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PubMed1982

Infectious cell entry mechanism of influenza virus.

Interaction between influenza virus WSN strain and MDCK cells was studied by using spin-labeled phospholipids and electron microscopy. Envelope fusion was negligibly small at neutral pH but greatly activated in acidic media in a narrow pH range around 5.0. The half-time was less than 1 min at 37 degrees C at pH 5.0. Virus binding was almost independent of the pH. Endocytosis occurred with a half-time of about 7 min at 37 degrees C at neutral pH, and about 50% of the initially bound virus was internalized after 1 h. Electron micrographs showed binding of virus particles in coated pits in the microvillous surface of plasma membrane and endocytosis into coated vesicles. Chloroquine inhibited virus replication. The inhibition occurred when the drug was added not later than 10 min after inoculation. Chloroquine caused an increase in the lysosomal pH 4.9 to 6.1. The drug did not affect virus binding, endocytosis, or envelope fusion at pH 5.0. Electron micrographs showed many virus particles remaining trapped inside vacuoles even after 30 min at 37 degrees C in the presence of drug, in contrast to only a few particles after 10 min in vacuoles and secondary lysosomes in its absence. Virus replication in an artificial condition, i.e., brief exposure of the inoculum to acidic medium followed by incubation in neutral pH in the presence of chloroquine, was also observed. These results are discussed to provide a strong support for the infection mechanism of influenza virus proposed previously: virus uptake by endocytosis, fusion of the endocytosed vesicles with lysosome, and fusion of the virus envelope with the surrounding vesicle membrane in the secondary lysosome because of the low pH. This allows the viral genome to enter the target cell cytoplasm.

Animals↗

PubMed1995

Unique features of internal initiation of hepatitis C virus RNA translation.

The question of whether hepatitis C virus (HCV) RNA is translated by a mechanism of internal ribosome entry has been examined by testing whether insertion of HCV sequences between the two cistrons of a dicistronic mRNA promotes translation of the downstream cistron in rabbit reticulocyte lysates. Deletion analysis showed that efficient internal initiation required a segment of the HCV genome extending from about nucleotides 40-370 and that deletions from the 3'-end of this element were highly deleterious. As the authentic initiation codon for HCV polyprotein synthesis is at nucleotide 342, this demonstrates that, besides 5'-UTR sequences, a short length of HCV coding sequences is required for internal initiation. This finding was confirmed in transfection assays of BT7-H cells and was shown to be independent of the nature of the downstream reporter cistron. The strong requirement for coding sequences is in sharp contrast to internal initiation of picornavirus RNA translation. As a probable correlate with this, it was also found that the efficiency of internal initiation was only marginally compromised when the authentic initiation codon was mutated to a non-AUG codon, again in sharp contrast with the picornaviruses. The finding that coding sequences are required for internal initiation has important implications for the design of experiments to test for internal initiation of translation of cellular mRNAs.

Animals↗

PubMed2003

Brain-derived neurotrophic factor inhibits human immunodeficiency virus-1/gp120-mediated cerebellar granule cell death by preventing gp120 internalization.

The human immunodeficiency virus type 1 (HIV-1) envelope protein gp120 has been implicated in the pathogenesis of HIV-1 dementia. Thus, inhibition of gp120 activity could reduce HIV toxicity in the brain. We have used primary cultures of rat cerebellar granule cells to examine mechanisms whereby gp120 causes cell death and to characterize neuroprotective agents. gp120 induced a time- and concentration-dependent apoptotic cell death, which was caspase-3-mediated but caspase-1 independent, and was totally blocked by the irreversible caspase-3-like protease inhibitor N-acetyl-Asp-Glu-Val-Asp-chloromethylketone. Caspase-3 activation was observed only in neurons that internalize gp120, indicating that internalization is key to gp120 toxicity. Because brain-derived neurotrophic factor (BDNF) prevents caspase-3-mediated neuronal cell death, we examined whether BDNF could prevent gp120-mediated apoptosis. Preincubation of neurons with BDNF before the addition of gp120 reduced caspase-3 activation, and consequently rescued 80% of neurons from apoptosis. Most importantly, BDNF reduced the levels of CXC chemokine receptor-4 (CXCR4), a receptor that mediates HIV-1 gp120-induced apoptosis. This effect correlated with the ability of BDNF to reduce gp120 internalization and apoptosis. Moreover, BDNF blocked the neurotoxic effect of stromal-derived factor-1alpha, a natural ligand for CXCR4, further establishing a correlation between neuroprotection and downregulation of CXCR4. We propose that BDNF may be a valid therapy to slow down the progression of HIV/gp120-mediated neurotoxicity.

Animals↗

PubMed1996

Translation initiation in GB viruses A and C: evidence for internal ribosome entry and implications for genome organization.

GB viruses A and C (GBV-A and GBV-C) are two recently described RNA viruses which appear to be members of the Flaviviridae. Although both viruses appear to contain long 5' nontranslated regions, the sites of polyprotein initiation and the presence of core-like proteins remain to be determined. Translation studies were undertaken to determine the mechanism and sites of polyprotein initiation in GBV-A and GBV-C. Rabbit reticulocyte lysates programmed with monocistronic RNAs containing 5' ends of GBV-A or GBV-C fused in-frame with the chloramphenicol acetyltransferase (CAT) open reading frame generated GBV-CAT fusion proteins in vitro. Site-specific mutagenesis and N-terminal sequencing located the sites of translation initiation immediately upstream of the putative signal sequence for the GBV E1 envelope glycoproteins. Efficient translation of the monocistronic GBV-CAT RNAs required the inclusion of GBV coding sequences. This, coupled with the presence of at least 523 nucleotides of 5' nontranslated RNA containing multiple AUG codons, suggests that translation initiation of these RNAs did not utilize a ribosome scanning mechanism. Translation of bicistronic RNAs containing 5' nontranslated sequences within the intercistronic space was consistent with the presence of a weakly active internal ribosome entry site in both GBV-A and GBV-C. Secondary structure predictions indicate that the 5' ends of these viruses assume similar complex structures distinct from those identified in the internal ribosome entry site-containing picornaviruses, pestiviruses, and hepatitis C viruses. The data indicate that GBV-A and GBV-C are unique members of the Flaviviridae that do not contain core-like proteins at the N termini of their putative polyproteins.

Amino Acid Sequence↗

PubMed1996

Structural analysis of the interaction of the pyrimidine tract-binding protein with the internal ribosomal entry site of encephalomyocarditis virus and foot-and-mouth disease virus RNAs.

Initiation of translation of a subset of eukaryotic mRNAs results from internal ribosomal entry. This process is exemplified by encephalomyocarditis virus (EMCV), which contains an internal ribosomal entry site (IRES) within its 5' nontranslated region that is approximately 450-nt long and consists of a series of stem-loops designated H-L. We have previously identified a cellular 58-kDa polypeptide that binds specifically to this IRES and that is implicated in its function as the pyrimidine tract-binding protein PTB. We have now mapped PTB binding sites directly on the IRES elements of EMCV and the related foot-and-mouth disease virus (FMDV) using structure-specific enzymatic probes and base-specific chemical probes. PTB bound to six sites on the EMCV IRES: site 1 (UCUU401) is upstream of domain H, site 2 is the basal helix of domain H (nt 407-410 and 440-443), site 3 (UCUUU423) is the apical loop of domain H, site 4 is the apical helix and adjacent internal bulged loop of domain K, site 5 (CUUUA750) is the apical loop of domain K, and site 6 (CCUUU815) is downstream of domain L. PTB bound to sites on the FMDV IRES that correspond precisely to EMCV sites 3, 5, and 6. These sites have the consensus sequence CUUU and form two groups that are located near to the 5' and 3' borders of these IRES elements. Their position, and the effects of mutation of them on IRES function are consistent with PTB's role in IRES-mediated initiation being to bind to multiple sites in the IRES, thereby stabilizing a specific active conformation.

Aphthovirus↗

PubMed2006

Long-distance RNA-RNA interactions between terminal elements and the same subset of internal elements on the potato virus X genome mediate minus- and plus-strand RNA synthesis.

Potexvirus genomes contain conserved terminal elements that are complementary to multiple internal octanucleotide elements. Both local sequences and structures at the 5' terminus and long-distance interactions between this region and internal elements are important for accumulation of potato virus X (PVX) plus-strand RNA in vivo. In this study, the role of the conserved hexanucleotide motif within SL3 of the 3' NTR and internal conserved octanucleotide elements in minus-strand RNA synthesis was analyzed using both a template-dependent, PVX RNA-dependent RNA polymerase (RdRp) extract and a protoplast replication system. Template analyses in vitro indicated that 3' terminal templates of 850 nucleotides (nt), but not 200 nt, supported efficient, minus-strand RNA synthesis. Mutational analyses of the longer templates indicated that optimal transcription requires the hexanucleotide motif in SL3 within the 3' NTR and the complementary CP octanucleotide element 747 nt upstream. Additional experiments to disrupt interactions between one or more internal conserved elements and the 3' hexanucleotide element showed that long-distance interactions were necessary for minus-strand RNA synthesis both in vitro and in vivo. Additionally, multiple internal octanucleotide elements could serve as pairing partners with the hexanucleotide element in vivo. These cis-acting elements and interactions correlate in several ways to those previously observed for plus-strand RNA accumulation in vivo, suggesting that dynamic interactions between elements at both termini and the same subset of internal octanucleotide elements are required for both minus- and plus-strand RNA synthesis and potentially other aspects of PVX replication.

Base Sequence↗

PubMed1997

Genetic analysis of internal ribosomal entry site on hepatitis C virus RNA: implication for involvement of the highly ordered structure and cell type-specific transacting factors.

Hepatitis C virus (HCV) carries an internal ribosomal entry site (IRES) within the 5' portion of the RNA. To identify structures that influence efficiency of the translation initiation, relative activities of modified IRESs were examined by using engineered bicistronic mRNAs, between the two cistrons of which various mutant IRESs were inserted. An IRES derived from genotype 2b is at least two times more efficient than one from genotype 1b in cultured cells. Activity ratios of genotype 2b IRES to 1b IRES differ in magnification among cultured cells, suggesting the difference in assortment of IRES-related host factors among individual cell types. Recombinant IRESs between the genotypes show similar or higher activities compared with 2b IRES in cell-free systems and show intermediate activities in cultured cells. Patterns of relative activities of those IRESs indicate that the IRES activity is not regulated by defined structure(s), although a cluster of different nucleotides is observed in the genome region of nucleotides 176-224 between the two alleles. The results suggest that a highly ordered structure formed by the entire 5' portion of the RNA is important for the IRES activity. The 5' border of HCV IRES was examined by using a series of deletion RNAs in various systems. The results strongly suggest that the border resides between nucleotide positions 28 and 45. Patterns of relative activities of the deletion IRESs differ in translation systems or cell types. These results imply that interactions of HCV RNA with the related transacting factor(s) may differ in the translation systems or cell types.

Base Sequence↗

PubMed1972

Structure of the ribonucleoprotein of influenza virus.

The ribonucleoprotein (RNP) internal components of influenza virus were separated into distinct size classes by sedimentation in glycerol gradients and examined by electron microscopy by using positive staining with uranyl acetate. The large RNP have a peak in length distribution at 90 to 110 nm, the medium, at 60 to 90 nm, and the small, at 30 to 50 nm. These lengths can be correlated with the estimated molecular weights of the ribonucleic acids contained in the various RNP size classes. The RNP structure appears to consist of a strand which is folded back on itself and coiled in a regular double-helical arrangement.

Animals↗

PubMedDate not supplied

[Isolation of the internal proteins of the influenza virus by preparative polyacrylamide gel electrophoresis for obtaining monospecific antisera].

The principal possibility of isolation of internal proteins (M and NP) of influenza type A (H1N1 and H3N2) and B viruses by SDS-PAG preparative electrophoresis and preparation of monospecific antisera to these proteins was demonstrated. The resulting preparations may be used for testing the biological objects by enzymeimmunoassay.

Animals↗

PubMed1997

Complementation of defective picornavirus internal ribosome entry site (IRES) elements by the coexpression of fragments of the IRES.

Mutant forms of the encephalomyocarditis virus (EMCV) internal ribosome entry site (IRES) have been produced and shown to be severely defective in directing internal initiation of protein synthesis within cells using the vaccinia/T7 RNA polymerase system. Mutants in different regions of the IRES were complemented in trans by coexpression of the intact EMCV IRES but not by coexpression of the related IRES elements from Theiler's murine encephalomyelitis virus (another cardiovirus) or from foot-and-mouth disease virus. Distinct, truncated regions of the EMCV IRES, insufficient to direct internal initiation, were also shown to complement defective EMCV IRES elements. It was necessary for the complementing molecule, whether truncated or full length, to be expressed in the positive sense orientation. RT-PCR analysis provided no support for the idea that any recombination event was responsible for the complementation. The data suggest that multiple activities are performed by distinct functional entities within the IRES in the process of internal initiation of protein synthesis. At least some of these different functions may be achieved by different molecules acting in trans.

Cell Line↗

PubMed2003

Novel partner proteins of adenovirus penton.

Each of the 12 vertices of the adenovirus virion is made of penton, the complex of two oligomeric proteins: a pentameric penton base anchored in the capsid and an antenna-like trimeric fiber extending outwards. Adenovirus penton plays an essential role in the infection of host cells because it is indispensable for virus attachment and internalization. The initial interactions of penton with the primary and secondary receptors are well described. In contrast with that, the role of the penton components downstream of the initial cell contact is not known. This work shows for the first time that two adenovirus structural proteins, fiber and base, are able to interact intimately with different classes of cellular targets. In the case of penton base, a protein responsible for virus internalization, the partners include three ubiquitin-protein ligases that are involved in protein turnover, cell cycle control and endocytosis. Another base protein partner, BAG3, is involved in controlling Hsc70 chaperone activity. Virus attachment protein, fiber, interacts with many different partners, some of them involved in signal transduction and cell growth. Further work will illustrate the implications of these interactions for both the viral and cellular life cycles.

Adenoviridae↗

PubMed2001

Efficacy of zanamivir against avian influenza A viruses that possess genes encoding H5N1 internal proteins and are pathogenic in mammals.

In 1997, an avian H5N1 influenza virus, A/Hong Kong/156/97 (A/HK/156/97), caused six deaths in Hong Kong, and in 1999, an avian H9N2 influenza virus infected two children in Hong Kong. These viruses and a third avian virus [A/Teal/HK/W312/97 (H6N1)] have six highly related genes encoding internal proteins. Additionally, A/Chicken/HK/G9/97 (H9N2) virus has PB1 and PB2 genes that are highly related to those of A/HK/156/97 (H5N1), A/Teal/HK/W312/97 (H6N1), and A/Quail/HK/G1/97 (H9N2) viruses. Because of their similarities with the H5N1 virus, these H6N1 and H9N2 viruses may have the potential for interspecies transmission. We demonstrate that these H6N1 and H9N2 viruses are pathogenic in mice but that their pathogenicities are less than that of A/HK/156/97 (H5N1). Unadapted virus replicated in lungs, but only A/HK/156/97 (H5N1) was found in the brain. After three passages (P3) in mouse lungs, the pathogenicity of the viruses increased, with both A/Teal/HK/W312/97 (H6N1) (P3) and A/Quail/HK/G1/97 (H9N2) (P3) viruses being found in the brain. The neuraminidase inhibitor zanamivir inhibited viral replication in Madin-Darby canine kidney cells in virus yield assays (50% effective concentration, 8.5 to 14.0 microM) and inhibited viral neuraminidase activity (50% inhibitory concentration, 5 to 10 nM). Twice daily intranasal administration of zanamivir (50 and 100 mg/kg of body weight) completely protected infected mice from death. At a dose of 10 mg/kg, zanamivir completely protected mice from infection with H9N2 viruses and increased the mean survival day and the number of survivors infected with H6N1 and H5N1 viruses. Zanamivir, at all doses tested, significantly reduced the virus titers in the lungs and completely blocked the spread of virus to the brain. Thus, zanamivir is efficacious in treating avian influenza viruses that can be transmitted to mammals.

Administration, Intranasal↗

PubMed2006

Prediction model of hepatocarcinogenesis for patients with hepatitis C virus-related cirrhosis. Validation with internal and external cohorts.

BACKGROUND/AIMS: To estimate hepatocarcinogenesis rates in patients with hepatitis C virus (HCV)-related cirrhosis, an accurate prediction table was created. METHODS: A total of 183 patients between 1974 and 1990 were assessed for carcinogenesis rate and risk factors. Predicted carcinogenesis rates were validated using a cohort from the same hospital between 1991 and 2003 (n=302) and an external cohort from Tokyo National Hospital between 1975 and 2002 (n=205). RESULTS: The carcinogenesis rates in the primary cohort were 28.9% at the 5th year and 54.0% at the 10th year. A proportional hazard model identified alpha-fetoprotein (>or=20 ng/ml, hazard ratio 2.30, 95% confidence interval 1.55-3.42), age (>or=55 years, 2.02, 95% CI 1.32-3.08), gender (male, 1.58, 95% CI 1.05-2.38), and platelet count (<100,000 counts/mm3, 1.54, 95% CI 1.04-2.28) as independently associated with carcinogenesis. When carcinogenesis rates were simulated in 16 conditions according to four binary variables, the 5th- and 10th-year rates varied from 9 to 64%, and 21-93%, respectively. Actual carcinogenesis rates in the internal and external validation cohorts were similar to those of the simulated curves. CONCLUSIONS: Simulated carcinogenesis rates were applicable to patients with HCV-related cirrhosis. Since, hepatocarcinogenesis rates markedly varied among patients depending on background features, we should consider stratifying them for cancer screening and cancer prevention programs.

Adult↗

PubMed1998

Turnover of thrombomodulin at the cell surface occurs at a similar rate to receptors that are not actively internalized.

Loss of thrombomodulin (TM) from the endothelial cell surface is thought to contribute to thrombosis encountered in malignant and inflammatory disease. Internalization or endocytosis of TM from the cell surface has been proposed to be one mechanism by which TM levels are reduced. Previous work has led to a view that TM is rapidly internalized using a non-conventional pathway, under the direction of a signal motif in its extracellular domain. This is contrary to the clathrin-dependent route taken by most rapidly internalized receptors that possess signals in their cytoplasmic domains. However the internalization rate of TM has never been directly compared to known internalizing or non-internalizing molecules. Here we show that the rate and amount of uptake of TM (5-10% per hour) is indistinguishable from the non-internalizing influenza virus haemagglutinin (HA), and is considerably less than the actively internalizing low density lipoprotein receptor, which reaches a steady state of approximately 50% internalized in 15 min. The low level rate of TM and HA internalization observed is comparable to the rate of normal plasma membrane turnover. Furthermore, this uptake of TM does not require a signal in its extracellular domain.

Amino Acid Sequence↗

PubMed1984

Determination of the internal volume of reconstituted sendai virus envelopes by quenching of calcein fluorescence.

Sendai virus envelopes (SVE) were isolated from Sendai virus particles by Triton X-100 solubilization and ultracentrifugation. The envelopes were reconstituted in the presence of the fluorescent dye calcein by gradual removal of the detergent with Bio-beads SM-2. The internal volume of reconstituted Sendai virus envelopes (RSVE) was determined by quenching the fluorescence of calcein with cobalt (II) ions. The internal volume of RSVE was found to be proportional to the initial SVE protein concentration in the reconstitution mixture, reaching about 18% of the total volume with 5.6 mg of SVE protein per ml. When radiolabelled cloned Epstein-Barr virus DNA fragment was included in the reconstitution mixture, the proportion of DNA associated with the vesicles much exceeded the trapping volume, indicating adsorption of DNA to the internal surface of RSVE. These determinations will allow optimization of the use of RSVE as gene-transfer vehicles.

DNA, Viral↗

PubMed2004

[Isolation of influenza A H1N2 virus from a returning traveller at Nagoya International Airport].

A reassortant influenza A H1N2 virus was isolated from a returning traveller arriving at Nagoya International Airport, Japan from Indonesia in May, 2002. A Hemagglutination inhibition test revealed that the virus was similar to a vaccine strain of A/NewCaledonia/20/99. A phylogenetic analysis demonstrated that the virus forms a cluster with other influenza A H1N2 viruses isolated in other countries. The reassortment event was theoretically assumed to have occurred between the 1999/2000 and 2000/2001 influenza seasons. Neither A H1N2 nor A H3N1 virus was detected from 256 isolates of AH1 or 177 of AH3 influenza viruses isolated in Aichi Prefecture, Japan between the 1999/2000 and 2001/2002 influenza seasons. This finding suggests the importance of influenza surveillance at an airport quarantine office to detect promptly a novel influenza virus penetrating to Japan.

Aviation↗

PubMed1997

Deletion analysis of both the long terminal repeat and the internal promoters of the human foamy virus.

Deletion analyses of the long terminal repeat (LTR) and internal promoters (IP) of human foamy virus (HFV) showed that a negative acting element resides in the U5 region of the 5' LTR reducing reporter gene expression tenfold. The basal activity of the IP was higher than that obtained with LTR promoter constructs and strongly elevated in permissive BHK-21 cells whereas semi-permissive COS-7 cells showed low basal activity. Since the basal activity of the IP is critical for initiating HFV gene expression by providing Bel 1 transactivator early after infection, the basal activity of the IP may be the crucial factor that contributes to whether cells are permissive for HFV infection or not. Deletion mutagenesis allowed to define the minimal IP region. A region strongly transactivated by Bel 1 extends from -136 to +58 relative to the cap site of the IP. The major Bel 1 response element of the IP required for transactivation is located upstream of the cap site between -136 and -88 relative to the internal cap site. A DNA fragment reported to be protected by recombinant Bel 1 was deleted with marginal reduction of Bel 1 transactivation. HFV gene expression directed by the IP and LTR promoters is thus multiply regulated by positive and negative acting response elements in cis and their binding partners in trans.

Animals↗

PubMed2006

A sensitive one-step real-time PCR for detection of avian influenza viruses using a MGB probe and an internal positive control.

BACKGROUND: Avian influenza viruses (AIVs) are endemic in wild birds and their introduction and conversion to highly pathogenic avian influenza virus in domestic poultry is a cause of serious economic losses as well as a risk for potential transmission to humans. The ability to rapidly recognise AIVs in biological specimens is critical for limiting further spread of the disease in poultry. The advent of molecular methods such as real time polymerase chain reaction has allowed improvement of detection methods currently used in laboratories, although not all of these methods include an Internal Positive Control (IPC) to monitor for false negative results. Therefore we developed a one-step reverse transcription real time PCR (RRT-PCR) with a Minor Groove Binder (MGB) probe for the detection of different subtypes of AIVs. This technique also includes an IPC. METHODS: RRT-PCR was developed using an improved TaqMan technology with a MGB probe to detect AI from reference viruses. Primers and probe were designed based on the matrix gene sequences from most animal and human A influenza virus subtypes. The specificity of RRT-PCR was assessed by detecting influenza A virus isolates belonging to subtypes from H1-H13 isolated in avian, human, swine and equine hosts. The analytical sensitivity of the RRT-PCR assay was determined using serial dilutions of in vitro transcribed matrix gene RNA. The use of a rodent RNA as an IPC in order not to reduce the efficiency of the assay was adopted. RESULTS: The RRT-PCR assay is capable to detect all tested influenza A viruses. The detection limit of the assay was shown to be between 5 and 50 RNA copies per reaction and the standard curve demonstrated a linear range from 5 to 5 x 108 copies as well as excellent reproducibility. The analytical sensitivity of the assay is 10-100 times higher than conventional RT-PCR. CONCLUSION: The high sensitivity, rapidity, reproducibility and specificity of the AIV RRT-PCR with the use of IPC to monitor for false negative results can make this method suitable for diagnosis and for the evaluation of viral load in field specimens.

Animals↗