Enhancement of rubella virus replication in swine testicle cells by co-infection with hog cholera virus.
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Animals↗
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Results for “Virus Replication”
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The DNA-A component of a plant geminivirus (Indian mung bean yellow mosaic virus) replicates in budding yeast cells.
Understanding the biochemistry of DNA replication of the plant DNA viruses is important for the development of antiviral strategies. Since DNA replication is little studied in plants, a genetically tractable, easily culturable, eukaryotic model system is required to pursue such studies in a facile manner. Here we report the development of a yeast model system that supports DNA replication of a chosen geminivirus strain, Indian mung bean yellow mosaic virus. The replication of plasmid DNA in the model system relies specifically on the virus-derived elements and factors. Usage of this model system revealed the role of at least one hitherto unknown viral factor for viral DNA replication. The episomal characteristic of single-strandedness of replicated plasmid DNA was shown, and the expression of viral genes was also confirmed. This model system is expected to shed light on the machinery and mechanism involved in geminiviral DNA replication in plants.
Base Sequence↗
The human herpesvirus 6 G protein-coupled receptor homolog U51 positively regulates virus replication and enhances cell-cell fusion in vitro.
Human herpesvirus 6 (HHV-6) is a ubiquitous T-lymphotropic betaherpesvirus that encodes two G protein-coupled receptor homologs, U12 and U51. HHV-6A U51 has been reported to bind to CC chemokines including RANTES, but the biological function of U51 remains uncertain. In this report, we stably expressed short interfering RNAs (siRNAs) specific for U51 in human T cells and then infected these cells with HHV-6. Viral DNA replication was reduced 50-fold by the U51 siRNA, and virally induced cytopathic effects were also inhibited. In contrast, viral replication and syncytium formation were unaltered in cells that expressed a scrambled derivative of the siRNA or an irrelevant siRNA and were restored to normal when a human codon-optimized derivative of U51 was introduced into cells containing the U51 siRNA. To examine the mechanism whereby U51 might contribute to viral replication, we explored the signaling characteristics of U51. None of the chemokines and opioids tested was able to induce G protein coupling by U51, and no evidence for opioid ligand binding by U51 was obtained. The effect of U51 on cell-cell fusion was also evaluated; these studies showed that U51 enhanced cell fusion mediated by the G protein of vesicular stomatitis virus. However, a U51-specific antiserum had no virus-neutralizing activity, suggesting that U51 may not be involved in the initial interaction between the virus particle and host cell. Overall, these studies suggest that HHV-6 U51 is a positive regulator of virus replication in vitro, perhaps because it may promote membrane fusion and facilitates cell-cell spread of this highly cell-associated virus.
Antibodies, Viral↗
Early- and intermediate-stage variants of simian immunodeficiency virus replicate efficiently in cells lacking CCR5.
Primate lentiviruses are thought to use the chemokine receptor CCR5 as the major coreceptor for entry into cells. Here we show that some variants of simian immunodeficiency virus (SIV) replicate efficiently in peripheral blood mononuclear cells (PBMCs) lacking a functional CCR5. There were differences in the replication patterns of sequential variants that evolved during SIVMne infection; the late-stage pathogenic variants were unable to replicate in PBMCs lacking CCR5, whereas the early- and intermediate-stage viruses replicated as well in PBMCs lacking CCR5 as they did in cells with wild-type CCR5. The coreceptor specificities of these sequential variants were compared using indicator cell lines expressing known SIV coreceptors. Among the known SIV coreceptors, there were none that were functional for the early and intermediate variants but not the late-stage variants, suggesting that the coreceptor used for replication in PBMCs may be a coreceptor that has not yet been described. Because some variants replicate with high efficiency in peripheral blood cells using this as yet uncharacterized cellular receptor, this coreceptor may be important for viral entry of some target cell populations in the host.
Animals↗
Role of RNA structure and RNA binding activity of foot-and-mouth disease virus 3C protein in VPg uridylylation and virus replication.
The uridylylation of the VPg peptide primer is the first stage in the replication of picornavirus RNA. This process can be achieved in vitro using purified components, including 3B (VPg) with the RNA dependent RNA polymerase (3Dpol), the precursor 3CD, and an RNA template containing the cre/bus. We show that certain RNA sequences within the foot-and-mouth disease virus (FMDV) 5' untranslated region but outside of the cre/bus can enhance VPg uridylylation activity. Furthermore, we have shown that the FMDV 3C protein alone can substitute for 3CD, albeit less efficiently. In addition, the VPg precursors, 3B(3)3C and 3B(123)3C, can function as substrates for uridylylation in the absence of added 3C or 3CD. Residues within the FMDV 3C protein involved in interaction with the cre/bus RNA have been identified and are located on the face of the protein opposite from the catalytic site. These residues within 3C are also essential for VPg uridylylation activity and efficient virus replication.
Amino Acid Sequence↗
Deletion or substitution of the aphthovirus 3' NCR abrogates infectivity and virus replication.
The 3' noncoding region (NCR) of the genomic picornaviral RNA is believed to contain major cis-acting signals required for negative-strand RNA synthesis. The 3' NCR of foot-and-mouth disease virus (FMDV) was studied in the context of a full-length infectious clone in which the genetic element was deleted or exchanged for the equivalent region of a distantly related swine picornavirus, swine vesicular disease virus (SVDV). Deletion of the 3' NCR, while maintaining the intact poly(A) tail as well as its replacement for the SVDV counterpart, abrogated virus replication in susceptible cells as determined by infectivity and Northern blot assays. Nevertheless, the presence of the SVDV sequence allowed the synthesis of low amounts of chimeric viral RNA at extended times post-transfection as compared to RNAs harbouring the 3' NCR deletion. The failure to recover viable viruses or revertants after several passages on susceptible cells suggests that the presence of specific sequences contained within the FMDV 3' NCR is essential to complete a full replication cycle and that FMDV and SVDV 3' NCRs are not functionally interchangeable.
3' Untranslated Regions↗
Ultrastructural study of the renal tubular system in acute experimental African swine fever: virus replication in glomerular mesangial cells and in the collecting ducts.
Despite the considerable attention given to kidney lesions in African swine fever (ASF), a number of questions remain to be answered. Structural and ultrastructural examination showed that a highly virulent isolate of ASF virus (Malawi 83) replicated in glomerular mesangial cells and renal collecting duct epithelial cells, with hyperplasia of the latter in infected pigs. Replication in mesangial cells may be due to their contact with the bloodstream, as well as to their phagocytic capacity and high metabolism rate. Virus replication in macrophages and endothelial cells of interstitial capillaries, and the necrosis of these infected cells gave rise to a large number of free virus in interstitial tissue. This, together with the lesser thickness of the basal membrane of collecting ducts in comparison to the rest of the tubular system, probably facilitates ASFV infection of tubular epithelial cells. Virus replication in these cells may account for the presence of virus in the urine of pigs with acute ASF where haematuria is not observed.
Acute Disease↗
Tissue culture of isolated human pancreatic islets infected with different strains of coxsackievirus B4: assessment of virus replication and effects on islet morphology and insulin release.
The aim was to study whether different strains of Coxsackievirus B4 (CBV-4) are able to infect human pancreatic islet cells in vitro and cause morphological and functional damages. Isolated islets maintained in tissue culture were infected with five well- characterised strains of CBV-4. Aliquots of the culture medium were analysed with regard to virus replication and insulin content. Infected and uninfected islets were examined by light microscopy to determine the degree of virus-induced cytopathic effect (CPE). The results showed that the islet cells were susceptible to infection by all the strains of CBV-4 although the outcome of the infection differed. The virus titres obtained at 48 and 72 hours post infection differed significantly between all the CBV-4 strains (p<0.001), indicating different ability to replicate in islet cells. Pronounced to weak CPE, which was partly due to the origin (donor) of the islets, was induced by four of the five CBV-4 strains. One strain (VD2921) replicated without causing CPE despite high virus titres. One (V89-4557) of the CBV-4 strains always revealed pronounced CPE. Infection by this strain also caused functional impairment that significantly affected insulin response to high glucose at 48 hours post infection (p<0.001). Replication of another CBV-4 strain (JVB) in the islet cells significantly increased the release of insulin compared to non-infected control cells (p<0.001) indicating damage of the beta-cells leading to leakage of insulin.
Adolescent↗
Characterization of hepatitis C virus replication in cloned cells obtained from a human T-cell leukemia virus type 1-infected cell line, MT-2.
We recently found that a human T-cell leukemia virus type 1-infected cell line, MT-2, could support the replication of hepatitis C virus (HCV) (N. Kato, T. Nakazawa, T. Mizutani, and K. Shimotohno, Biochem. Biophys. Res. Commun. 206:863-869, 1995). In order to develop a culture system in which HCV replicates more efficiently, we examined the efficiency of HCV replication in cloned MT-2 cell lines by the limiting dilution method. Consequently, we obtained five clones in which intracellular positive-stranded HCV RNA could be detected until at least 21 days postinoculation (p.i.), as opposed to 15 days p.i. in uncloned MT-2 cells. MT-2C, one of the five clones which supported HCV replication up to 30 days p.i., was used for further characterization of HCV replication. Semiquantitative analysis of HCV by PCR revealed that RNA synthesis in infected cells increased after inoculation, reached a maximum level at 4 days p.i., and maintained this level until at least 11 days p.i. The 5' untranslated region of negative-stranded HCV RNA was also detected in the infected cells by two different methods with strand specificity. These results suggest that HCV replicated and multiplied in the MT-2C cells. HCV-infected MT-2C cells that were treated with antibiotics, such as G418 and hygromycin B, sustained HCV RNA for a longer period than did untreated cells. We demonstrated inhibitory effects on HCV replication by an antisense oligonucleotide complementary to the HCV core encoding region and by interferon-alpha. Furthermore, cell-free viral transmission was demonstrated by this culture system. These results suggest that our cell culture system will be useful for studying the mechanism of HCV replication, for screening antiviral agents, and for developing HCV vaccines.
Cell Line, Transformed↗
A cis-acting element that directs circular adeno-associated virus replication and packaging.
A novel pathway of adeno-associated virus (AAV) replication marked by the assembly of circular monomer duplex intermediates (cAAV) has been recently discovered. In the present report we identify a single AD domain of the inverted terminal repeat as a minimal origin of cAAV replication. A small internal palindrome (BB'), necessary for optimal Rep-inverted terminal repeat interaction, does not contribute to the efficiency of cAAV replication, while the terminal resolution site is an essential cis-acting element. Furthermore, recombinant cAAV vectors that encompass only the AD domain replicate exclusively in a circular form and no detectable linear duplex replicative intermediates are generated, suggesting that both pathways of AAV replication are independent and can be separated. In addition, we show that cAAVs are efficient templates for encapsidation of single-stranded DNA genomes, an observation that assigns a biological role for these novel replication species. Together, these findings shed new light on the current model of AAV replication and packaging.
Base Sequence↗
Hepatitis B virus replication is regulated by the acetylation status of hepatitis B virus cccDNA-bound H3 and H4 histones.
BACKGROUND & AIMS: HBV covalently closed circular DNA (cccDNA), the replicative intermediate responsible for persistent HBV infection of hepatocytes, is the template for transcription of all viral mRNAs. Nuclear cccDNA accumulates as a stable episome organized into minichromosomes by histone and nonhistone proteins. In this study we investigated, by a newly developed sensitive and specific assay, the relationship between viral replication and HBV chromatin assembly, transcription, and interaction with viral and cellular regulatory proteins. METHODS: To achieve this aim we coupled a quantitative chromatin immunoprecipitation (ChIP) technique to an established method that allows the amplification of virion-encapsidated HBV genomes after transfection of linear HBV DNA into human hepatoma HuH7 cells. The cccDNA-ChIP technique was also applied to study HBV minichromosome transcriptional regulation in liver tissue from HBV-infected patients. RESULTS: The use of anti-acetyl-H4/-H3 specific antibodies to immunoprecipitate transcriptionally active chromatin revealed that HBV replication is regulated by the acetylation status of the cccDNA-bound H3/H4 histones. Class I histone deacetylases inhibitors induced an evident increase of both cccDNA-bound acetylated H4 and HBV replication. Finally, histones hypoacetylation and histone deacetylase 1 recruitment onto the cccDNA in liver tissue correlated with low HBV viremia in hepatitis B patients. CONCLUSIONS: We developed a ChIP-based assay to analyze, in vitro and ex vivo, the transcriptional regulation of HBV cccDNA minichromosome. Our results provide new insights on the regulation of HBV replication and identify the enzymatic activities that modulate the acetylation of cccDNA-bound histones as new therapeutic targets for anti-HBV drugs.
Acetylation↗
Monoclonal antibodies specific for wild mouse neurotropic retrovirus: detection of comparable levels of virus replication in mouse strains susceptible and resistant to paralytic disease.
We used AKR/J mice to produce monoclonal antibodies specific for a neurotropic ecotropic (WM-E) virus initially isolated from wild mice. The rationale for this approach involved the observation that these mice were immunologically hyporesponsive to endogenous ecotropic virus (Akv) but fully responsive to type-specific determinants of WM-E. Hybridoma cell lines derived from mice immunized with both denatured and viable virus produced antibodies with specificity for three viral membrane-associated polypeptides, gp70, p15(E), and p15gag. Epitopes specific for WM-E virus were detected in each of these polypeptides. Cross-reactivity with Friend ecotropic virus (Friend murine leukemia virus) was observed with some gp70- and p15gag-specific antibodies, but no reactivity with endogenous Akv ecotropic virus was seen. The majority of these antibodies did not react with either xenotropic or mink cell focus-forming viruses. Two WM-E-specific anti-gp70 antibodies reacting with different determinants had virus-neutralizing activity in the absence of complement, suggesting that the respective epitopes may participate in receptor binding or virus penetration events. We used these monoclonal antibodies in initial studies to examine the replication of WM-E virus in neonatally inoculated AKR/J mice which are fully resistant to the paralytic disease induced by this virus. Since these mice express high levels of endogenous ecotropic virus, standard assays for ecotropic virus cannot be used to study this question. We present evidence that the resistance to disease does not involve a resistance to virus replication, since these mice expressed levels of viremia and virus replication in spleen and lumbar spinal cord comparable to susceptible NFS/N mice at a time when the latter began to manifest clinical signs of lower-motor-neuron pathology.
Animals↗
In vivo administration to rhesus monkeys of a CD4-specific monoclonal antibody capable of blocking AIDS virus replication.
Monoclonal antibodies (mAbs) specific for CD4 are potent inhibitors of HIV replication in vitro. These agents may be useful prophylactically or in chronic HIV infection if they can be administered without inducing immunosuppression. In the present study, we explored the safety of a CD4-specific murine mAb in rhesus monkeys. The mAb 5A8, which binds to domain 2 of the CD4 molecule, inhibits AIDS virus replication noncompetitively at a postvirus binding step. This antibody, which had a similar affinity for rhesus monkey and human CD4 cells, efficiently inhibited in vitro replication of both HIV-1 and the simian immunodeficiency virus of macaques. A single 3-mg/kg injection of mAb 5A8 into normal rhesus monkeys coated all circulating and lymph node CD4 cells for 4-6 days. CD4 cells were not cleared from circulation nor was the CD4 molecule modulated from the lymphocyte surface. In fact, administration of mAb 5A8 resulted in an approximately one-to twofold increase in absolute number of circulating CD4 cells. Repeated administration in normal rhesus monkeys resulted in CD4 lymphocyte coating with mAbs for > 9 days without CD4 cell clearance or modulation. While coated with mAbs, PBLs of these monkeys retained normal in vitro proliferative responses to mitogens and these animals generated normal humoral responses in vivo to tetanus toxoid. Loss of cell coating with mAbs in normal monkeys corresponded to the appearance of anti-mouse immunoglobulin antibodies. Thus, administration of certain anti-CD4 mAbs capable of blocking HIV replication can achieve coating of the entire CD4 cell pool in rhesus monkeys without inducing significant cell loss or immunosuppression.
Animals↗
Electron-microscopic morphology of virus replication in cells of the murine Borstel-leukemia X 429.
Intraperitoneal injections of cell-free filtrates of Borstel-leukemia X 429 (immature-cell myeloid leukemia induced by 200 rad whole-body X-irradiation) were given to 330 1-week-old NMRI mice of both sexes. Leukemia developed in 21% after injection of cell-free filtrates from leukemic spleens and in 20% after injection of cell-free filtrates from leukemic lymph nodes. The mean time lapse from injection to the appearance of the leukemia was 12.8 months. The virus replication in the newly formed leukemic cells, was studied by electron microscopy. Special attention was given to the relation between A-particles and mature type-C viruses. The leukemia cells contain intracytoplasmic and intracisternal A-particles consisting of two concentric spherical shells with an external diameter of 70 nm. A-particles present mainly as closed "virus fields" in the vicinity of the cell nucleus, frequently in the Golgi area. The intracisternal A-particles appear inside the lumen of expanded vesicles of the cytoplasma. Typ-C viruses develop on the cytoplasmic membrane and/or on the outer cell membrane by condensation of crescent-shaped electron-dense zones. All layers of the later mature virus are recognisable in these early morphological stages. After the virus has been detached from the cell it collapses, and the outer membrane appears wrinkled. No spatial correlation between the storage sites of the A-particles and the sites of formation of mature type-C viruses are demonstrable.
Animals↗
Expression of chimeric tRNA-driven antisense transcripts renders NIH 3T3 cells highly resistant to Moloney murine leukemia virus replication.
NIH 3T3 cells infected with Moloney murine leukemia virus (MoMLV) express high levels of virus-specific RNA. To inhibit replication of the virus, we stably introduced chimeric tRNA genes encoding antisense templates into NIH 3T3 cells via a retroviral vector. Efficient expression of hybrid tRNA-MoMLV antisense transcripts and inhibition of MoMLV replication were dependent on the use of a particular type of retroviral vector, the double-copy vector, in which the chimeric tRNA gene was inserted in the 3' long terminal repeat. MoMLV replication was inhibited up to 97% in cells expressing antisense RNA corresponding to the gag gene and less than twofold in cells expressing antisense RNA corresponding to the pol gene. RNA and protein analyses suggest that inhibition was exerted at the level of translation. These results suggest that RNA polymerase III-based antisense inhibition systems can be used to inhibit highly expressed viral genes and render cells resistant to viral replication via intracellular immunization strategies.
Animals↗
Glycoprotein 110, the Epstein-Barr virus homolog of herpes simplex virus glycoprotein B, is essential for Epstein-Barr virus replication in vivo.
The Epstein-Barr virus (EBV) glycoprotein gp110 has substantial amino acid homology to gB of herpes simplex virus but localizes differently within infected cells and is essentially undetectable in virions. To investigate whether gp110, like gB, is essential for EBV infection, a selectable marker was inserted within the gp110 reading frame, BALF4, and the resulting null mutant EBV stain, B95-110HYG, was recovered in lymphoblastoid cell lines (LCLs). While LCLs infected with the parental virus B95-8 expressed the gp110 protein product following productive cycle induction, neither full-length gp110 nor the predicted gp110 truncation product was detectable in B95-110HYG LCLs. Infectious virus could not be recovered from B95-110HYG LCLs unless gp110 was provided in trans. Rescued B95-110HYG virus latently infected and growth transformed primary B lymphocytes. Thus, gp110 is required for the production of transforming virus but not for the maintenance of transformation of primary B lymphocytes by EBV.
B-Lymphocytes↗
The DNA binding domain of herpes simplex virus type 1 origin binding protein is a transdominant inhibitor of virus replication.
The origin binding protein (OBP) of herpes simplex virus (HSV) type 1 specifically interacts with two high-affinity sites in each HSV DNA replication origin. The sequence-specific DNA binding activity of OBP maps to the carboxy-terminal one-third of the protein. For a single binding site, recombinantly expressed forms of this DNA binding domain have the same sequence specificity and binding affinity as the full-length OBP. However, unlike the full-length protein, truncated OBP does not bind HSV replication origins in a cooperative manner. To determine if cooperative interactions between DNA-bound OBP molecules are essential for viral DNA replication, the 317-amino-acid carboxy-terminal DNA binding domain of OBP was expressed in chick embryo fibroblasts. Cells were infected with HSV type 1, and viral DNA synthesis and virus production were monitored. We found that cells expressing truncated OBP were severely restricted for virus replication and that HSV DNA synthesis was undetectable. The results demonstrate that the amino-terminal two-thirds of OBP is essential for HSV DNA replication and that the OBP DNA binding domain acts as a transdominant inhibitor of viral DNA replication. The results also suggest that this experimental approach could be used to generate a refined map of essential OBP functions and that the approach may be generally applicable to the analysis of the multifunction HSV DNA replication complex.
Animals↗
Why do human hepatitis viruses replicate so poorly in cell cultures?
The five viruses which classically cause hepatitis in man represent diverse families of viruses and share in common only a striking hepatotropism and substantial restrictions to replication in conventional cell cultures. Hepatitis A virus is unique among these viruses in that it is amenable to propagation in cell culture, but replication of this virus is much slower and less efficient than replication of other picornaviruses. This probably reflects less efficient cap-independent viral translation, as well as restrictions at other points in the replication cycle. We speculate that the significantly restricted replication of hepatitis viruses in cell culture reflects evolutionary forces controlling their transmission and propagation through human populations.
Base Sequence↗