Growth activation of influenza virus by trypsin and effect of T-705 (favipiravir) on trypsin-optimized growth condition.

Influenza virus is activated by proteolytic cleavage of hemagglutinin by trypsin. After determining the optimal trypsin concentration, intracellular and extracellular influenza A/PR/8/34 (H1N1) and A/Victoria/361/2011 (H3N2) virus productions were compared in cultures treated with T-705 (favipiravir) and GS 4071 (an active form of oseltamivir). Although both drugs efficiently inhibited extracellular viral RNA release in a dose-dependent manner, T-705 inhibited it to the level of the inoculum without trypsin treatment, while GS 4071 inhibited it to a final level 10 times higher than that without trypsin. T-705 inhibited intracellular viral RNA production to the level of input virus in both trypsin-treated and untreated cells. In contrast, GS 4071 dose-dependently inhibited intracellular viral RNA production in cells treated with trypsin but allowed viral RNA synthesis. The level of maximum inhibition by GS 4071was 10 times higher than that of cells without trypsin and 1,000 times greater than the inoculum titer in cells without trypsin. T-705 inhibited both intracellular and extracellular virus production 1,000 and 10 times more strongly, respectively, than GS 4071. T-705 has powerful anti-influenza activity in the absence of trypsin and even in the trypsin-optimized growth condition, suggesting the therapeutic advantage in treatment of influenza complicated with bacterial pneumonia. Keywords: influenza; T-705; Tamiflu; trypsin; bacterial trypsin-like protease.

Persistent Primary Cytomegalovirus Infection After Deceased Donor Kidney Transplant: Ganciclovir Susceptibility of Human Cytomegalovirus With UL97 D605E Mutation: A Case Report.

BACKGROUND: Cytomegalovirus (CMV) could cause rejection in immunocompromised patients during early post-renal transplant stage. The American Transplant Society guidelines recommend prophylactic therapy with ganciclovir (GCV) for 3 to 6 months to prevent CMV infections in adult renal transplant patients. However, there is no recommended CMV treatment regimen for pediatric patients. MAIN FINDINGS: We performed deceased donor kidney transplant from an anti-CMV antibody-positive donor to an anti-CMV antibody-negative 15-year-old female recipient with end-stage renal disease caused by bilateral renal hypoplasia. One month after transplant, increase in positive cells in the CMV antigenemia assay indicated a primary CMV infection in the patient, who immediately received GCV. She was switched to foscarnet after 4 months of anti-CMV therapy because of clinical GCV resistance. CMV was isolated from the peripheral blood mononuclear cells but neutralizing antibody was not detected. Isolated CMV was susceptible to GCV and foscarnet, although it carried the UL97 D605E mutation, assumed to be associated with GCV resistance. CONCLUSIONS: The primary CMV infection presented a phenotypic clinical drug resistance, but all recovered CMV isolates were drug-susceptible even if isolated after prolonged anti-CMV therapy, indicating that immune status was more important for recovery from primary CMV infection than anti-CMV therapy.

T-705 (Favipiravir) suppresses tumor necrosis factor α production in response to influenza virus infection: A beneficial feature of T-705 as an anti-influenza drug.

Influenza virus infection induces the production of various cytokines, which play important roles in the pathogenesis of infection. Among the cytokines induced by influenza, tumor necrosis factor α (TNF-α) production has been correlated with the severity of lung lesions. We investigated the effects of T-705 (Favipiravir, 6-fluoro-3-hydroxy-2-pyrazinecarboxamide) on cytokine production due to influenza virus infection in vitro and in vivo, compared with oseltamivir or GS 4071, an active form of oseltamivir. TNF-α production in mouse macrophage-derived P388D1 cells infected with the influenza virus was lower following treatment with T-705 at concentrations of 0.3 to 100 µg/ml than treatment with GS 4071 at the same concentrations. The effect of treatment with T-705 on the cytokine production induced by the influenza virus infection was investigated in mouse influenza virus infection model. At 48 h post-infection (p.i.) T-705 significantly suppressed the viral load in the lungs and TNF-α production in the airways of infected mice even when viral loads were high. Furthermore, T-705 suppressed only TNF-α production from the early phase of infection. In this study, T-705 showed the antiviral activity of reducing pulmonary viral load compared with oseltamivir, thereby suppressing the TNF-α production. This feature of T-705 is benefit against severe influenza infection.

Evaluation of nanodispersion of iron oxides using various polymers.

In order to create Fe2O3 and Fe2O3·H2O nanoparticles, various polymers were used as dispersing agents, and the resulting effects on the dispersibility and nanoparticulation of the iron oxides were evaluated. It was revealed that not only the solution viscosity but also the molecular length of the polymers and the surface tension of the particles affected the dispersibility of Fe2O3 and Fe2O3·H2O particles. Using the dispersing agents 7.5% hydroxypropylcellulose-SSL, 6.0% Pharmacoat 603, 5.0% and 6.5% Pharmacoat 904 and 7.0% Metolose SM-4, Fe2O3 nanoparticles were successfully fabricated by wet milling using Ultra Apex Mill. Fe2O3·H2O nanoparticles could also be produced using 5.0% hydroxypropylcellulose-SSL and 4.0 and 7.0% Pharmacoat 904. The index for dispersibility developed in this study appears to be an effective indicator of success in fabricating nanoparticles of iron oxides by wet milling using Ultra Apex Mill.

A single mutation responsible for temperature-sensitive entry and assembly defects in the VP1-2 protein of herpes simplex virus.

Evidence for an essential role of the herpes simplex virus type 1 (HSV-1) tegument protein VP1-2 originated from the analysis of the temperature-sensitive (ts) mutant tsB7. At the nonpermissive temperature (NPT), tsB7 capsids accumulate at the nuclear pore, with defective genome release and substantially reduced virus gene expression. We compared the UL36 gene of tsB7 with that of the parental strain HFEM or strain 17 and identified four amino acid substitutions, 1061D → G, 1453Y → H, 2273Y → H, and 2558T → I. We transferred the UL36 gene from tsB7, HFEM, or strain 17 into a KOS background. While KOS recombinants containing the HFEM or strain 17 UL36 gene exhibited no ts defect, recombinants containing the tsB7 UL36 VP1-2 exhibited a 5-log deficiency at the NPT. Incubation at the NPT resulted in little or no virus gene expression, though limited expression could be detected in a highly delayed fashion. Using shift-down regimes, gene expression recovered and recapitulated the time course normally observed, indicating that the initial block was in a reversible pathway. Using temperature shift-up regimes, a second defect later in the replication cycle was also observed in the KOS.ts viruses. We constructed a further series of recombinants which contained subsets of the four substitutions. A virus containing the wild-type (wt) residue at position 1453 and with the other three residues being from tsB7 VP1-2 exhibited wt plaquing efficiency. Conversely, a virus containing the three wt residues but the single Y → H change at position 1453 from tsB7 exhibited a 4- to 5-log drop in plaquing efficiency and was defective at both early and late stages of infection.

Characterization of the herpes simplex virus (HSV)-1 tegument protein VP1-2 during infection with the HSV temperature-sensitive mutant tsB7.

VP1-2, encoded by the UL36 gene of herpes simplex virus (HSV), is a large structural protein, conserved across the family Herpesviridae, that is assembled into the tegument and is essential for virus replication. Current evidence indicates that VP1-2 is a central component in the tegumentation and envelopment processes and that it also possesses important roles in capsid transport and entry. However, any detailed mechanistic understanding of VP1-2 function(s) remains limited. This study characterized the replication of HSV-1 tsB7, a temperature-sensitive mutant restricted at the non-permissive temperature due to a defect in VP1-2 function. A tsB7 virus expressing green fluorescent protein-fused VP16 protein was used to track the accumulation and location of a major tegument protein. After infection at the permissive temperature and shift to the non-permissive temperature, the production of infectious virus ceased. VP1-2 accumulated in altered cytosolic clusters, together with VP16 and other virion proteins. Furthermore, correlating with the results of immunofluorescence, electron microscopy demonstrated abnormal cytosolic capsid clustering and a block in envelopment. As VP1-2 encompasses a ubiquitin-specific protease domain, the occurrence of ubiquitin-conjugated proteins during tsB7 infection was also examined at the non-permissive temperature. A striking overaccumulation was observed of ubiquitin-specific conjugates in cytoplasmic clusters, overlapping and adjacent to the VP1-2 clusters. These results are discussed in relation to the possible functions of VP1-2 in the assembly pathway and the nature of the defect in tsB7.

Heparanase expression and activity influences chondrogenic and osteogenic processes during endochondral bone formation.

Endochondral bone formation is a highly orchestrated process involving coordination among cell-cell, cell-matrix and growth factor signaling that eventually results in the production of mineralized bone from a cartilage template. Chondrogenic and osteogenic differentiation occur in sequence during this process, and the temporospatial patterning clearly requires the activities of heparin binding growth factors and their receptors. Heparanase (HPSE) plays a role in osteogenesis, but the mechanism by which it does so is incompletely understood. We used a combination of ex vivo and in vitro approaches and a well described HPSE inhibitor, PI-88 to study HPSE in endochondral bone formation. In situ hybridization and immunolocalization with HPSE antibodies revealed that HPSE is expressed in the peri-chondrium, peri-osteum, and at the chondro-osseous junction, all sites of key signaling events and tissue morphogenesis. Transcripts encoding Hpse also were observed in the pre-hypertrophic zone. Addition of PI-88 to metatarsals in organ culture reduced growth and suggested that HPSE activity aids the transition from chondrogenic to osteogenic processes in growth of long bones. To study this, we used high density cultures of ATDC5 pre-chondrogenic cells grown under conditions favoring chondrogenesis or osteogenesis. Under chondrogenic conditions, HPSE/Hpse was expressed at high levels during the mid-culture period, at the onset of terminal chondrogenesis. PI-88 addition reduced chondrogenesis and accelerated osteogenesis, including a dramatic up-regulation of osteocalcin levels. In normal growth medium, addition of PI-88 reduced migration of ATDC-5 cells, suggesting that HPSE facilitates cartilage replacement by bone at the chondro-osseous junction by removing the HS component of proteoglycans, such as perlecan/HSPG2, that otherwise prevent osteogenic cells from remodeling hypertrophic cartilage.

Molecular complexity in establishing uterine receptivity and implantation.

Implantation is the process by which the blastocyst comes into intimate physical and physiological contact with the uterine endometrium. This process is governed by an intimate cross-talk between the activated blastocyst and the receptive uterus. An increased understanding of mammalian implantation has been gained through the use of the mouse model. This review highlights the more recently defined signaling cascades involved in this dialogue, focusing specifically on cyclooxygenase-2-derived prostaglandins, endocannabinoids, Wnt proteins, homeotic transcription factors, and immunophilins. Unraveling the nature of these signals and discovering additional molecular cascades may lead to strategies to correct implantation failure and improve pregnancy rates in women.

Herpes simplex virus encodes a virion-associated protein which promotes long cellular processes in over-expressing cells.

BACKGROUND: Herpes simplex virus (HSV) possesses a number of accessory genes which are dispensable for replication in cell culture. A previous study showed that the UL21 gene product of HSV type 1 is a virion component that is not necessary for viral replication. The function of the gene product remains unknown. RESULTS: We found that the HSV-1 UL21 gene product, a capsid-associated tegument protein with an apparent molecular mass of 62 kDa, promotes the outgrowth of long cellular processes when it is over-expressed in non-neural cells. The UL21 protein co-localizes and physically associates with microtubules in the long processes. Analysis using mutant proteins implicates a proline-rich region in promotion of the processes. CONCLUSIONS: The results suggest that the UL21 protein, like tau and other MAPs, promotes the process by directly or indirectly interacting with microtubules and facilitates the intracellular transport of the virus.

Expression of NAD(+)-dependent isocitrate dehydrogenase in brown adipose tissue.

cDNA clones significantly expressed in brown adipose tissue (BAT) but not in white adipose tissue (WAT) of rats were isolated by use of a PCR-select cDNA subtraction kit. Of the isolated clones, structural features of two of them, 2-58 and 2-67, were studied in detail. The results indicated that these clones were cDNAs encoding alpha- and beta-subunits of rat NAD(+)-dependent isocitrate dehydrogenase (NAD(+)-ICDH). Previous biochemical study suggested the importance of NAD(+)-ICDH in metabolism in BAT; however, transcript levels of individual subunits of this enzyme in BAT had never been analyzed. In the present study, using these newly isolated cDNAs, we clearly demonstrate that the expression of three subunits of NAD(+)-ICDH was the most remarkable in BAT among the various tissues analyzed.

Impaired induction of protective immunity by highly virulent herpes simplex virus type 2 in a murine model of genital herpes.

We investigated the immune events in the vagina of mice intravaginally infected with highly virulent herpes simplex virus type 2 (HSV-2) strain 186, and compared them with those induced by HSV type 1 strain KOS, a widely known laboratory strain. Although there was no significant difference between 186 and KOS in the viral replication in the initial stage of infection, inadequate and delayed clearance of virus from the vaginal mucosa was observed in 1 86-challenged mice. The induction of antigen-presenting cells (APC) such as dendritic cells (DC) and macrophages (Mphi) in the vagina was slow in 186-challenged mice, and the number of T cells in the vagina in 186-challenged mice was much lower than that in KOS-challenged mice. Furthermore, the level of IL-12 as well as that of IFN-gamma was significantly lower in 186-challenged mice than in KOS-challenged mice, while the level of IL-4 in 186-challenged mice was higher than that in KOS-challenged mice. On the basis of these observations, we suggest that the weak activation of epithelial cells and the delayed induction of APC by 186-infection may be involved in the inadequate activation of T cells and the ineffective virus clearance from the vaginal mucosa.

Synthesis, subcellular localization and VP16 interaction of the herpes simplex virus type 2 UL46 gene product.

We developed a rabbit polyclonal antiserum reactive against a recombinant 6x His-UL46 fusion protein expressed in Escherichia coli, and using this antiserum identified the UL46 gene product of herpes simplex virus type 2 (HSV-2) to be phosphoproteins with apparent molecular masses of 82-, 84-, and 86-kDa in infected Vero cells. The UL46 protein was produced in the late phase of infection in a manner highly dependent on viral DNA synthesis, and was mainly distributed at the edge of the nucleus in the cytoplasm. Although its kinetics of production and its progress of distribution were different from those of the major tegument protein VP16 (the UL48 gene product or alpha-trans-inducing factor (alphaTIF)), most of the UL46 protein colocalized with VP16 in the late phase of infection, and copurified with it in column chromatography. Moreover, our data showed that the HSV-2 UL46 protein, when coexpressed with VP16, enhanced alpha4 promotor-regulated gene expression in a transient luciferase reporter assay, while the expression of the UL46 protein alone suppressed it.

A protective role of interleukin-15 in a mouse model for systemic infection with herpes simplex virus.

To define the role of cytokine binding to the IL-2/IL-15R beta chain in protective immunity against systemic infection with herpes simplex virus type 2 (HSV-2), IL-2/IL-15 receptor(R)beta knock-out mice were inoculated intraperitoneally with HSV-2 strain 186. IL-2/IL-15R beta-deficient mice were susceptible to systemic HSV-2 infection compared with their heterozygous littermates. The emergence of natural killer (NK) cells was impaired in IL-2/IL-15R beta knock-out mice, but CD4(+) T cell receptor (TCR) alpha beta(+) T cells were normally detected in the peritoneal cavity after infection with HSV-2. However, the generation of HSV-2-specific CD4(+) T helper (Th) 1 cells producing interferon-gamma was impaired in IL-2/IL-15R beta knock-out mice following HSV-2 infection. The serum IL-15 level in control mice was increased in the early stage after HSV-2 infection but was not detectable in IL-2/IL-15R beta knock-out mice. In vivo administration of recombinant IL-15 protected normal mice from HSV-2-induced lethality, accompanied by increases in numbers of NK cells and HSV-2-specific Th1 cells. Taken together, these results suggest that IL-15, using the IL-2/IL15R beta chain, plays an important role in mounting protective immunity during the course of systemic HSV-2 infection.

Memory-type CD8+ T cells protect IL-2 receptor alpha-deficient mice from systemic infection with herpes simplex virus type 2.

IL-2Ralpha-deficient (IL-2Ralpha(-/-)) mice exhibit an impaired activation-induced cell death for T cells and develop abnormal T cell activation with age. In our study, we found that IL-2Ralpha(-/-) mice at the age of 5 wk contained an increased number of CD44(+)CD69(-)CD8(+) T cells in lymph nodes, which expressed a high intensity of IL-2Rbeta and vigorously proliferated in response to a high dose of IL-15 or IL-2. The T cells produced a large amount of IFN-gamma in response to IL-15 plus IL-12 in a TCR-independent bystander manner. When IL-2Ralpha(-/-) mice were inoculated i.p. with HSV type 2 (HSV-2) 186 strain, they showed resistance to the infection accompanied by an increased level of serum IL-15. The depletion of CD8(+) T cells by in vivo administration of anti-CD8 mAb rendered IL-2Ralpha(-/-) mice susceptible to HSV-2-induced lethality. These results suggest that memory-type CD8(+) T cells play a novel role in the protection against HSV-2 infection in IL-2Ralpha(-/-) mice.

The UL34 gene product of herpes simplex virus type 2 is a tail-anchored type II membrane protein that is significant for virus envelopment.

The UL34 gene of herpes simplex virus type 2 (HSV-2) is highly conserved in the herpesvirus family. The UL34 gene product was identified In lysates of HSV-2-infected cells as protein species with molecular masses of 31 and 32.5 kDa, the latter being a phosphorylated product. Synthesis of these proteins occurred at late times post-infection and was highly dependent on viral DNA synthesis. Immunofluorescence assays revealed that the UL34 protein was localized in the cytoplasm in a continuous net-like structure, closely resembling the staining pattern of the endoplasmic reticulum (ER), in both HSV-2-infected cells and in cells transiently expressing UL34 protein. Deletion mutant analysis showed that this colocalization required the C terminus of the UL34 protein. The UL34 protein associated with virions but not with A, B or C capsids. We treated virions, HSV-2-infected cells and cells expressing the UL34 protein with a protease in order to examine the topology of the UL34 protein. In addition, we constructed UL34 deletion mutant proteins and examined their intracellular localization. Our data strongly support the hypothesis that the UL34 protein is inserted into the viral envelope as a tail-anchored type II membrane protein and is significant for virus envelopment.

The role of the UL41 gene of herpes simplex virus type 1 in evasion of non-specific host defence mechanisms during primary infection.

The UL41 gene product (vhs) of herpes simplex virus (HSV) is packaged in the virion, and mediates host protein synthesis shutoff at the early stage of the virus replication cycle. In order to clarify the role of vhs in virus replication and virulence, we isolated a completely UL41-deficient mutant (the VRDelta41 strain) and its revertant (the VRDelta41R strain). In the mouse encephalitis model, the replication of strain VRDelta41 was inhibited after 2 days post-infection, resulting in low virulence, by gamma-ray-sensitive cells such as lymphocytes and/or neutrophils. The result suggested that some cytokines, produced in VRDelta41-inoculated brains, activate and induce the migration of gamma-ray-sensitive cells to the infection site. Therefore, cytokines produced by HSV-1-infected human cells were screened, and potent inductions of interleukin (IL)-1beta, IL-8 and macrophage inflammatory protein-1alpha by VRDelta41 infection were observed. Moreover, the VRDelta41 strain showed 20- and 5-fold higher sensitivity to interferon-alpha and -beta compared to the wild-type strain, respectively. These results indicate that one important role of vhs in vivo is evasion from non-specific host defence mechanisms during primary infection through suppression of cytokine production in HSV-infected cells and reduction of the anti-HSV activity of interferon-alpha and -beta.

[The functions of alpha herpesvirus gene products].

Herpes simplex virus type 1(HSV-1), type 2(HSV-2) and varicella-zoster virus (VZV) belong to alphaherpesvirus family. These herpesviruses have large DNA genomes which contain at least 69 genes. These viral genes are divided into two groups based on whether they are essential or dispensable for virus growth in cell culture. The essential gene products include transcriptional factor, viral DNA replication, DNA cleavage/packaging and virion components, while the dispensable gene products include deoxyribonucleotides metabolism, protein kinases, tegument proteins. This article briefly reviews the functions of alphaherpesvirus gene products, particularly roles of viral encoded protein kinases, tegument proteins and host immune evasion.

Specific elevation of transcript levels of particular protein subtypes induced in brown adipose tissue by cold exposure.

To understand the difference in metabolic flow in rat brown adipose tissue (BAT) from that in white adipose tissue (WAT) at the molecular level, we examined the steady-state transcript levels of 39 proteins in both adipose tissues with and without cold exposure by Northern blot analysis. In addition to the transcript levels of uncoupling protein isoforms, those of proteins involved in the transport and catabolism of fatty acids and glucose in BAT were elevated by cold exposure, suggesting the stimulation of utilization of fatty acids and glucose as fuels in BAT. As to these changes, the muscle-type subtypes were remarkable; and therefore, they were suggested to be responsible for the cold exposure-induced acceleration of energy expenditure in BAT. Furthermore, of the isoforms of beta-adrenergic receptor (beta-AR) and CCAAT enhancer binding protein (C/EBP), transcript levels of beta(1)-AR and C/EBPbeta in BAT were increased by the cold exposure. Possible roles of these proteins in energy metabolism in BAT were discussed.

Mitochondrial distribution and function in herpes simplex virus-infected cells.

In this study, mitochondria migrated to a perinuclear region in the cytoplasm in herpes simplex virus (HSV)-infected cells. HSV infection did not promote the expression of cytochrome c oxidase subunit 2 but did promote that of stress-responsive HSP60, both of which are known to be components of mitochondria. The levels of cellular ATP and lactate and mitochondrial membrane potential were maintained for at least 6 h but decreased at the late stage of infection. It was also found that the UL41 and UL46 gene products, both of which are known to be tegument proteins, accumulated in the perinuclear region. The clustering of mitochondria and the accumulation of tegument proteins were completely blocked by the addition of nocodazole and vinblastine. These results suggest that mitochondria respond to the stimulation of HSV infection, migrating with tegument proteins along microtubules to a site around the nucleus, and maintain function until at least the middle stage of infection.

Expression of herpes simplex virus type 2 US3 affects the Cdc42/Rac pathway and attenuates c-Jun N-terminal kinase activation.

BACKGROUND: Although the US3 gene product of herpes simplex virus (HSV) has been identified as a serine/threonine protein kinase (PK), the functions are poorly understood. RESULTS: We found that US3 PK of HSV-2 induced disruption of actin filaments, cell rounding and accumulation of binucleate cells in HEp2 cells. Cell rounding was abrogated by expression of either kinase-dead forms of US3 PK or a mutant protein lacking the acidic cluster in the kinase regulatory domain. Co-expression of dominant active forms of Cdc42/Rac inhibited cell rounding, suggesting that a signal transduction pathway involving Cdc42/Rac may play a role in the morphological changes induced by the kinase. Cdc42 and Rac, members of the Rho family of small GTPases, function as molecular switches changing actin cytoskeletal organization, influencing transcription and controlling apoptotic cell death. By computed homology search, we noticed significant similarities between US3 PK and p21-activated kinase (PAK), which is activated by the Cdc42 or Rac. We also found that the expression of US3 suppressed the activation of c-Jun N-terminal kinase (JNK), a kinase that is downstream of PAK. CONCLUSIONS: These observations suggest that the US3 PK affects the Cdc42/Rac pathway and can act as an upstream suppressor of JNK in the stress-activated signalling pathway.