Sendai Virus Replication Research Articles

New synthetic glutathione derivatives with increased antiviral activities.

A series of glutathione (GSH) derivatives with aliphatic chains of different lengths, coupled by peptides bound to the alpha-NH2 group of Glu, were synthesized. When added to several cell lines, the C6 (n-hexanoyl), C8 (n-octanoyl) and C12 (n-dodecanoyl) derivatives were toxic while the C2 (nethanoyl) and C4 (n-butanoyl) derivatives were not. Preliminary experiments were performed to investigate the potential antiviral activity of the C2 and C4 derivatives compared to GSH. The C4 derivative was the most potent and fully characterized. GSH-C4 is a poor substrate of GSH metabolizing enzymes; once oxidized by disulphide-bound formation, C4 is slowly reduced by GSH-reductase. GSH-C4 completely abrogated Sendai virus replication at 7.5 mM with an EC50 of 3.6 mM, compared to 7.5 mM for GSH. GSH-C4 completely inhibited herpes simplex virus (HSV-1) virus production in Vero cells at 10 mM, while the same dose of GSH caused only a 2.5 log10 reduction. Furthermore, the GSH-C4 treatment (7.5 mM) was able to markedly reduce the cytopathic effect of HSV-1 in Vero cells. Thus, GSH derivatives with increased hydrophobic properties are more effective antiviral agents against Sendai and HSV-1 viruses than GSH, suggesting their usefulness in antiviral therapy.

Differential effects of Sendai virus infection on mediator synthesis by mesangial cells from two mouse strains

Recently, we observed that the severity of glomerulonephritis in an experimental model of immunoglobulin A nephropathy (IgAN) induced by Sendai virus differs between C57BL/6 and BALB/c mouse strains. The determinants of differing renal insufficiency are not understood. In the present study, we examine the capacity for mesangial cells to support Sendai viral replication and assess the direct effects of Sendai virus on the production of selected cytokines, chemokines, and eicosanoids by mesangial cells, comparing C57BL/6 to BALB/c mouse strains. Sendai virus replication was measured by viral plaque assay using LLCMK2 cells. Production of cytokines [interleukin-6 (IL-6) and tumor necrosis factor-alpha (TNF-alpha)], chemokines (JE and KC), and eicosanoids [prostaglandin E2 (PGE2) and thromboxane B2 (TxB2)] in culture medium was evaluated by sandwich enzyme-linked immunosorbent assay (ELISA) or competitive enzyme immunoassay (EIA) after 48 hours' incubation with infectious or inactivated Sendai virus. Sendai virus replicates equally well in mesangial cells from both strains, and infection evokes increased IL-6, JE, KC, and PGE2 production in relation to viral dose. BALB/c mesangial cells produce significantly more IL-6 and JE than those from C57BL/6, and the dose response for KC is steeper in BALB/c mesangial cells than those from C57BL/6. Synthesis of PGE2 in BALB/c mesangial cells is higher than that of C57BL/6 mesangial cells, both under basal conditions and in response to infectious Sendai virus, again in a dose-dependent manner. There is no TNF-alpha or thromboxane response to viral stimulation. We conclude that different mesangial cell responses to this common mucosal viral pathogen might influence the severity of IgAN in our model system.

Primary replication of a recombinant Sendai virus vector in macaques.

An efficient antigen expression system using a recombinant Sendai virus (SeV) has been established recently and its potential to induce resistance against immunodeficiency virus infections in macaques has been shown. SeV replication has been well characterized in mice, the natural host, but not in primates, including humans. Here, primary SeV replication was investigated in macaques. After intranasal immunization with a recombinant SeV expressing simian immunodeficiency virus Gag protein, SeV-Gag, robust gag expression was observed in the nasal mucosa and much lower but significant levels of gag expression were observed in the local retropharyngeal and submandibular lymph nodes (LN). Expression peaked within a week and lasted at least up to 13 days after immunization. SeV-Gag was isolated from nasal swabs consistently at day 4 but not at all at day 13. Gag expression was undetectable in the lung as well as in remote lymphoid tissues, such as the thymus, spleen and inguinal LN, indicating that the spread of the virus was more restricted in macaques than in mice. SeV-specific T cells were detectable in SeV-immunized macaques at day 7. Finally, no naive macaques showed significant levels of anti-SeV antibodies in the plasma, even after living in a cage together with an acutely SeV-infected macaque for 5 weeks, indicating that SeV transmission from SeV-infected macaques to naive ones was inefficient. None of the SeV-immunized macaques displayed appreciable clinical manifestations. These results support the idea that this system may be used safely in primates, including humans.

Synthesis, biological evaluation, and pharmacophore generation of uracil, 4(3H)-pyrimidinone, and uridine derivatives as potent and selective inhibitors of parainfluenza 1 (Sendai) virus.

Several new 6-oxiranyl-, 6-oxiranylmethyluracils, and pyrimidinone derivatives, synthesized by lithiation-alkylation sequence of 1,3,6-trimethyluracil, 1,3-dimethyl-6-chloromethyluracil, and 2-alkoxy-6-methyl-4(3H)-pyrimidinones, showed a potent and selective antiviral activity against Sendai virus (SV) replication. To gain insight into the structural features required for SV inhibition activity, the new compounds were submitted to a pharmacophore generation procedure using the program Catalyst. The resulting pharmacophore model showed high correlation and predictive power. It also rationalized the relationships between structural properties and biological data of these inhibitors of SV replication.

Role of Primary Constitutive Phosphorylation of Sendai Virus P and V Proteins in Viral Replication and Pathogenesis

Functional analysis of the primary constitutive phosphorylation of Sendai virus P and V proteins was performed using both in vitro and in vivo systems. Sendai virus minigenome transcription and replication in transfected cells were not significantly affected in the presence of primary phosphorylation deficient P protein (S249A, S249D, P250A) as measured by either the luciferase activity or the Northern blot analysis. Similarly, recombinant Sendai viruses lacking the primary phosphorylation in P grew to titers close to the wild-type virus in cell cultures and in the natural host of Sendai virus, the mouse. Mutant viruses showed no altered pathogenesis in mice lungs. Oligomerization of P by binding WT P or mutant P to GST-P (WT) Sepharose beads revealed that the primary phosphorylation was not crucial for P protein oligomerization. Similar to P protein primary phosphorylation, the V protein primary phosphorylation at serine249 was not essential for minigenome transcription and replication, as both WT and mutant V proteins were found equally inhibitory to the minigenome replication. These results show that the primary phosphorylation of P protein has no essential role in Sendai virus transcription, replication, and pathogenesis.

Increased replication of sendai virus in morphine-treated epithelial cells: evidence for the involvement of the intracellular levels of glutathione

This paper shows that morphine increases Sendai virus replication in cultured epithelial cells. The effect was maximal when it was added before viral infection. Morphine also reduced the intracellular level of glutathione, namely, the oxidative and most abundant cell thiol. Altered intracellular redox status has recently been proposed as a factor influencing viral infection. Support for this view was provided by our data showing that inhibition of de novo glutathione synthesis, using L-buthionine sulfoximine, increased virus replication. These findings provide the first evidence that morphine increases the susceptibility to virus infection by altering the intracellular levels of glutathione.

Sendai virus infection induces apoptosis through activation of caspase-8 (FLICE) and caspase-3 (CPP32).

Sendai virus (SV) infection and replication lead to a strong cytopathic effect with subsequent death of host cells. We now show that SV infection triggers an apoptotic program in target cells. Incubation of infected cells with the peptide inhibitor z-VAD-fmk abrogated SV-induced apoptosis, indicating that proteases of the caspase family were involved. Moreover, proteolytic activation of two distinct caspases, CPP32/caspase-3 and, as shown for the first time in virus-infected cells, FLICE/caspase-8, could be detected. So far, activation of FLICE/caspase-8 has been described in apoptosis triggered by death receptors, including CD95 and tumor necrosis factor (TNF)-R1. In contrast, we could show that SV-induced apoptosis did not require TNF or CD95 ligand. We further found that apoptosis of infected cells did not influence the maturation and budding of SV progeny. In conclusion, SV-induced cell injury is mediated by CD95- and TNF-R1-independent activation of caspases, leading to the death of host cells without impairment of the viral life cycle.

A potent and selective inhibition of parainfluenza 1 (Sendai) virus by new 6-oxiranyl-, 6-methyloxiranyluracils, and 4(3H)-pyrimidinone derivatives.

Several new 6-oxiranyl-, 6-methyloxiranyluracils, and pyrimidinone derivatives, synthesized by the lithiation-alkylation sequence of 1,3,6-trimethyluracil, 1,3-dimethyl-6-chloromethyluracil, and 2-alkoxy-6-methyl-4(3H)-pyrimidinones, showed a potent and selective antiviral activity against the parainfluenza 1(Sendai) virus replication.

The activity of Sendai virus genomic and antigenomic promoters requires a second element past the leader template regions: a motif (GNNNNN)3 is essential for replication.

The paramyxovirus genome, a nonsegmented, negative-polarity, single-stranded RNA of approximately 15 kb, contains six transcription units flanked at the 3' and 5' ends by a short (approximately 50- to 60-nucleotide) extracistronic sequence, dubbed the positive and negative leader regions. These leader template regions, present at the 3' end of the genome and the antigenome, have been shown to contain essential signals governing RNA replication activity. Whether they are sufficient to promote replication is still open to question. By using a series of Sendai virus defective interfering RNAs carrying a nested set of deletions in the promoter regions, it is shown here that for both the genomic and antigenomic promoters, a 3'-end RNA sequence of 96 nucleotides is required to allow replication. Sequence comparison of active and inactive promoters led to the identification of a set of three nucleotide hexamers (nucleotides 79 to 84, 85 to 90, and 91 to 96) containing a repeated motif RXXYXX [shown as 5'-3' positive-strand]. Sequential mutation of each hexamer into its complementary sequence confirmed their essential role. The three hexamers are required, and their relative positioning is important, since displacing them by 6 nucleotides destroyed promoter function. RNAs carrying degenerate nucleotides in the three hexamers were used as replication templates. They led to the selection of actively replicating RNA species exclusively carrying the basic motif (GNNNNN)3 from nucleotides 79 to 96. These results clearly show that, apart from the region from nucleotides 1 to 31, previously identified as governing Sendai virus replication activity, a second element, spanning at the most nucleotides 79 to 96, appears essential. Thus, the paramyxovirus replication promoters are not confined to the leader template regions, as seems to be the case for the rhabdoviruses.

Phosphorylation of the Sendai Virus M Protein Is Not Essential for Virus Replication Eitherin Vitroorin Vivo

A large proportion of intracellular Sendai virus (SeV) M proteins is phosphorylated, but in mature virions the M protein is not phosphorylated or dephosphorylated. Phosphorylated M protein in cells is bound to the cytoskeletal components more firmly than unphosphorylated M protein. Thus it has been hypothesized that M protein phosphorylation plays an important role in the virus life cycle, especially in the step of maturation. Here, a transient expression-mutation experiment of the M gene demonstrated that a change of the Ser residue at the 70th position from the N-terminus to Ala (S70A) totally abolished M protein phosphorylation, strongly suggesting that this residue is phosphorylated. The mutated M gene was then placed in the corresponding region in the cDNA plasmid which generates a full-length antigenome SeV RNA, and a mutant SeV M-S70A was successfully recovered from the cDNA. This mutant virus was indeed defective in M protein phosphorylation but did not differ at all from the wild-type SeV recovered from the parental cDNA either in the replication kinetics and plaque morphology in cultured cells or inin vivoreplication and pathogenicity for mice. We thus concluded that no phosphorylation of the M protein was required for SeV replication eitherin vitroorin vivo.

Loss of GSH, Oxidative Stress, and Decrease of Intracellular pH as Sequential Steps in Viral Infection

Madin-Darby canine kidney cells infected with Sendai virus rapidly lose GSH without increase in the oxidized products. The reduced tripeptide was quantitatively recovered in the culture medium of the cells. Since the GSH loss in infected cells was not blocked by methionine, a known inhibitor of hepatocyte GSH transport, a nonspecific leakage through the plasma membrane is proposed. UV-irradiated Sendai virus gave the same results, confirming that the major loss of GSH was due to membrane perturbation upon virus fusion. Consequent to the loss of the tripeptide, an intracellular pH decrease occurred, which was due to a reversible impairment of the Na+/H+ antiporter, the main system responsible for maintaining unaltered pHi in those cells. At the end of the infection period, a rise in both pHi value and GSH content was observed, with a complete recovery in the activity of the antiporter. However, a secondary set up of oxidative stress was observed after 24 h from infection, which is the time necessary for virus budding from cells. In this case, the GSH decrease was partly due to preferential incorporation of the cysteine residue in the viral proteins and partly engaged in mixed disulfides with intracellular proteins. In conclusion, under our conditions of viral infection, oxidative stress is imposed by GSH depletion, occurring in two steps and following direct virus challenge of the cell membrane without the intervention of reactive oxygen species. These results provide a rationale for the reported, and often contradictory, mutual effects of GSH and viral infection.

Partial Characterization of a Sendai Virus Replication Promoter and the Rule of Six

We have used a cDNA copy of a natural, internally deleted, Sendai virus defective interfering genome to study the effect of insertions and deletions (which maintain the hexamer genome length) on the ability of viral genomes to be amplified in a transfected cell system. The insertion of 18 nt at nt72(in the 5′ untranslated region of the N gene, just downstream of thele+region) was found to be lethal, whereas similar insertions further from the genome ends were well tolerated. Curiously, the insertion of 6 nt on either side of thele+/N junction (at nt47and nt67) was well tolerated, but the insertion of 12 nt at either site, or of 6 nt at both sites, largely ablated genome amplification. These results suggest that an element of this replication promoter is located downstream of nt67, in the 5′ untranslated region of the first gene. Remarkably, the addition of 6 nt by the insertion of 2, 3, or 4 nt at nt47plus the insertion of 4, 3, or 2 nt, respectively, at nt67was poorly tolerated, presumably because the hexamer phase of the intervening sequence was altered with respect to the N subunits of the template. These results suggest that the rule of six operates, at least in part, at the level of the initiation of antigenome synthesis.

Protein factors required for in vitro transcription of Sendai virus genome.

To elucidate the mechanism of transcription and replication of Sendai virus, we developed an efficient and faithful in vitro transcription system using purified virus particles. The in vitro RNA synthesis was almost entirely dependent on the addition of eukaryotic cell extracts, including those from various cultured mammalian cells, mammalian tissues, and even from plant cells. The RNA products were almost identical to authentic mRNA species synthesized in the infected cells, in their size distribution, the presence of 3'-poly(A) tail and the presence of methylated 5'-cap structure (m7GpppAm). Ribonuclease protection experiments after annealing the in vitro RNA with viral genomic RNA (vRNA) indicated that the virion-associated RNA-dependent RNA polymerase transcribes correct regions of the RNA genome in vitro. The active component(s) that is required for Sendai virus mRNA synthesis was partially purified from bovine brain and was separated into at least two complementary fractions, one of which could be replaced by highly purified cellular tubulin. When viral ribonucleoprotein complexes were used instead of virus particles in the in vitro transcription, only Sendai virus-infected cell extracts supported mRNA synthesis, and extracts from uninfected cells or cells infected with other viruses were found to be inert. These results suggest that, in addition to the general factors which are present ubiquitously in eukaryotic cells, a factor(s) specific to Sendai virus-infection is required for Sendai virus transcription.

Transport of Envelope Proteins of Sendai Virus, HN and F0, Is Blocked at Different Steps by Thapsigargin and Other Perturbants to Intracellular Ca2+11

The effects of thapsigargin (Tg), a specific inhibitor of Ca(2+)-ATPase of the endoplasmic reticulum (ER), on replication of Sendai virus (HVJ) in BALB3T3 cells were examined. In the presence of Tg, the cells infected with HVJ did not release viral particles to the culture medium. Tg inhibited almost completely the expression of viral envelope proteins, HN and F0, on the cell surface, although it did not affect the synthesis of viral proteins. Two other inhibitors of Ca(2+)-ATPase of the ER, 2,5-di(tert-butyl)-1,4-benzohydroquinone (BHQ) and cyclopiazonic acid (CPA), as well as Ca(2+)-ionophores such as A23187 and ionomycin, also inhibited the expression of HN protein on the cell surface. Tg seemed to inhibit the intracellular transport or maturation of the viral membrane proteins by perturbing intracellular distribution of Ca2+ ions. In the presence of Tg, HN protein remained sensitive to endoglycosidaseH (endoH) for 3 h after its synthesis. On the other hand, F0 protein became resistant to endoH and sensitive to neuraminidase even in the presence of Tg. These results indicate that the transport of HN protein is blocked at the ER or the cis-Golgi region, while that of F0 protein is blocked at the post-Golgi stage in the presence of Tg.

Inhibition of Sendai virus replication by delta 12-prostaglandin J2: induction of heat shock protein synthesis and alteration of protein glycosylation.

delta 12-Prostaglandin J2 (delta 12-PGJ2), a natural dehydration product of prostaglandin D2 present in human body fluids, was shown to suppress Sendai virus replication in monkey kidney cells, at doses non-toxic to uninfected cells. Dramatic inhibition of virus production could be obtained at doses of delta 12-PGJ2 which did not inhibit cellular or viral protein synthesis, suggesting an effect on virus assembly and/or maturation. At the active concentration, delta 12-PGJ2 caused a decrease in glucosamine incorporation into the virus glycoproteins HN and F, and in at least one host cell polypeptide, while it did not affect most cellular glycoproteins and it induced the glycosylation of a 47-kDa cellular polypeptide. These effects were accompanied by the induction of heat shock protein synthesis, which was found to differ in its specificity and kinetics from induction by prostaglandin A1.

The effect of experimental infection of mouse preimplantation embryos with paramyxovirus Sendai.

Zona-intact and zona-free mouse embryos at the morula stage were exposed to Sendai virus in vitro, and then transferred to the uteri of recipient foster mothers. Both embryos developed into expanded blastocyst stage after 48 hr of culture. Zona-intact embryos were resistant to infection and subsequent transfer resulted in the development of fetuses, indicating that the zona pellucida plays a role of a barrier to virus infection. On the other hand, zona-free embryos were susceptible to infection and only one fetus out of 64 transfers developed to term. Implantation sites were scarcely observed in the uteri of the foster mothers that received zona-free embryos, suggesting that most of the embryos did not develop after embryo transfer. Sendai virus was shed in the culture fluid of the zona-free embryos indicating viral replication in the embryonic cells. By immunofluorescence assay, viral antigens were detected in the embryos, tissues of the fetus and implantation site derived from the zona-free embryos. These findings indicate that replication of Sendai virus in the embryonic cells interfere with early embryonic development and fetal growth of the embryo.

Suppression of virus replication by prostaglandin A is associated with heat shock protein synthesis.

The antiviral action of cyclopentenone prostaglandins (PGs) is generally associated with alterations in the synthesis and/or maturation of specific virus proteins. In particular, inhibition of Sendai virus (SV) replication in African green monkey kidney cells by PGA1 has been shown to be a cell-mediated event, due to alterations in SV protein glycosylation and accompanied by the induction of a cellular polypeptide of Mr 74K. In this report we identify this protein as a heat shock protein (HSP) related to the major 70K HSP group (HSP70). Induction of HSP70 synthesis by PGA1 was found to be dose-dependent, and an accumulation of HSP70 comparable to that occurring after heat shock could be obtained at concentrations of PGA1 that did not inhibit macromolecular synthesis in uninfected cells, but caused a dramatic block of virus replication in SV-infected cells. Induction of HSP70 by PGA1 occurred at the transcriptional level and was not affected by SV infection. HSP70 synthesis was evident between 2 and 3 h after PGA1 treatment, maximal at 12 h and went back to control levels by 26 h after the addition of PGA1, thus preceding virus protein synthesis. Finally, of several PGs tested, only those which possess antiviral activity induced the synthesis of HSP70. These results, together with the observation that suppression of HSP70 synthesis by actinomycin D also abolishes the PGA1-induced alteration of SV glycoproteins, suggest that HSP70 could play a role in the block of virus replication by cyclopentenone PGs.

The infection of mouse preimplantation embryos to sendai virus (parainfluenza I)

To provide information on the susceptibility of mouse embryos to Sendai virus, it was investigated if viral replication occurs in the preimplantation embryo at different stages of development, with or without the zona pellucida (ZP). Mice were induced to superovulate, and embryos were collected on Days 2, 3 and 4 after mating. The ZP was removed by digestion with 0.5% pronase. Embryos were exposed to Sendai virus, washed, and allowed to develop in fresh culture medium. The presence of viral antigen in the embryonic cells was examined by the fluorescent antibody test (FAT). Specific immunofluorescence was demonstrated in the ZP-free morula and ZP-intact blastocyst. However, viral antigen was not detected in the ZP-intact two-cell, four-cell, eight-cell or morula stage embryos. Infected embryos developed normally to expanded blastocysts. These findings show that mouse embryonic cells are permissive hosts to Sendai virus replication and that the ZP played the role of a barrier against the virus.

Inhibition of Virus Protein Glycosylation as the Mechanism of the Antiviral Action of Prostaglandin A in Sendai Virus-infected Cells

Prostaglandin A (PGA) inhibits Sendai virus replication at doses non-toxic to uninfected cells. In this report, the antiviral action of PGA was found to be associated with specific alterations of viral protein synthesis. SDS-PAGE analysis of [35S]methionine-labelled proteins showed that while the non-glycosylated viral polypeptides (P, NP and M) were normally synthesized in PGA1-treated cells, the viral glycoproteins HN and F0 were not detected. Two new polypeptides of Mr respectively 4000 and 1000 lower than the HN and F0 proteins were instead detected. The results suggest that these new polypeptides are defectively glycosylated forms of HN and F0. In fact PGA1 was found selectively to inhibit glucosamine incorporation into Sendai virus-infected cells, but not in uninfected cells. Moreover, in infected cells the inhibition of glucosamine incorporation appeared to be selective towards viral polypeptides. This effect was not due to a decreased uptake of glucosamine from the cells after PGA1 treatment. The results also show that the PGA1-induced alteration of the HN protein caused a loss of its biological function and prevented the insertion of this protein into the cell membrane, thereby blocking virus maturation. Finally, a polypeptide of Mr 74K, the synthesis of which was induced by PGA1, appeared to be a possible mediator of PGA1 antiviral action.

PGJ2, a new antiviral prostaglandin: inhibition of Sendai virus replication and alteration of virus protein synthesis.

Prostaglandin J2 (PGJ2) was found to suppress dramatically Sendai virus replication in African green monkey kidney cells in culture. PGJ2 was not toxic at the active dose to uninfected cells and did not significantly inhibit macromolecular synthesis, but it specifically stimulated the synthesis of a polypeptide of 74,000 mol. wt. In Sendai virus-infected cells, PGJ2 partially inhibited virus protein synthesis and caused an alteration in the mobility of the virus glycoprotein HN in SDS-PAGE, corresponding to a decrease of about 4000 in its mol. wt. We propose that the PGJ2-induced alteration in the molecular structure of the HN protein prevents the insertion of this protein into the cell membrane thereby blocking virus maturation. The alpha,beta-unsaturated carbonyl group in the cyclopentane ring of the PGJ2 molecule may be necessary for antiviral activity.