TMPRSS12 Is an Activating Protease for Subtype B Avian Metapneumovirus.

The entry of avian metapneumovirus (aMPV) into host cells initially requires the fusion of viral and cell membranes, which is exclusively mediated by fusion (F) protein. Proteolysis of aMPV F protein by endogenous proteases of host cells allows F protein to induce membrane fusion; however, these proteases have not been identified. Here, we provide the first evidence that the transmembrane serine protease TMPRSS12 facilitates the cleavage of subtype B aMPV (aMPV/B) F protein. We found that overexpression of TMPRSS12 enhanced aMPV/B F protein cleavage, F protein fusogenicity, and viral replication. Subsequently, knockdown of TMPRSS12 with specific small interfering RNAs (siRNAs) reduced aMPV/B F protein cleavage, F protein fusogenicity, and viral replication. We also found a cleavage motif in the aMPV/B F protein (amino acids 100 and 101) that was recognized by TMPRSS12. The histidine, aspartic acid, and serine residue (HDS) triad of TMPRSS12 was shown to be essential for the proteolysis of aMPV/B F protein via mutation analysis. Notably, we observed TMPRSS12 mRNA expression in target organs of aMPV/B in chickens. Overall, our results indicate that TMPRSS12 is crucial for aMPV/B F protein proteolysis and aMPV/B infectivity and that TMPRSS12 may serve as a target for novel therapeutics and prophylactics for aMPV. IMPORTANCE: Proteolysis of the aMPV F protein is a prerequisite for F protein-mediated membrane fusion of virus and cell and for aMPV infection; however, the proteases used in vitro and vivo are not clear. A combination of analyses, including overexpression, knockdown, and mutation methods, demonstrated that the transmembrane serine protease TMPRSS12 facilitated cleavage of subtype B aMPV (aMPV/B) F protein. Importantly, we located the motif in the aMPV/B F protein recognized by TMPRSS12 and the catalytic triad in TMPRSS12 that facilitated proteolysis of the aMPV/B F protein. This is the first report on TMPRSS12 as a protease for proteolysis of viral envelope glycoproteins. Our study will shed light on the mechanism of proteolysis of aMPV F protein and pathogenesis of aMPV.

Mutation of the f-protein cleavage site of avian paramyxovirus type 7 results in furin cleavage, fusion promotion, and increased replication in vitro but not increased replication, tissue tropism, or virulence in chickens.

We constructed a reverse genetics system for avian paramyxovirus serotype 7 (APMV-7) to investigate the role of the fusion F glycoprotein in tissue tropism and virulence. The AMPV-7 F protein has a single basic residue arginine (R) at position -1 in the F cleavage site sequence and also is unusual in having alanine at position +2 (LPSSR↓FA) (underlining indicates the basic amino acids at the F protein cleavage site, and the arrow indicates the site of cleavage.). APMV-7 does not form syncytia or plaques in cell culture, but its replication in vitro does not depend on, and is not increased by, added protease. Two mutants were successfully recovered in which the cleavage site was modified to mimic sites that are found in virulent Newcastle disease virus isolates and to contain 4 or 5 basic residues as well as isoleucine in the +2 position: (RRQKR↓FI) or (RRKKR↓FI), named Fcs-4B or Fcs-5B, respectively. In cell culture, one of the mutants, Fcs-5B, formed protease-independent syncytia and grew to 10-fold-higher titers compared to the parent and Fcs-4B viruses. This indicated the importance of the single additional basic residue (K) at position -3. Syncytium formation and virus yield of the Fcs-5B virus was impaired by the furin inhibitor decanoyl-RVKR-CMK, whereas parental APMV-7 was not affected. APMV-7 is avirulent in chickens and is limited in tropism to the upper respiratory tract of 1-day-old and 2-week-old chickens, and these characteristics were unchanged for the two mutant viruses. Thus, the acquisition of furin cleavability by APMV-7 resulted in syncytium formation and increased virus yield in vitro but did not alter virus yield, tropism, or virulence in chickens.

RNA interference in vitro and in vivo using DsiRNA targeting the nucleocapsid N mRNA of human metapneumovirus.

Human metapneumovirus causes respiratory diseases with outcomes that can be severe in children, the immunocompromised, and the elderly. Synthetic small interfering RNAs (siRNAs) that silence targeted genes can be used as therapeutic agents. Currently, there is no specific therapy for hMPV. In this study, we designed Dicer-substrate siRNAs (DsiRNAs) that target metapneumovirus sequences on the mRNAs of the N, P, and L genes. In vitro, six DsiRNAs were shown to inhibit virus replication using cell proliferation tests. Of those, the DsiRNA that targets the most conserved mRNA sequence was then resynthesized in Evader™ format with heavy 2'-O-methyl modification of the guide strand. In a murine model, the prophylactic administration of this Evader™ DsiRNA was effective at partially inhibiting viral replication of hMPV (13×10(3) vs. 29×10(3)PFU/g of lung; p<0.01), which was not the case for the control, a mismatched DsiRNA. Inhibition was achieved without inducing cytokines or off-target effects. Moreover, the specificity of the siRNA mechanism of action was demonstrated in vitro and in vivo using 5'-RACE methodology. This in vivo approach of using a DsiRNA against hMPV is an important step in the development of synthetic siRNA as a therapeutic agent for this virus.

A recombinant sialidase fusion protein effectively inhibits human parainfluenza viral infection in vitro and in vivo.

BACKGROUND: The first step in infection by human parainfluenza viruses (HPIVs) is binding to the surface of respiratory epithelial cells via interaction between viral receptor-binding molecules and sialic acid-containing receptors. DAS181, a recombinant sialidase protein containing the catalytic domain of Actinomyces viscosus sialidase, removes cell surface sialic acid, and we proposed that it would inhibit HPIV infection. METHODS: Depletion of sialic acid receptors by DAS181 was evaluated by lectin-binding assays. Anti-HPIV activity in cultured cell lines and in human airway epithelium was assessed by the reduction in viral genomes and/or plaque forming units on treatment. In vivo efficacy of intranasally administered DAS181 was assessed using a cotton rat model. RESULTS: DAS181-mediated desialylation led to anti-HPIV activity in cell lines and human airway epithelium. Intranasal DAS181 in cotton rats, a model for human disease, significantly curtailed infection. CONCLUSIONS: Enzymatic removal of the sialic acid moiety of HPIV receptors inhibits infection with all tested HPIV strains, both in vitro and in cotton rats. Enzyme-mediated removal of sialic acid receptors represents a novel antiviral strategy for HPIV. The results of this study raise the possibility of a broad spectrum antiviral agent for influenza virus and HPIVs.

Viral respiratory infection causes airway hyperresponsiveness and decreases histamine N-methyltransferase activity in guinea pigs.

We investigated the effects of viral respiratory infection by Sendai virus on bronchial responses to aerosolized histamine in anesthetized guinea pigs and on the activity of histamine N-methyltransferase (HMT). We measured the change in total pulmonary resistance induced by histamine in the presence or absence of a specific HMT inhibitor, SKF 91488, in noninfected and infected animals. In the absence of SKF 91488, the bronchoconstrictor response to histamine was greater in infected than in noninfected animals. SKF 91488 (10(-2) M, 90 breaths) potentiated the responses to histamine in noninfected animals, and the magnitude of augmented responses to histamine by SKF 91488 was similar to that by viral infection. Furthermore, SKF 91488 did not further potentiate the responses to histamine in infected animals. However, responses to aerosolized acetylcholine were unaffected by viral infection and SKF 91488. The HMT activity decreased by 56% in the trachea, 86% in the bronchi, and 52% in the parenchymal tissue in the infected animals. In contrast to HMT activity, acetylcholinesterase activity was unaffected by viral infection. These results suggest that respiratory infection by Sendai virus causes enhanced bronchial responsiveness to histamine by decreasing HMT activity in airways.

Sendai virus infection changes the subcellular localization of tryptase Clara in rat bronchiolar epithelial cells.

Tryptase Clara activates the infectivity of Sendai and influenza viruses proteolytically. In this study, we investigated changes in the subcellular localization of tryptase Clara in rat bronchioles with progression of Sendai virus infection. Tryptase Clara and Sendai virus F2 antigen were localized by light and electron immunohistochemical studies. In the uninfected rat lung, tryptase Clara was specifically localized in the secretory granules of respiratory bronchiolar epithelial nonciliated cells, but not in bronchiolar ciliated, or alveolar cells. In the initial stage of Sendai virus infection with slight pathological changes, however, anti-tryptase Clara was highly reactive in luminal peripheral membranes of both nonciliated and ciliated epithelial cells of the bronchioles together with some Sendai virus envelope glycoprotein, F2 antigen. In the progressed stage, tryptase Clara was hard to detect, with heavy accumulation of F2 antigen in the epithelial cells. These immunohistochemical results support our previous findings that in the bronchial lavage fluid tryptase Clara is significantly increased both in amount and activity after viral infection. These results suggest that Sendai virus stimulates the secretion of tryptase Clara from nonciliated bronchiolar epithelial cells to the airway lumen. Accumulation of tryptase Clara on the luminal surface of the bronchiolar epithelial cells and/or in the airway lumen may produce favourable conditions for proteolytic viral activation and multiplication.

[Enzyme activity of interferon system in virus diseases]. / Aktivnost' fermentov sistemy interferona pri virusnykh zabolevaniiakh.

The enzymes activities of interferon system at viral infections of different etiology/influenza, parainfluenza, hepatitis B with delta infection and urticaria chronica with respiratory and herpes infection/has been studied and evaluation of the noted changes in the enzymes activity is discussed. It is shown that the same pathological changes in enzymes activities of interferon system were observed at different viral infections.

Neutral endopeptidase and neurogenic inflammation in rats with respiratory infections.

Neuropeptides such as substance P are implicated in inflammation mediated by sensory nerves (neurogenic inflammation), but the roles in disease of these peptides and the peptidases that degrade them are not understood. It is well established that inflammation is a prominent feature of several airway diseases, including viral infections, asthma, bronchitis, and cystic fibrosis. These diseases are characterized by cough, airway edema, and abnormal secretory and bronchoconstrictor responses, all of which can be elicited by substance P. The effects of substance P and other peptides that may be involved in inflammation are decreased by endogenous neutral endopeptidase (NEP; also called enkephalinase, EC 3.4.24.11), which is a peptidase that degrades substance P and other peptides. In the present study, we report that rats with histories of infections caused by common respiratory tract pathogens (parainfluenza virus type 1, rat corona-virus, and Mycoplasma pulmonis) not only have greater susceptibility to neurogenic inflammatory responses than do pathogen-free rats but also have a lower activity of NEP in the trachea. This reduction in NEP activity may cause the increased susceptibility to neurogenic inflammation by allowing higher concentrations of substance P to reach tachykinin receptors in the trachea. Thus decreased NEP activity may exacerbate some of the pathological responses in animals with respiratory tract infections.

Reversal of virus-induced alveolar macrophage bactericidal dysfunction by cyclooxygenase inhibition in vitro.

Virus infection of alveolar macrophages (AM) both in vivo and in vitro has been associated with a decreased ability of these cells to kill bacteria, together with enhanced production of metabolites of arachidonic acid. These metabolites, especially PGE2, may be inhibitory to some phagocyte functions. Primary cultures of bovine AM obtained by bronchoalveolar lavage of normal cattle were infected in vitro with parainfluenza-3 (PI3 virus) virus. Killing of Staphylococcus epidermidis by AM was determined on days 1-4 post-infection (p.i.) PI3 virus-infected AM killed significantly fewer bacteria on day 4 p.i. compared to uninfected controls (12.1 +/- 1.3% infected vs. 52.7 +/- 7.2% controls, P less than or equal to 0.05). Bacterial killing by virus-infected AM, but not control AM, was significantly enhanced on day 4 p.i. by addition of cyclooxygenase inhibitors 1 hr prior to bactericidal assay (28.0 +/- 4.5% indomethacin, 36.0 +/- 4.1% mefenamic acid, 38.6 +/- 7.3% piroxicam, 37.0 +/- 6.4% NDGA, 44.9 +/- 7.7% ETYA, P less than or equal to 0.05). Phagocytosis of opsonized sheep erythrocytes and superoxide generation by virus-infected AM were not significantly increased by cyclooxygenase inhibition. Phagosome-lysosome fusion was severely impaired in virus-infected AM. Pretreatment of virus-infected AM with indomethacin significantly enhanced the percentage of cell expressing fusion activity. This data suggests that in vitro bactericidal dysfunction associated with virus infection of AM is partially the result of enhanced production of prostaglandins or thromboxane by AM and/or an abnormal response to normal levels of endogenously produced cyclooxygenase metabolites. The data further indicate the presence of cyclooxygenase sensitive (phagosome-lysosome fusion) and insensitive (phagocytic) components of virus-induced bactericidal dysfunction in AM.

Considerations about the possible function of ceruloplasmin in influenza and parainfluenza virus infections.

The experimental data reveal that ceruloplasmin is a serum nonspecific factor acting during the early and late stages of infection with some respiratory viruses. This complex action seems to be based both on the enzymatic activity and on the copper-glycoprotein structure of ceruloplasmin. The changes in progen antigenicity produced by ceruloplasmin are probably involved in the mechanism of the antigenic shift and drift.

Allantoic fluid protease activity during influenza virus infection.

Neutral protease activity of allantoic fluid from embryonated chicken eggs was quantified during the course of influenza virus infection. Antigenic subtypes of influenza A viruses selected for study were H1N1 strains PR/8/34, Brazil/8/78, FM/1/47, the H3N2 strain Bangkok/1/80 and the H5N9 Turkey/ /Ontario/66 as well as the Sendai strain of parainfluenza type 1 virus. Three different types of profiles of allantoic fluid proteases could be readily distinguished after infection of eggs with various virus strains. In all profiles, periodic peaks of protease activity always preceded the partial shut down of protamine cleaving proteases which paralleled the production of near maximum titers of infectious virus. To determine the mechanism involved in this reduction of proteolytic activity, infectious allantoic fluids were analysed for the presence of protease inhibitors. Acid heat treated 48 hour virus-infected allantoic fluids of different influenza strains could inhibit the activities of subtilisin and allantoic fluid proteolytic enzymes.

Effects of viral pneumonia on lung macrophage lysosomal enzymes.

During viral pneumonitis in mice, lung fluid protein and free lysosomal enzyme activity are increased while macrophage lysosomal enzymes are decreased.