsiveness after inhalation of vMC0 by healthy mice. This novel murine model of picornaviral airway infection and inflammation should be useful for defining mechanisms of HRV pathogenesis in humans.

Synthesis of the allergen ovomucoid by a replicating Mengo virus

Summary.Interferons induced by viral infections can have powerful immuno- modulatory effects, and several epidemiologic studies have found an association between certain viral infections and reduced prevalence of allergy. We hypothesized that allergenic proteins could be synthesized by a replicating virus, and this construct could be useful as an immunomodulator. To test this hypothesis, we cloned an allergenic protein (ovomucoid [OVM]) into a murine picornavirus (Mengo virus) vector. This plasmid has a multicloning site surrounded by auto-catalytic sequences so that a foreign protein will be cleaved from viral proteins during replication. OVM sequences were cloned in the context of full-length viral genome cDNA, T7 RNA transcripts of this plasmid were transfected into HeLa cells, and recombinant virus plaques appeared on the second passage. Sequence analysis of recombinant viruses derived from individual plaques demonstrated that three viral isolates contained up to 2/3 of the OVM coding sequence, which was retained by the viruses after 5 additional passages in HeLa cells. The experiments verify the stable expression of immunoreactive OVM subunits by replicating viruses. These virus/allergen constructs could provide a tool to evaluate whether intracellular presentation of allergenic proteins in the context of a viral infection could prevent allergic sensitization upon re-challenge.