Early Stage Of Viral Replication Research Articles

Mode of antiviral action of silver nanoparticles against HIV-1

BackgroundSilver nanoparticles have proven to exert antiviral activity against HIV-1 at non-cytotoxic concentrations, but the mechanism underlying their HIV-inhibitory activity has not been not fully elucidated. In this study, silver nanoparticles are evaluated to elucidate their mode of antiviral action against HIV-1 using a panel of different in vitro assays.ResultsOur data suggest that silver nanoparticles exert anti-HIV activity at an early stage of viral replication, most likely as a virucidal agent or as an inhibitor of viral entry. Silver nanoparticles bind to gp120 in a manner that prevents CD4-dependent virion binding, fusion, and infectivity, acting as an effective virucidal agent against cell-free virus (laboratory strains, clinical isolates, T and M tropic strains, and resistant strains) and cell-associated virus. Besides, silver nanoparticles inhibit post-entry stages of the HIV-1 life cycle.ConclusionsThese properties make them a broad-spectrum agent not prone to inducing resistance that could be used preventively against a wide variety of circulating HIV-1 strains.

A Single Amino Acid Substitution in HIV-1 Reverse Transcriptase Significantly Reduces Virion Release

HIV-1 protease (PR) mediates the proteolytic processing of virus particles during or after virus budding. PR activation is thought to be triggered by appropriate Gag-Pol/Gag-Pol interaction; factors affecting this interaction either enhance or reduce PR-mediated cleavage efficiency, resulting in markedly reduced virion production or the release of inadequately processed virions. We previously showed that a Gag-Pol deletion mutation involving the reverse transcriptase tryptophan (Trp) repeat motif markedly impairs PR-mediated virus maturation and that an alanine substitution at W401 (W401A) or at both W401 and W402 (W401A/W402A) partially or almost completely negates the enhancement effect of efavirenz (a nonnucleoside reverse transcriptase inhibitor) on PR-mediated virus processing efficiency. These data suggest that the Trp repeat motif may contribute to the PR activation process. Here we demonstrate that due to enhanced Gag cleavage efficiency, W402 alanine or leucine substitution significantly reduces virus production. However, W402 replacement with phenylalanine does not significantly affect virus particle assembly or processing, but it does markedly impair viral infectivity in a single-cycle infection assay. Our results demonstrate that a single amino acid substitution at HIV-1 RT can radically affect virus assembly by enhancing Gag cleavage efficiency, suggesting that in addition to contributing to RT biological function during the early stages of virus replication, the HIV-1 RT tryptophan repeat motif in a Gag-Pol context may play an important role in suppressing the premature activation of PR during late-stage virus replication.

Effect of the fluorescent tracer Fast Blue on the Pseudorabies virus infection

Event Abstract Back to Event Effect of the fluorescent tracer Fast Blue on the Pseudorabies virus infection Gabor Marton1, Dora Tombacz2, Judit Toth2, Andrea Szabo1, Zsolt Boldogkoi2 and Antal Nógrádi1* 1 Department of Ophthalmology, University of Szeged, Hungary 2 Department of Medical Biology, University of Szeged, Hungary The Pseudorabies virus (Prv) inoculated into peripheral tissue infects neurons and spreads through synaptically linked cells. Retrograde fluorescent tracers can be used for the identification of the infected first-order neurons. The question can be raised whether these fluorescent tracers have an effect on the Prv infection. In this study we applied the fluorescent tracer Fast Blue to the cut sciatic nerve 24 or 48 hours after inoculation of Prv (Bartha strain) into hindlimb muscles of SD rats. The virus infection was studied also in the trigeminal tract: the Kaplan strain of the virus was inoculated into the cornea of SD rats and after 2 or 6 hours FB was applied into the cornea. In cultured mouse neuroblastoma cells the expression of different viral kinetic genes was investigated in the presence of FB. The application of the FB 24 hours after the PrV injection into the hindlimb muscles prevented the infection of lumbar motoneurons, while FB treatment applied 48 hours after virus inoculation was not able to inhibit infection of lumbar motoneurons. In the trigeminal nuclei the number of the virus infected neurons significantly decreased when FB was applied 2 or 6 hours after the inoculation of the Prv into the cornea. In the neuroblastoma cultures treated with FB the expression of the viral RNAs and amount of the viral DNAs was repressed. Our results show that Fast Blue delivered to the primarily infected rat neurons at an early stage of virus replication is able to prevent infection of motoneurons. The infection of the trigeminal nuclear neurons can also be effectively inhibited using FB. The molecular investigations confirmed our morphological findings. Conference: 12th Meeting of the Hungarian Neuroscience Society, Budapest, Hungary, 22 Jan - 24 Jan, 2009. Presentation Type: Poster Presentation Topic: Developmental neurobiology and subcortical functions Citation: Marton G, Tombacz D, Toth J, Szabo A, Boldogkoi Z and Nógrádi A (2009). Effect of the fluorescent tracer Fast Blue on the Pseudorabies virus infection. Front. Syst. Neurosci. Conference Abstract: 12th Meeting of the Hungarian Neuroscience Society. doi: 10.3389/conf.neuro.01.2009.04.065 Copyright: The abstracts in this collection have not been subject to any Frontiers peer review or checks, and are not endorsed by Frontiers. They are made available through the Frontiers publishing platform as a service to conference organizers and presenters. The copyright in the individual abstracts is owned by the author of each abstract or his/her employer unless otherwise stated. Each abstract, as well as the collection of abstracts, are published under a Creative Commons CC-BY 4.0 (attribution) licence (https://creativecommons.org/licenses/by/4.0/) and may thus be reproduced, translated, adapted and be the subject of derivative works provided the authors and Frontiers are attributed. For Frontiers’ terms and conditions please see https://www.frontiersin.org/legal/terms-and-conditions. Received: 02 Mar 2009; Published Online: 02 Mar 2009. * Correspondence: Antal Nógrádi, Department of Ophthalmology, University of Szeged, Szeged, Hungary, nogradi.antal@med.u-szeged.hu Login Required This action requires you to be registered with Frontiers and logged in. To register or login click here. Abstract Info Abstract The Authors in Frontiers Gabor Marton Dora Tombacz Judit Toth Andrea Szabo Zsolt Boldogkoi Antal Nógrádi Google Gabor Marton Dora Tombacz Judit Toth Andrea Szabo Zsolt Boldogkoi Antal Nógrádi Google Scholar Gabor Marton Dora Tombacz Judit Toth Andrea Szabo Zsolt Boldogkoi Antal Nógrádi PubMed Gabor Marton Dora Tombacz Judit Toth Andrea Szabo Zsolt Boldogkoi Antal Nógrádi Related Article in Frontiers Google Scholar PubMed Abstract Close Back to top Javascript is disabled. Please enable Javascript in your browser settings in order to see all the content on this page.

The large form of human 2′,5′-Oligoadenylate Synthetase (OAS3) exerts antiviral effect against Chikungunya virus

Chikungunya virus (CHIKV) becomes one of the most important mosquito-borne alphavirus in the medical field. CHIKV is highly sensitive to antiviral activity of Type-I interferons (IFN-α/β). Here, we investigated the role of IFN-induced 2′,5′-Oligoadenylate Synthetase (OAS) family in innate immunity to CHIKV. We established inducible human epithelial HeLa cell lines expressing either the large form of human OAS, OAS3, or the genetic variant OAS3-R844X which is predicted to lack about 20% of the OAS3 protein from the carboxy terminus. HeLa cells respond to ectopic OAS3 expression by efficiently inhibiting CHIKV growth. The characteristic of the antiviral effect was a blockade in early stages of virus replication. Thus, OAS3 pathway may represent a novel antialphaviral mechanism by which IFN-α/β controls CHIKV growth. HeLa cells expressing the truncated form of OAS3 were less resistant to CHIKV infection, raising the question on the involvement of OAS3 genetic polymorphism in human susceptibility to alphavirus infection.

Chromatin Tethering of Incoming Foamy Virus by the Structural Gag Protein

Retroviruses hijack cellular machineries to productively infect their hosts. During the early stages of viral replication, proviral integration relies on specific interactions between components of the preintegration complex and host chromatin-bound proteins. Here, analyzing the fate of incoming primate foamy virus, we identify a short domain within the C-terminus of the structural Gag protein that efficiently binds host chromosomes, by interacting with H2A/H2B core histones. While viral particle production, virus entry and intracellular trafficking are not affected by mutation of this domain, chromosomal attachment of incoming subviral complexes is abolished, precluding proviral integration. We thus highlight a new function of the structural foamy Gag protein as the main tether between incoming subviral complexes and host chromatin prior to integration.

Minimal Region Sufficient for Genome Dimerization in the Human Immunodeficiency Virus Type 1 Virion and Its Potential Roles in the Early Stages of Viral Replication

It has been suggested that the dimer initiation site/dimer linkage sequence (DIS/DLS) region of the human immunodeficiency virus type 1 (HIV-1) RNA genome plays an important role at various stages of the viral life cycle. Recently we found that the duplication of the DIS/DLS region on viral RNA caused the production of partially monomeric RNAs in virions, indicating that this region indeed mediates RNA-RNA interaction. In this report, we followed up on this finding to identify the necessary and sufficient region for RNA dimerization in the virion of HIV-1. The region thus identified was 144 bases in length, extending from the junction of R/U5 and U5/L stem-loops to the end of SL4. The trans-acting responsive element, polyadenylation signal, primer binding site, upper stem-loop of U5/L, and SL2 were not needed for the function of this region. The insertion of this region into the ectopic location of the viral genome did not affect the level of virion production by transfection. However, the resultant virions contained monomerized genomes and showed drastic reductions in infectivity. A reduction was observed especially in the reverse transcription process. An attempt to generate a replication-competent virus with monomerized genome was performed by the long-term culture of mutant virus-infected cells. All recovered viruses were wild-type revertants, indicating a fatal defect of the mutation. These results suggest that genome dimerization or DIS/DLS itself also plays an important role in the early stages of virus infection.

Interaction of Human Immunodeficiency Virus Type 1 Integrase with Cellular Nuclear Import Receptor Importin 7 and Its Impact on Viral Replication

Similar to all other viruses, human immunodeficiency virus type 1 (HIV-1) depends heavily on cellular factors for its successful replication. In this study we have investigated the interaction of HIV-1 integrase (IN) with several host nuclear import factors using co-immunoprecipitation assays. Our results indicate that IN interacts specifically with host importin 7 (Imp7) in vivo, but does not interact with importin 8 (Imp8) or importin alpha (Rch1). In contrast, another HIV-1 karyophilic protein MAp17, which is capable of binding Rch1, fails to interact with Imp7, suggesting that IN and Map17 may interact with different cellular pathways during HIV-1 replication. Genetic analysis revealed that the C-terminal domain of IN is the region responsible for interaction between IN with Imp7, and an IN mutant (K240A,K244A/R263A,K264A) disrupted the Imp7 binding ability of the protein, indicating that both regions ((235)WKGPAKLLWKG and (262)RRKAK) within the C-terminal domain of IN are required for efficient IN/Imp7 interaction. Using a vesicular stomatitis virus G glycoprotein pseudotyped HIV single-cycle replication system, we showed that the IN/Imp7 interaction-deficient mutant was unable to mediate viral replication and displayed impairment at both viral reverse transcription and nuclear import steps. Moreover, transient knockdown of Imp7 in both HIV-1 producing and target cells resulted in a 2.5-3.5-fold inhibition of HIV infection. Altogether, our results indicate that HIV-1 IN specifically interacts with Imp7, and this viral/cellular protein interaction contributes to efficient HIV-1 infection.

Bupleurum kaoi inhibits Coxsackie B virus type 1 infection of CCFS-1 cells by induction of type I interferons expression

Coxsackie B virus type 1 (CVB 1) infection is known to cause high morbidity and mortality in children, however, there is no effective drug for treating this disease. The present study aimed to examine the antiviral activity of Bupleurum kaoi (BK), a popular herbal drug for treating viral and bacterial infections, against CVB 1 infection and its mechanisms of action. Our data showed that BK neutralized the CVB 1-induced cytopathic effect in human neonatal foreskin fibroblast cell line (CCFS-1/KMC), with IC 50 and EC 50 values around 12.38 μg/ml and 50.93 μg/ml, respectively. Its CC 50 and SI values were 883.56 μg/ml and 17.34, respectively. These results suggest that BK possessed anti-CVB 1 activity, and showed no effect on CCFS-1 cell viability and growth at concentration 250 μg/ml. The time-of-addition studies showed that BK (50, 100 and 200 μg/ml) added at various time of preinfection (−1 to −3 h), coinfection (0 h) and postinfection (1–3 h) could inhibit CVB 1 infection. Interestingly, BK also showed an inhibition on viral replication through the induction of IFN-α/β expression. In conclusion, BK possessed antiviral activity against CVB 1 infection. It interfered the early stage of viral replication and viral replication after infection through the induction of type I interferon expression.

Chlorella viruses contain genes encoding a complete polyamine biosynthetic pathway

Two genes encoding the putative polyamine biosynthetic enzymes agmatine iminohydrolase (AIH) and N-carbamoylputrescine amidohydrolase (CPA) were cloned from the chloroviruses PBCV-1, NY-2A and MT325. They were expressed in Escherichia coli to form C-terminal (His)6-tagged proteins and the recombinant proteins were purified by Ni2+-binding affinity chromatography. The biochemical properties of the two enzymes are similar to AIH and CPA enzymes from Arabidopsis thaliana and Pseudomonas aeruginosa. Together with the previously known virus genes encoding ornithine/arginine decarboxlyase (ODC/ADC) and homospermidine synthase, the chloroviruses have genes that encode a complete set of functional enzymes that synthesize the rare polyamine homospermidine from arginine via agmatine, N-carbamoylputrescine and putrescine. The PBCV-1 aih and cpa genes are expressed early during virus infection together with the odc/adc gene, suggesting that biosynthesis of putrescine is important in early stages of viral replication. The aih and cpa genes are widespread in the chlorella viruses.

ANTIVIRAL EFFECTS OF SAIKOSAPONINS ON HUMAN CORONAVIRUS 229E IN VITRO

SUMMARY 1Saikosaponins represent a group of oleanane derivatives, usually as glucosides, that are found in a number of plant families. Saikosaponins isolated from medicinal plants such as Bupleurum spp., Heteromorpha spp. and Scrophularia scorodonia have been reported to possess various biological activities, specifically antihepatitis, antinephritis, antihepatoma, anti‐inflammation, immunomodulation and antibacterial effects.2The aim of the present study was to examine the anticoronaviral activity of saikosaponins (A, B2, C and D) and their mode of action. Using the 2,3‐bis[2‐methoxy‐4‐nitro‐5‐sulfophenyl]‐5‐[(phenylamino) carbonyl‐2H‐tetrazolium hydroxide] (XTT) assay, results showed that all saikosaponins tested demonstrated antiviral activity at concentrations of 0.25–25 µmol/L, with the strongest activity being noted for saikosaponin B2 (IC50 = 1.7 ± 0.1 µmol/L). Interestingly, both saikosaponins A (50% cellular cytotoxicity (CC50) concentration = 228.1 ± 3.8 µmol/L; selectivity index (SI) = 26.6) and B2 (CC50 = 383.3 ± 0.2 µmol/L; SI = 221.9) exhibited no cytotoxic effects on target cells at concentrations that achieved antiviral activity. In the time‐of‐addition studies, saikosaponin B2, at 6 µmol/L, significantly inhibited human coronavirus 229E infection following its addition at various time pre‐infection (−4 to −1 h), coinfection (0 h) and post‐infection (1–4 h). Furthermore, saikosaponin B2 also showed an inhibitory effect on viral attachment and penetration.3The present results indicate that saikosaponin B2 has potent anticoronaviral activity and that its mode of action possibly involves interference in the early stage of viral replication, such as absorption and penetration of the virus.

Xiao Chai Hu Tang inhibits CVB 1 virus infection of CCFS-1 cells through the induction of Type I interferon expression

Coxsackie B virus type 1 (CVB 1) infection is known to cause high morbidity and mortality in children, however, there is no effective drug for treating this disease. The present study aimed to examine the antiviral activity of Xiao Chai Hu Tang (XCHT), a popular herbal drug for treating viral and bacterial infections, against CVB 1 infection and its mechanisms of action. Our data showed that XCHT neutralized the CVB 1-induced cytopathic effect in human neonatal foreskin fibroblast cell line (CCFS-1/KMC), with IC 50 (virus-induced cytopathic effect by 50%) and EC 50 (concentration of 50% effectiveness) values around 12.39 and 50.93 μg/ml, respectively. Its CC 50 (concentration of 50% cellular cytotoxicity) and SI (selective index) values were 945.75 μg/ml and 18.92, respectively. These results suggest that XCHT possessed anti-CVB 1 activity, and showed no effect on CCFS-1 cell viability and growth at concentration 250 μg/ml. The time-of-addition studies showed that XCHT (50, 100 and 200 μg/ml) added at various time of preinfection (− 1 to − 3 h), coinfection (0 h) and postinfection (1∼3 h) could inhibit CVB 1 infection. Interestingly, XCHT also showed an inhibition on viral replication through the induction of IFN-α/β expression. In conclusion, XCHT possessed antiviral activity against CVB 1 infection. It interfered the early stage of viral replication (prophylactic effect) and viral replication after infection (therapeutic effect) through the induction of Type I interferon expression.

Antihuman Immunodeficiency Virus Type 1 (HIV-1) Activity of Rare Earth Metal Complexes of 4-Hydroxycoumarins in Cell Culture.

The cerium Ce(III), lanthanum La(III), and neodymium Nd(III) complexes with 4-hydroxy-3-(3-oxo-1-phenylbutyl)-2H-1-benzopyran-2-one (warfarin) (W) and 3,3′-benzylidenebis[4-hydroxycoumarin] (1) were synthesized and studied for the first time for cytotoxicity (on MT-2 cells) and as anti-HIV agents under acute and chronic infection. The complexes were characterized by different physicochemical methods: mass spectrometry, 1H NMR, 13C NMR, and IR spectroscopy. The spectra of the complexes were interpreted on the basis of comparison with the spectrum of the free ligands. Anti-HIV effect of the complexes/ligands was measured in MT-2 cells by microtiter infection assay. Detection of endogenous reverse transcriptase (RT) activity and RT processivity by PCR indicative for proviral DNA synthesis demonstrated that anti-HIV activity has not been linked to early stages of viral replication. No effect on late steps of viral replication has been found using cells chronically producing HIV-1LAI virus. La(W) demonstrated anti-HIV activity (IC50=21.4 μM) close to maximal nontoxic concentration. Nd(W), Ce(1), and Nd(1) demonstrated limited anti-HIV potency, so none of the complexes seems appropriate to be used in clinic. Further targeting of HIV-1 inhibition by La(W) is under progress.

Human cytomegalovirus entry into epithelial and endothelial cells depends on genes UL128 to UL150 and occurs by endocytosis and low-pH fusion.

Human cytomegalovirus (HCMV) replication in epithelial and endothelial cells appears to be important in virus spread, disease, and persistence. It has been difficult to study infection of these cell types because HCMV laboratory strains (e.g., AD169 and Towne) have lost their ability to infect cultured epithelial and endothelial cells during extensive propagation in fibroblasts. Clinical strains of HCMV (e.g., TR and FIX) possess a cluster of genes (UL128 to UL150) that are largely mutated in laboratory strains, and recent studies have indicated that these genes facilitate replication in epithelial and endothelial cells. The mechanisms by which these genes promote infection of these two cell types are unclear. We derived an HCMV UL128-to-UL150 deletion mutant from strain TR, TRdelta4, and studied early events in HCMV infection of epithelial and endothelial cells, and the role of genes UL128 to UL150. Analysis of wild-type TR indicated that HCMV enters epithelial and endothelial cells by endocytosis followed by low-pH-dependent fusion, which is different from the pH-independent fusion with the plasma membrane observed with human fibroblasts. TRdelta4 displayed a number of defects in early infection processes. Adsorption and entry of TRdelta4 on epithelial cells were poor compared with those of TR, but these defects could be overcome with higher doses of virus and the use of polyethylene glycol (PEG) to promote fusion between virion and cellular membranes. High multiplicity and PEG treatment did not promote infection of endothelial cells by TRdelta4, yet virus particles were internalized. Together, these data indicate that genes UL128 to UL150 are required for HCMV adsorption and penetration of epithelial cells and to promote some early stage of virus replication, subsequent to virus entry, in endothelial cells.

A β-galactose-specific lectin isolated from the marine worm Chaetopterus variopedatus possesses anti-HIV-1 activity

A 30 kDa β-galactose-specific lectin named CVL was isolated from the polychaete marine worm Chaetopterus variopedatus (Annelida) and its anti-HIV-1 activity in vitro was determined. Results showed that CVL inhibited cytopathic effect induced by HIV-1 and the production of viral p24 antigen. The EC 50 values were 0.0043 and 0.057 μM, respectively. Time-of-addition analysis of anti-HIV-1 activity indicated its action was at the early stage of virus replication. CVL could blocked the cell-to-cell fusion process of HIV infected and uninfected cells with an EC 50 of 0.073 μM. The inhibition of HIV-1 entry into host cells was demonstrated by using fluorescence-based real-time quantify PCR. At CVL concentration of 0.33 μM and 0.07 μM, 86% and 21% virus attachment were blocked, respectively. The anti-HIV-1 action of CVL might relate to blockade of HIV-1 entry into cells.

Altered patterns of cellular gene expression by JC virus

The restricted tropism of the polyomavirus JC virus (JCV) makes pathogenesis-related studies challenging. Because of the difficulties in cultivating JCV, most pathogenesis studies have employed SV40-derived cell lines or JCV/SV40 chimeras that utilize composite enhancer sequences from both viruses. Our objective was to study the early genes expressed by JCV to better understand viral gene regulation. Subconfluent monolayers of primary human fetal glial (PHFG) cells were transfected with ligated full-length JCV DNA and replication was confirmed at days 5 and 10. Total cellular RNA was extracted on day 10 from JCVand mock-transfected PHFG cells. 20 μg RNA was hybridized to an Affymetrix U133A chip, and the data was analyzed using Microarray Suite 5.0. Of the 400 differentially expressed genes, up-regulated genes clustered into 3 major functional groups: cell proliferation, cell-communication, and IFN-responsive genes. Upregulation of IFNinducible genes such as, MxA, Stat1, OAS1 and cig5 was confirmed by realtime PCR. Based on Western blot analysis cig5 and MxA protein levels were significantly elevated. Correlation of T-antigen and agnoprotein gene expression with both mRNA and protein accumulation of antiviral genes such as, MxA and cig5 at an early stage of viral replication suggests that induction of IFN-inducible genes constitutes one of the first cellular response to JCV replication and viral transcription, and its regulation is orchestrated via virusinduced binding of transcription factors. The global analysis of changes in mRNA expression levels following viral infection offers a catalog of genes that are modulated as a result of the host-pathogen interaction and provides a new approach for the investigation of PML pathogenesis.

Potent, selective and cell-mediated inhibition of human herpesvirus 6 at an early stage of viral replication by the non-nucleoside compound CMV423

CMV423 (2-chloro-3-pyridin-3-yl-5,6,7,8-tetrahydroindolizine-1-carboxamide) is a new antiviral agent with potent and selective in vitro activity against the β-herpesvirus human cytomegalovirus (HCMV), but not against α- or γ-herpesviruses. Here we report that its activity also extends to human herpesvirus 6 (HHV-6) and 7 (HHV-7). When compared in vitro to ganciclovir and foscarnet (the standard drugs recommended for treatment of HHV-6 infections), CMV423 showed a superior selectivity, due to its high activity (antiviral ic 50: 53 nM) and low cytotoxicity ( cc 50: 144 μM), both in continuous cell lines and in CBLCs infected with HHV-6. From mechanistic experiments at the level of viral mRNA and protein expression, we learned that CMV423 targets an event following viral entry but preceding viral DNA replication. Its antiviral action was dependent on the cell line used, implying involvement of a cellular component. When compared to a panel of known protein kinase inhibitors, CMV423 was found to share anti-HHV-6 characteristics with herbimycin A, which affects tyrosine kinase activity through heat shock protein 90 (Hsp90) inhibition. We demonstrated that high concentrations of CMV423 have an inhibitory effect on the total cellular protein tyrosine kinase activity, and that CMV423 and herbimycin A, when combined, act synergistically against HHV-6. The activities of cyclin-dependent kinases, protein kinases A and C, and the HHV-6-encoded pU69 kinase were not affected. We, therefore, conclude that CMV423 exerts its activity against HHV-6 through inhibition of a cellular process that is critical at early stages of viral replication and that may affect protein tyrosine kinase activity.

The ubiquitin-vacuolar protein sorting system is selectively required during entry of influenza virus into host cells.

Influenza virus enters cells by endocytosis, and requires the low pH of the late endosome for successful infection. Here, we investigated the requirements for sorting into the multivesicular body pathway of endocytosis. We show that treatment of host cells with the proteasome inhibitors MG132 and lactacystin directly affects the early stages of virus replication. Unlike other viruses, such as retroviruses, influenza virus budding was not affected. The requirement for proteasome function was not shared by two other pH-dependent viruses: Semliki Forest virus and vesicular stomatitis virus. With MG132 treatment, incoming influenza viruses were retained in endosomes that partially colocalized with mannose 6-phosphate receptor, but not with classical markers of early or late endosomes. Colocalization was also observed with Rme-1, which is part of the recycling pathway of endocytosis. In addition, influenza virus entry was dependent on the vacuolar protein sorting pathway, as over-expression of dominant-negative hVPS4 caused arrest of viruses in endosome-like populations that partially colocalized with the hVPS4 protein. Overall, we conclude that influenza virus selectively requires the ubiquitin/vacuolar protein sorting pathway for entry into host cells, and that it must communicate with a specific cellular machinery for intracellular sorting during the initial phase of virus infection.

Cardiac Myosin and the TH1/TH2 Paradigm in Autoimmune Myocarditis

Myocarditis and dilated cardiomyopathy may represent acute and chronic stages of a progressive organ-specific autoimmune disease of the myocardium. 1-3 Myocarditis is an inflammatory disease of the heart that is characterized by a cellular infiltrate in the myocardium, and dilated cardiomyopathy is a chronic heart muscle disease characterized by ventricular hypertrophy. 1-4 Dilated cardiomyopathy is a primary cause of severe heart failure with subsequent transplantation or death within several years after diagnosis. 1,5 The direct result of cellular infiltration of the myocardium is necrosis and loss of myocytes leading to the development of contractile dysfunction and ventricular dilatation. The loss of myocytes and formation of scar tissue in the myocardium would lead to loss of contractile function and ventricular enlargement. In a small percentage of individuals, loss of myocyte function and development of dilated cardiomyopathy can result from mutations in or viral proteolytic digestion of dystrophin or dystrophin-associated glycoproteins that lead to cytoskeletal disruption and loss of overall contractile function in the heart. 4,6-8 However, the most common cause of myocarditis is viral infection. An exhaustive list of myocarditis-inducing agents including microbial pathogens and toxins is provided by Brown and O’Connell 2 in a review of myocarditis and dilated cardiomyopathy. Among the most common viral causes of human myocarditis are coxsackieviruses. 2,9-11 Mouse models of coxsackievirus-induced myocarditis were studied by Woodruff and colleagues 12-14 who demonstrated that lymphocytes from mouse models of myocarditis could destroy cardiomyocytes in culture. Gauntt and colleagues 15 also investigated the role of virulent myocarditic versus nonvirulent amyocarditic coxsackieviruses in mouse models of myocarditis. In their studies, there appeared to be several stages of disease in their mouse model. The first early stage of viral replication and cell lysis in the heart showed no evidence of heart failure or cellular inflammation, however, with the onset of specific immune responses, cellular infiltration of the myocardium was observed in susceptible mice. The cellular infiltrates in the myocardium were minimal to severe and led to the loss of myocytes by necrosis. The development of dilated cardiomyopathy is thought to represent the third and more chronic stage of heart disease that develops after the cellular inflammation. 16 It would follow that once the damage has been done to the cardiomyocyte function either by elimination of large numbers of myocytes because of inflammation, necrosis, and scarring or by disruption of the cytoskeleton so that the myocyte is dysfunctional, then anti-inflammatory therapy would not be effective. Anti-inflammatory therapies would only be efficacious during the time of inflammatory onset. In fact, immunosuppressive therapy was not effective in the Myocarditis Trial. 2 By the time myocarditis symptoms present, many cases may have already advanced to a stage in which necrosis and scarring have already led to ventricular dilatation and contractile dysfunction. For comparison, in rheumatic carditis, the valve is insidiously and permanently damaged by autoimmune attack after streptococcal pharyngitis. In rheumatic carditis, valve injury may not be evident until a heart murmur is perceived that reflects the scarred valve. In myocarditis, the end-stage of disease may represent a physical, structural defect as a result of autoimmune-mediated damage during the inflammatory stage of the disease.

Histopathology and cytopathology of white spot syndrome virus (WSSV) in cultured Penaeus monodon from peninsular Malaysia with emphasis on pathogenesis and the mechanism of white spot formation.

Since 1994, white spot syndrome virus (WSSV) has been detected in cultured shrimp Penaeus monodon in Peninsular Malaysia. The gross signs, target organs and histo-cytopathology for the viral infection were studied and it was found to infect most organs and tissues including oocytes, but not hepatopancreatocytes and epithelial cells of the midgut, which were regarded as refractory tissues. Based on a time-sequence of ultrastructural cytopathology, 4 cytopathic profiles and 6 phases of viral morphogenesis were described. The virions were elliptical to short rods with trilamilar envelopes that measured 305 +/- 30 x 127 +/- 11 nm. Viral nucleosomes were often present singly in infected nuclei and were associated with the early stages of viral replication. The structure of WSSV pathognomonic white, cuticular lesions was examined at the microscopic and ultrastructural levels and the mechanism of their formation appeared to be related to the disruption of exudate transfer from epithelial cells to the cuticle via cuticular pore canals.

The alpha chemokine, interleukin 8, inhibits the antiviral action of interferon alpha.

Interferon (IFN) exhibits a potent antiviral activity in vitro and plays a major role in the early defense against viruses. Like IFN, the proinflammatory chemokine, interleukin (IL)-8, is induced by viruses and appears in circulation during viral infections. In an in vitro cytopathic effect assay for IFN, we found that IL-8 can inhibit IFN-alpha activity in a dose-dependent manner. This action was reversed by specific monoclonal antibodies to IL-8. The chemokine was able to attenuate the IFN-mediated inhibition of viral replication as determined by measuring infectious virus yield. IL-8 also diminished the ability of IFN to inhibit an early stage of viral replication since IL-8 attenuated the inhibition of the formation of viral proteins. It appeared that IL-8 interfered with a late rather than an early step of IFN-mediated pathway such as early gene expression. The IL-8 inhibitory action on IFN-alpha antiviral activity was associated with reduced 2',5'-A oligoadenylate synthetase activity, a pathway well correlative with the anti- encephalomyocarditis virus action of IFN-alpha. Understanding pathways that antagonize IFN action may lead to novel approaches to potentiate endogenous and therapeutic IFN.