Effects of steroid hormones on replication of murine sarcoma virus in mouse embryo cultures.

The androgenic steroid hormone, testosterone, inhibited focus formation by the murine Moloney sarcoma virus in mouse embryo cells. The inhibition of focus formation was enhanced by cyclic AMP. Although focus formation was inhibited, there was no inhibition of viral replication. The glucogenic adrenal corticosteroids, cortisol and dexamethasone, and 17-beta-estradiol and progesterone did not affect focus formation by MuSV(M).     

Superhelix density of replicating simian virus 40 DNA molecules

Simian virus 40 replicating DNA molecules were isolated and fractionated according to the extent of replication by isopynic centrifugation in ethidium bromide-CsCl. Electron microscopic examination of the replicating molecules in the presence of ethidium bromide revealed that the sense of the superhelix in replicating molecules is the same as that of simian virus 40 DNA I. Replicating DNA molecules of differing extents of replication were also analyzed by sedimentation in varying concentrations of ethidium bromide. It was observed that the superhelix density of the unreplicated portion of replicating molecules was greater than that of DNA I and that it increased as the degree of replication increased. In contrast with the increase in superhelix density that was related to the extent of replication, all replicating molecules contained a rather constant number (2 to 5) of additional superhelical turns per molecule, irrespective of the extent of replication. This suggests that a region (or regions) of about 20 to 50 nucleotides may exist in a denatured state in replicating molecules, presumably at the replicating forks of the molecule.     

Focus formation by the human immunodeficiency virus (HIV) in the immobilized MT-4 cell culture and its application to the evaluation of anti-HIV agents.

Immunofluorescence studies were performed on the infection of monolayer cultures of immobilized MT-4 cells with human immunodeficiency virus type 1 (HIV-1). By using the anti-viral p24 monoclonal antibody, we could observe formation of foci of p24 antigen-positive cells within 3 to 4 days when the infection was initiated with a relatively small amount of the virus. Frequency of the focus formation was in proportion to the dose of input virus (ranging from 0.001 to 0.1 PFU/cell), which allowed us to apply this phenomenon to the assay of anti-HIV agents as well as to the estimation of relative infectivity of the virus stocks. When antiviral agents were added to the infected cultures, number of foci as well as the size of each focus was reduced in a concentration-dependent manner. The dose required for reducing the number of foci by 50% was calculated to be 6 ng/ml and 8 ng/ml for tunicamycin (TM) and azidothymidine (AZT), respectively. These values are comparable to those obtained by other current assay methods. In addition, focus reduction assay is also useful in searching for such antiviral agents that would inhibit or block the early step of viral replication cycle.     

18beta-glycyrrhetinic acid inhibits rotavirus replication in culture

ABSTRACT: BACKGROUND: Glycyrrhizin (GA) and primary metabolite 18beta-glycyrrhetinic acid (GRA) are pharmacologically active components of the medicinal licorice root, and both have been shown to have antiviral and immunomodulatory properties. Although these properties are well established, the mechanisms of action are not completely understood. In this study, GA and GRA were tested for the ability to inhibit rotavirus replication in cell culture, toward a long term goal of discovering natural compounds that may complement existing vaccines. METHODS: Epithelial cells were treated with GA or GRA various times pre- or post-infection and virus yields were measured by immunofluorescent focus assay. Levels of viral proteins VP2, VP6, and NSP2 in GRA treated cells were measured by immunoblot to determine if there was an effect of GRA treatment on the accumulation of viral protein. RESULTS: GRA treatment reduced rotavirus yields by 99% when added to infected cultures post- virus adsorption, whereas virus yields in GA treated cultures were similar to mock treated controls. Time of addition experiments indicated that GRA-mediated replication inhibition likely occurs at a step or steps subsequent to virus entry. The amounts of VP2, VP6 and NSP2 were substantially reduced when GRA was added to cultures up to two hours post-entry. CONCLUSIONS: GRA, but not GA, has significant antiviral activity against rotavirus replication in vitro, and studies to determine whether GRA attenuates rotavirus replication in vivo are underway.     

Influenza-virus-induced signaling cascades: targets for antiviral therapy?

Influenza viruses continue to pose a severe threat worldwide, causing thousands of deaths and an enormous economic loss every year. The major problem in fighting influenza is the high genetic variability of the virus, resulting in the rapid formation of variants that escape the acquired immunity against previous virus strains, or have resistance to antiviral agents. Every virus depends on its host cell and, hence, cellular functions that are essential for viral replication might be suitable targets for antiviral therapy. As a result, intracellular signaling cascades induced by the virus, in particular mitogen-activated protein kinase pathways, have recently come into focus.     

Assembly of infectious hepatitis C virus particles

A hallmark of the hepatitis C virus (HCV) replication cycle is its tight link with host cell lipid synthesis. This is best illustrated by the peculiar pathway used for the assembly of infectious HCV particles. Research in the past few years has shown that formation of HC-virions is closely connected to lipid droplets that could serve as an assembly platform. Moreover, HCV particle production appears to be strictly linked to very-low-density lipoproteins. In this review, we focus on new insights into the molecular aspects of the architecture and assembly of this unique type of virus particle.     

Lack of influence of mitochondrial genetical information on the multiplication of Herpes simplex virus in HeLa cells

Mitochondrial DNA synthesis in HeLa cells is inhibited by 0.2 μg ethidium bromide/ml whereas nuclear DNA synthesis is essentially unimpaired under the same conditions. The action of ehtidium bromide on mitochondrial DNA appears to be completed within 18 hours of exposure to the drug. Total cellular macromolecular synthesis under ethidium bromide is initially decreased and at later times slightly stimulated. Ethidium bromide pretreatment of HeLa cells did not significantly affect the multiplication of Herpes simplex virus as compared with that in control cells.     

Interactions of Polyoma and Mouse DNAs III. Mechanism of Polyoma Pseudovirion Formation

In primary mouse kidney cell cultures infected with polyoma virus, the processes leading to virion and pseudovirion formation were studied. By photometric DNA quantitation, we followed the kinetics of mouse and polyoma DNA synthesis and the formation of low-molecular-weight fragmented mouse DNA (mouse f-DNA). Virus was harvested at different times and analyzed for its proportion of pseudovirions. The following correlations between the intracellular events and the production of virions and pseudovirions were found. (i) Syntheses of cellular and viral DNA were closely linked, both in time and in rates of synthesis. (ii) An increase of mouse f-DNA could only be detected several hours after the onset of mouse and polyoma DNA replication; its formation coincided in time with the appearance of progeny virus. (iii) The proportion of pseudovirions was not dependent on the amount of mouse f-DNA formed, but seemed to be inversely related to the amount of viral DNA synthesized. This was borne out by experiments in which DNA synthesis was partially inhibited by mitomycin C or after a synchronized onset of DNA replication. Under these conditions, virus preparations with a two- to threefold increased proportion of pseudovirions were obtained as compared with those from uninhibited cultures. Virus isolated from the remaining monolayer always had a higher proportion of pseudovirions than virus isolated at the same time from the supernatant medium only; also, the proportion of pseudovirions increased slightly with time after infection. Thus, according to the experimental conditions used, polyoma virus preparations with a low (10 to 20%) or a high (60 to 80%) proportion of pseudovirions can be obtained.     

Viral-specific antigen synthesis following de novo RD-114 virus infection of stationary cells

The cell cycle dependence of retrovirus replication was studied. Canine sarcoma (D-17) cells were infected de novo with the xenotropic feline retrovirus RD-114 under conditions previously reported to simultaneously inhibit virus replication and cell DNA synthesis and/or cell division. RD-114 viral antigen synthesis was observed under conditions previously reported to be inhibitory to avian and murine oncornavirus replication, including confluency and serum deprivation, X-irradiation, mitomycin C pretreatment, colchicine, and ethidium bromide treatments of cells. Several mechanisms that could account for viral antigen synthesis under the restrictive conditions used are discussed.     

短发夹结构RNA干扰新城疫病毒的增殖

 以新城疫病毒（NDV）NP基因为标靶,构建3个细胞内表达发夹样结构小干扰RNA（shRNA）的质粒载体,在鸡胚成纤维细胞（CEF）和鸡胚上进行了RNAi试验,筛选出一个有效抑制病毒复制的小分子ndv1.用阳离子脂质体转染试剂Silent-fect 将ndv1转染CEF,以不相关shRNA质粒载体HK为阴性对照,4 h后接种NDV,与对照相比,干涉组在病毒感染后3 h NP基因的表达量降低2.3倍,6 h 降低21.1倍,9 h降低9.8倍;ndv1能在48 h内完全阻断NDV在CEF中的增殖,延缓病变出现时间,减轻病变程度.将Silent-fect-ndv1混合物与NDV同时注入10日龄SPF鸡胚绒毛尿囊腔,能使10⁵ ELD₅₀NDV感染后17 h鸡胚尿囊液中病毒增殖量减少94.4%,使10⁶ ELD₅₀NDV感染后17　h鸡胚尿囊液中病毒增殖量减少62.5%.实验结果证实,在CEF中存在RNAi机制,抑制NDV NP基因的表达能有效阻断该病毒增殖,说明NP基因在NDV复制过程中起重要作用.实验结果为进一步利用RNAi技术在CEF和鸡胚中研究病毒基因组功能及筛选抗病毒小分子奠定了基础.     

Knockdown of the cellular protein LRPPRC attenuates HIV-1 infection

HIV-1 exploits numerous host cellular pathways for productive infection. To identify novel factors involved in HIV-1 replication, HIV-1 integrase and matrix protein complexes were captured at 4 hours post infection for proteomic analysis using an affinity purification system. Leucine-rich PPR-motif containing (LRPPRC) protein, a cellular protein involved in mitochondrial function, cell metabolism, and cell-cycle progression was identified as one of the candidate HIV-1 factors. Co-immunoprecipitation RT-PCR experiments confirmed that LRPPRC associated with HIV-1 nucleic acids during the early steps of virus infection. To establish if LRPPRC was critical for HIV-1 infection, three independent LRPPRC knockdown cell lines were constructed (2.7, 3.6, and 4.1). Subcellular fractionation of these cell lines revealed differential knockdown of LRPPRC in subcellular compartments. LRPPRC was knocked down in the insoluble/cytoskeletal fractions of all three cell lines, but the 3.6 and 4.1 cells also showed a reduction in nuclear LRPPRC. Additionally, several cellular factors were downregulated and/or disrupted by loss of LRPPRC. HIV-1 infection was reduced in all three cell lines, but virus production and RNA encapsidation were unaffected, suggesting that LRPPRC was critical for the afferent stage of virus replication. Two of the three cell lines (3.6, 4.1) were refractory for murine leukemia virus infection, a virus dependent on cellular proliferation for productive infection. Consistent with this, these two cell lines exhibited reduced cellular growth with no loss of cellular viability or change in cell cycle phenotype. The early steps of virus infection were also differentially affected among the cell lines. A reduced level of preintegration complex formation was observed in all three cell lines, but viral DNA nuclear import was reduced only in the 3.6 and 4.1 cells. Combined, these data identify LRPPRC as a HIV-1 factor that is involved in HIV-1 replication through more than one mechanism.     

Identification of a ribonucleoprotein intermediate of tomato mosaic virus RNA replication complex formation

The replication of eukaryotic positive-strand RNA virus genomes occurs in the membrane-bound RNA replication complexes. Previously, we found that the extract of evacuolated tobacco BY-2 protoplasts (BYL) is capable of supporting the translation and subsequent replication of the genomic RNAs of plant positive-strand RNA viruses, including Tomato mosaic virus (ToMV). Here, to dissect the process that precedes the formation of ToMV RNA replication complexes, we prepared membrane-depleted BYL (mdBYL), in which the membranes were removed by centrifugation. In mdBYL, ToMV RNA was translated to produce the 130-kDa and 180-kDa replication proteins, but the synthesis of any ToMV-related RNAs did not occur. When BYL membranes were added back to the ToMV RNA-translated mdBYL after the termination of translation with puromycin, ToMV RNA was replicated. Using a replication-competent ToMV derivative that encodes the FLAG-tagged 180-kDa replication protein, it was shown by affinity purification that a complex that contained the 130-kDa and 180-kDa proteins and ToMV genomic RNA was formed after translation in mdBYL. When the complex was mixed with BYL membranes, ToMV RNA was replicated, which suggests that this ribonucleoprotein complex is an intermediate of ToMV RNA replication complex formation. We have named this ribonucleoprotein complex the "pre-membrane-targeting complex." Our data suggest that the formation of the pre-membrane-targeting complex is coupled with the translation of ToMV RNA, while posttranslationally added exogenous 180-kDa protein and replication templates can contribute to replication and can be replicated, respectively. Based on these results, we discuss the mechanisms of ToMV RNA replication complex formation.     

Tetrameric ring formation of Epstein-Barr virus polymerase processivity factor is crucial for viral replication

The Epstein-Barr virus BMRF1 DNA polymerase processivity factor, which is essential for viral genome replication, exists mainly as a C-shaped head-to-head homodimer but partly forms a ring-shaped tetramer through tail-to-tail association. Based on its molecular structure, several BMRF1 mutant viruses were constructed to examine their influence on viral replication. The R256E virus, which has a severely impaired capacity for DNA binding and polymerase processivity, failed to form replication compartments, resulting in interference of viral replication, while the C95E mutation, which impairs head-to-head contact in vitro, unexpectedly hardly affected the viral replication. Also, surprisingly, replication of the C206E virus, which is expected to have impairment of tail-to-tail contact, was severely restricted, although the mutant protein possesses the same in vitro biochemical activities as the wild type. Since the tail-to-tail contact surface is smaller than that of the head-to-head contact area, its contribution to ring formation might be essential for viral replication.     

Effects of Temperature and pH on Survival of Free Nuclear Polyhedrosis Virus of Autographa californica

The effects of temperature and low pH on replication and survival of nonoccluded Autographa californica nuclear polyhedrosis virus were investigated. No virus replication or formation of polynuclear inclusion bodies occurred at 37°C. The virus was immediately inactivated upon exposure to pH 2.0 and was inactivated within 1 h at pH 4.0. The virus titer slowly declined, a 3-orders of magnitude reduction in virus titer, at pH 5.0 during a 4-h exposure. Virus survival at pH 6.0 was equal to that of the control in cell culture medium 199 MK (pH 7.12).     

Complete replication of poliovirus in vitro: preinitiation RNA replication complexes require soluble cellular factors for the synthesis of VPg-linked RNA.

Translation of poliovirion RNA in HeLa S10 extracts resulted in the formation of RNA replication complexes which catalyzed the asymmetric replication of poliovirus RNA. Synthesis of poliovirus RNA was detected in unfractionated HeLa S10 translation reactions and in RNA replication complexes isolated from HeLa S10 translation reactions by pulse-labeling with [32P]CTP. The RNA replication complexes formed in vitro contained replicative-intermediate RNA and were enriched in viral protein 3CD and the membrane-associated viral proteins 2C, 2BC, and 3AB. Genome-length poliovirus RNA covalently linked to VPg was synthesized in large amounts by the replication complexes. RNA replication was highly asymmetric, with predominantly positive-polarity RNA products. Both anti-VPg antibody and guanidine HCl inhibited RNA replication and virus formation in the HeLa S10 translation reactions without affecting viral protein synthesis. The inhibition of RNA synthesis by guanidine was reversible. The reversible nature of guanidine inhibition was used to demonstrate the formation of preinitiation RNA replication complexes in reaction mixes containing 2 mM guanidine HCl. Preinitiation complexes sedimented upon centrifugation at 15,000 x g and initiated RNA replication upon their resuspension in reaction mixes lacking guanidine. Initiation of RNA synthesis by preinitiation complexes did not require active protein synthesis or the addition of soluble viral proteins. Initiation of RNA synthesis by preinitiation complexes, however, was absolutely dependent on soluble HeLa cytoplasmic factors. Preinitiation complexes also catalyzed the formation of infectious virus in reaction mixes containing exogenously added capsid proteins. The titer of infectious virus produced in such trans-encapsidation reactions reached 4 x 10(7) PFU/ml. The HeLa S10 translation-RNA replication reactions represent an efficient in vitro system for authentic poliovirus replication, including protein synthesis, polyprotein processing, RNA replication, and virus assembly.     

Complexes of Sendai virus NP-P and P-L proteins are required for defective interfering particle genome replication in vitro.

We present evidence that the formation of NP-P and P-L protein complexes is essential for replication of the genome of Sendai defective interfering (DI-H) virus in vitro, using extracts of cells expressing these viral proteins from plasmids. Optimal replication of DI-H nucleocapsid RNA required extracts of cells transfected with critical amounts and ratios of each of the plasmids and was three- to fivefold better than replication with a control extract prepared from a natural virus infection. Extracts in which NP and P proteins were coexpressed supported replication of the genome of purified DI-H virus which contained endogenous polymerase proteins, but extracts in which NP and P were expressed separately and then mixed were inactive. Similarly, the P and L proteins must be coexpressed for biological activity. The replication data thus suggest that two protein complexes, NP-P and P-L, are required for nucleocapsid RNA replication and that these complexes must form during or soon after synthesis of the proteins. Biochemical evidence in support of the formation of each complex includes coimmunoprecipitation of both proteins of each complex with an antibody specific for one component and cosedimentation of the subunits of each complex. We propose that the P-L complex serves as the RNA polymerase and NP-P is required for encapsidation of newly synthesized RNA.     

GSK-3-associated signaling is crucial to virus infection of cells

Signal transduction pathways play important roles in virus infection, replication, and associated pathogenesis. Some of the best understood cell signaling networks are crucial to virus infections such the mitogen-activated protein kinase (MAPK), phosphoinositide 3-kinase (PI3K), protein kinase C (PKC), and the WNT/β-catenin pathways. Glycogen synthase kinase-3 (GSK-3) is a lesser known signaling molecule in the field of virus research. Interestingly, GSK-3 forms the crux of multiple cell signaling pathways. However, recent studies indicate that GSK-3 may perform key roles in the response to viral infection, replication and pathogenesis. The effects of activated or inactivated forms of GSK-3 on virus infection are still not yet clearly understood phenomenon. The comprehension of the molecular mechanisms underlying the regulation of GSK-3-associated signaling pathways in terms of different stages of virus replication could be important not only to understand the pathogenesis of virus, but also possibly leading to new therapeutic targets. This review will focus on recent advances in understanding the roles of GSK-3 on viral replication, pathogenesis and the immune responses.     

Complex of the herpes simplex virus initiator protein UL9 with DNA as a platform for the design of a new type of antiviral drugs

The protein binding to the origin of replication of the herpes simplex virus type 1 is DNA helicase encoded by the UL9 gene of the herpes virus. The protein specifically binds to two binding sites in the viral DNA replication origins OriS or OriL. In order to determine the role of the UL9 protein in the initiation of replication and find efficient inhibitors of the UL9 activity, we have synthesized a recombinant UL9 protein expressed in E. coli cells. It was found that the recombinant UL9 protein binds to Boxes I and II in OriS and possesses DNA helicase and ATPase activities. In the complex with a fluorescent analog of ATP, two molecules of the ATP analog bind to one protein dimer molecule. It was also found that the UL9 protein in the dimer form can bind simultaneously to two DNA fragments, each containing specific binding sites for the protein. The interaction of the recombinant UL9 protein with the 63-mer double- and single-stranded oligonucleotides OriS and OriS*, which correspond to the origin of replication of herpes simplex virus, has been investigated. From the titrations of OriS and OriS* with ethidium bromide in the presence and absence of the UL9 protein, the equilibrium affinity constants of the protein binding to OriS and OriS* have been determined. A DNase I footprinting study showed that bis-netropsins exhibit preference for binding to the AT cluster in the origin of replication OriS and inhibit the fluctuation opening of AT base pairs in the AT cluster. The drugs also prevent formation of an intermediate conformation of OriS* that involves a disordered tail at the 3′ end and stable Box I-Box III hairpin to which the UL9 helicase selectively binds. The stabilization by bis-netropsins of the AT-rich hairpin at its 3′ end can inhibit the helicase activity. It was concluded that the antiviral activity of bis-netropsins may be associated with the inhibitory effects of bis-netropsins on these two stages of the reaction catalyzed by helicase UL9.