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PubMed1981

A region of the polyoma virus genome between the replication origin and late protein coding sequences is required in cis for both early gene expression and viral DNA replication.

Deletion mutants within the Py DNA region between the replication origin and the beginning of late protein coding sequences have been constructed and analysed for viability, early gene expression and viral DNA replication. Assay of replicative competence was facilitated by the use of Py transformed mouse cells (COP lines) which express functional large T-protein but contain no free viral DNA. Viable mutants defined three new nonessential regions of the genome. Certain deletions spanning the PvuII site at nt 5130 (67.4 mu) were unable to express early genes and had a cis-acting defect in DNA replication. Other mutants had intermediate phenotypes. Relevance of these results to eucaryotic "enhancer" elements is discussed.

Animals↗

PubMed2004

Impact of E1a modifications on tumor-selective adenoviral replication and toxicity.

Replicating adenoviral vectors are capable of multiplying up to a thousandfold in the target cell, a property that might prove to be of tremendous potential for cancer therapy. However, restricting viral replication and toxicity to cancer cells is essential to optimize safety. It has been proposed that modifications of the E1a protein that impair binding to Rb or p300 will prevent S-phase induction in normal cells, resulting in selective viral replication in tumor cells. However, it remains uncertain which of the several possible E1a modifications would be most effective at protecting normal cells without compromising the oncolytic effect of the vector. In this study, we have expressed several E1a-deletion mutants at high levels using the CMV promoter and tested them for their ability to facilitate S-phase induction, viral replication, and cytotoxicity in both normal and cancer cells. Deletion of the Rb-binding domain within E1a only slightly decreased the ability of the virus to induce S phase in growth-arrested cells. The effect of this deletion on viral replication and cytotoxicity was variable. There was reduced cytotoxicity in normal bronchial epithelial cells; however, in some normal cell types there was equal viral replication and cytotoxicity compared with wild type. Deletions in both the N-terminus and the Rb-binding domain were required to block S-phase induction effectively in growth-arrested normal cells; in addition, this virus demonstrated reduced viral replication and cytotoxicity in normal cells. An equally favorable replication and cytotoxicity profile was induced by a virus expressing E1a that is incapable of binding to the transcriptional adapter motif (TRAM) of p300. All viruses were equally cytotoxic to cancer cells compared with wild-type virus. In conclusion, deletion of the Rb-binding site alone within E1a may not be the most efficacious means of targeting viral replication and toxicity. However, deletion within the N-terminus in conjunction with a deletion within the Rb-binding domain, or deletion of the p300-TRAM binding domain, induces a more favorable cytotoxicity profile.

Adenoviridae↗

PubMed1997

DnaJ/hsp40 chaperone domain of SV40 large T antigen promotes efficient viral DNA replication.

The amino-terminal domain of SV40 large tumor antigen (TAg) is required for efficient viral DNA replication. However, the biochemical activity associated with this domain has remained obscure. We show here that the amino-terminal domain of TAg shares functional homology with the J-domain of DnaJ/hsp40 molecular chaperones. DnaJ proteins function as cofactors by regulating the activity of a member of the 70-kD heat shock protein family. Genetic analyses demonstrated that amino-terminal sequences of TAg comprise a novel J-domain that mediates a specific interaction with the constitutively expressed hsc70 and show that the J-domain is also required for efficient viral DNA replication in vivo. Furthermore, we demonstrated that the J-domain of two human DnaJ homologs, HSJ1 or DNAJ2, could substitute functionally for the amino-terminus of TAg in promoting viral DNA replication. Together, our findings suggest that TAg uses its J-domain to support SV40 DNA replication in a manner that is strikingly similar to the use of Escherichia coli DnaJ by bacteriophage lambda in DNA replication. However, TAg has evolved a more efficient strategy of DNA replication through an intrinsic J-domain to associate directly with a partner chaperone protein. Our observations provide evidence of a role for chaperone proteins in the process of eukaryotic DNA replication.

Amino Acid Sequence↗

PubMed2004

Loss of viral control in early HIV-1 infection is temporally associated with sequential escape from CD8+ T cell responses and decrease in HIV-1-specific CD4+ and CD8+ T cell frequencies.

The outcome of human immunodeficiency virus type 1 (HIV-1) infection is related to the set-point plasma virus load (pVL) that emerges after primary HIV-1 infection (PHI). This set-point pVL generally remains stable but eventually increases with progression to disease. However, the events leading to loss of viremic control are poorly understood. Here, we describe an individual who presented with symptomatic PHI and subsequently progressed rapidly, after an initial period of 1 year during which viral replication was well controlled. Escalation of viral replication in this atypical case was preceded by the emergence of escape variants in many epitopes targeted by dominant CD8+ T cell responses and a marked decrease in HIV-1-specific CD4+ and CD8+ T cell frequencies. There were no changes in viral tropism, replication kinetics, or neutralizing antibody titers. These findings demonstrate the temporal relationship between viral escape from CD8+ T cell activity, decrease in HIV-1-specific T cell frequencies, and loss of control of viral replication.

Adult↗

PubMed2004

The density of coreceptors at the surface of CD4+ T cells contributes to the extent of human immunodeficiency virus type 1 viral replication-mediated T cell death.

Chemokine receptors serve as coreceptors for HIV-1 entry into CD4(+) T cells. Several reports have mentioned that density of CCR5 expression modulates in vitro viral replication and in vivo the course of the disease. Our goal was to investigate the impact of coreceptor density at the surface of a CD4(+) cell line on HIV-1 entry, replication, spreading, and programmed cell death. We engineered a CEM cell line that expresses constitutively CD4 and CXCR4 and CCR5 after transfection. This model allows us to compare the effect of the X4 and R5 strains to induce T cell death in the same T cell host. We show here that the extent of T cell death correlates with the rate of virus replication. X4 induces faster T cell death than R5 that depends at least in part on the higher density of CXCR4 compared to CCR5. Furthermore, sorting CEM populations expressing low, intermediate, and high densities of CCR5 molecules but constant amount of CD4, we found that the capacity to induce T cell death depends at least in part on the level of CCR5 when low amount of virus was used to infect the CEM cells. Moreover, viral transcription, assessed by cell-associated HIV-1 RNA/DNA ratio, was increased in CCR5high as compared to CCR5low cells, while inhibition of replication by zidovudine was more effective in CCR5low cells. Our data indicate that the density of chemokine receptors expressed on CD4(+) T cells may be a critical parameters for the cytopathic effect of HIV strains and may have major impact on CD4 T cell depletion during HAART.

Apoptosis↗

PubMed1995

Rep protein of tomato yellow leaf curl geminivirus has an ATPase activity required for viral DNA replication.

The Rep protein of geminiviruses is the sole viral protein required for their DNA replication. The amino acid sequence of Rep protein contains an NTP binding consensus motif (P-loop). Here we show that purified Rep protein of tomato yellow leaf curl virus expressed in Escherichia coli exhibits an ATPase activity in vitro. Amino acid exchanges in the P-loop sequence of Rep causes a substantial decrease or loss of the ATPase activity. In vivo, mutant viruses carrying these Rep mutations do not replicate in plant cells. These results show that ATP binding by the Rep protein of geminiviruses is required for its function in viral DNA replication.

Adenosine Triphosphatases↗

PubMed2006

Antisense oligonucleotide inhibition of hepatitis C virus genotype 4 replication in HepG2 cells.

BACKGROUND: Hepatitis C (HCV) viral infection is a serious medical problem in Egypt and it has a devastating impact on the Egyptian economy. It is estimated that over 15% of Egyptians are infected by the virus and thus finding a cure for this disease is of utmost importance. Current therapies for hepatitis C virus (HCV) genotype 4 with interferon/ribavirin have not been successful and thus the development of alternative therapy for this genotype is desperately needed. RESULTS: Although previous studies utilizing viral subgenomic or full cDNA fragments linked to reporter genes transfected into adhered cells or in a cell free system showed promise, demonstration of efficient viral replication was lacking. Thus, we utilized HepG2 cells infected with native HCV RNA genomes in a replication competent system and used antisense phosphorothioate Oligonucleotides (S-ODN) against stem loop IIId and the AUG translation start site of the viral polyprotein precursor to monitor viral replication. We were able to show complete arrest of intracellular replication of HCV-4 at 1 uM S-ODN, thus providing a proof of concept for the potential antiviral activity of S-ODN on native genomic replication of HCV genotype 4. CONCLUSION: We have successfully demonstrated that by using two S-ODNs [(S-ODN1 (nt 326-348) and S-ODN-2 (nt 264-282)], we were able to completely inhibit viral replication in culture, thus confirming earlier reports on subgenomic constructs and suggesting a potential therapeutic value in HCV type 4.

Journal Article↗

PubMed2004

Higher CD4+ T cell counts associated with low viral pol replication capacity among treatment-naive adults in early HIV-1 infection.

BACKGROUND: Infection with primary drug-resistant human immunodeficiency virus type 1 (HIV-1) has been associated with higher CD4(+) T cell counts in drug-naive patients, suggesting that altered viral pol replication capacity (RC) associated with drug resistance diminishes immune injury in vivo, independent of exposure to drugs. METHODS: Virus replication over a single cycle was measured by use of a viral test vector containing patient-derived HIV-1 protease and reverse transcriptase gene segments. RESULTS: Among 191 recently infected patients, pol RC ranged widely, with only 6% of the variance explained by drug-resistance mutations. Patients infected with a virus with a low pol RC (</=43% of the reference virus) had significantly higher CD4(+) T cell counts at study entry, independent of drug resistance and plasma HIV-1 RNA level, and over time, both before and during combination antiretroviral therapy. CONCLUSIONS: Viral pol RC may influence HIV-1 disease progression by affecting the amount and tissue distribution of viral replication. The pol RC value of 43% may represent a threshold below which HIV-1 has lowered virulence and is less able to deplete CD4(+) T cell counts.

Adult↗

PubMed1983

Immunological characterization of the role of adenovirus terminal protein in viral DNA replication.

The function of the adenovirus-coded terminal protein and its precursor in viral DNA replication was studied by raising an antiserum against the adenovirus type 5 (Ad5) terminal protein isolated from virions. This antiserum reacted with both the terminal protein and its precursor as measured by a radioimmunoassay. In an in vitro DNA replication system employing nuclear extracts the addition of antiserum inhibits replication when a DNA-terminal protein complex from adenovirions is used as template. The replication of a 3.8% terminal fragment of the Ad2 genome with a protein-free origin (derived from the plasmid XD-7) is also inhibited by the antiserum. This observation confirms a role of the terminal protein precursor in DNA replication. The antiserum completely inhibited the formation of a covalent complex between the precursor terminal protein and dCMP, which is essential for initiation. A function of the terminal protein in the elongation reaction was shown by the inhibitory effect of antiserum on DNA chain elongation in isolated nuclei from Ad5-infected cells. Also in the in vitro DNA replication system employing nuclear extracts the elongation reaction is strongly reduced by addition of the antiserum. These results indicate that the terminal protein and/or its precursor are not only involved in initiation of DNA replication but also in DNA chain elongation.

Adenovirus Infections, Human↗

PubMed1986

Therapy with cyclosporine in experimental murine myocarditis with encephalomyocarditis virus.

To explain the progression from infectious viral myocarditis to congestive cardiomyopathy an infection/immune hypothesis has been proposed stating that the primary viral process incites an excessive or disordered immunologic response against the myocardium. To test whether one form of immunosuppressive therapy might ameliorate this process, we used cyclosporine in a murine preparation of infectious myocarditis (encephalomyocarditis [EMC] virus), which has been shown to result in a congestive cardiomyopathy pathologically similar to that seen in man. Eight-week-old male DBA-2 mice were infected with EMC virus and randomized to a treatment or control group. Cyclosporine (25 mg/kg/day) was administered subcutaneously for 3 weeks, starting (1) at 1 week after infection during viral replication, and (2) at 3 weeks after infection, after the period of active viral replication. In mice treated during viral replication there was a significantly higher mortality rate compared with that of control mice (15/21 vs 9/29, p = .01). There was no evident reduction in myocardial pathology (inflammation, necrosis, or calcification) in the treated compared with the control groups. In mice treated after the period of viral replication, there was no improvement in mortality (8/22 vs 2/19, NS) compared with control. Treated mice showed no reduction in myocardial histopathologic lesions. Furthermore, treated mice had significantly greater heart weight/body weight ratios (1.3 +/- 0.4% vs 1.0 +/- 0.3%, p less than .005), lung weight/body weight ratios (1.1 +/- 0.5% vs 0.8 +/- 0.3%, p less than .05), and liver weight/body weight ratios (6.0 +/- 0.8% vs 5.4 +/- 0.6%, p less than .005) than control mice, suggesting more severe myocardial failure.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1992

Zidovudine inhibits hepatitis B virus replication.

Hepatitis B virus DNA polymerase is a viral enzyme that can use viral DNA as well as viral RNA as a template for DNA synthesis. Since both activities are essential for the production of new virus particles, blocking of this enzyme should reduce viral replication. In the present study the in vitro effect of zidovudine triphosphate on hepatitis B virus DNA polymerase activity and the in vivo effect of zidovudine on viral replication in chronic HBsAg-positive patients are investigated. Zidovudine triphosphate inhibited in vitro DNA polymerase activity by 50% at a concentration of 0.3 microM. Serum DNA polymerase activity was significantly reduced in 7 patients who received zidovudine (200 mg orally 4 times daily) for one week. A dose-response effect was suggested by the results found for 6 patients who received 100 mg, 200 mg and 300 mg orally 4 times daily for one week with 2 drug-free weeks between each course. We conclude that zidovudine may be of value for non-responders to alpha-interferon therapy or patients with high initial levels of viral replication prior to the start of interferon treatment.

Administration, Oral↗

PubMed2005

Adenovirus vectors deleted for genes essential for viral DNA replication.

Adenovirus (Ad) gene therapy vectors made replication defective by deletion of the E1 region (first-generation vectors) induce high-level inflammation that leads to loss of both transduced gene expression and transduced cells. First-generation vectors were initially considered to be incapable of viral DNA replication, but it is necessary to delete one or more of the genes, all in the E2 transcription unit, that encode proteins essential for Ad DNA replication to completely eliminate viral DNA replication. Vectors deleted for one or more of the E2 genes (second-generation vectors) induce reduced levels of inflammation in certain animal models and offer promise for understanding the mechanisms by which adenovirus vectors induce inflammation and how inflammation can be inhibited. While first-generation vectors dominated the initial human gene therapy trials using adenovirus vectors, second-generation vectors may offer greater promise and greater safety for clinical studies.

Adenoviridae↗

PubMed2002

The replication of human immunodeficiency virus type 1 in macrophages is enhanced after phagocytosis of apoptotic cells.

Clearance of apoptotic cells increases macrophage secretion of antiinflammatory mediators and might modulate viral replication in human immunodeficiency virus (HIV) type 1-infected macrophages. To study this, primary macrophages were infected with HIV-1 and exposed to apoptotic cells. It was found that phagocytosis of apoptotic cells potently enhanced HIV-1 growth. The peptide Arg-Gly-Asp-Ser, which binds to integrin receptors, inhibited the uptake of apoptotic cells and the subsequent enhancement of HIV-1 replication. Viral replication was preceded by increased secretion of transforming growth factor (TGF)-beta1 and partially reverted by anti-TGF-beta1 antibodies. Moreover, anti-TGF-beta1 antibodies inhibited HIV-1 replication in macrophages not exposed to apoptotic cells. A positive correlation was observed between TGF-beta1 production and HIV-1 growth, and the addition of TGF-beta1 amplified HIV-1 replication in macrophages from low TGF-beta1 producers. The findings suggest that TGF-beta1 favors HIV-1 replication in macrophages and that the clearance of apoptotic cells by HIV-1-infected macrophages contributes to persistent viremia in patients infected with HIV-1.

Apoptosis↗

PubMed2026

hnRNPC facilitates coronavirus replication by directly binding the frameshift-stimulatory element of viral genomic RNA.

Translation of key viral replicative proteins in coronaviruses requires a programmed -1 ribosomal frameshifting (-1 PRF) event controlled by the viral frameshift-stimulatory element (FSE). Although previous studies have analyzed host factor dependencies of coronaviruses, how host cellular factors alter -1 PRF efficiency and affect viral replication remains poorly understood. Here, using RNA pull-down combined with LC-MS/MS analysis, we identified heterogeneous nuclear ribonucleoprotein C (hnRNPC) as a major interacting protein of FSE RNA. Coronavirus infection triggers hnRNPC mRNA decay, alters hnRNPC protein levels, and induces its cytoplasmic relocalization, where it appears to bind directly to FSE RNA through residues Asn7 and Asn83. This binding is associated with increased -1 PRF efficiency and may facilitate coronavirus replication. Deletion mapping analysis shows that hnRNPC preferentially binds U-rich regions of the FSE RNA. Finally, we demonstrated that the small molecule Elbasvir directly binds hnRNPC, disrupting the interaction between hnRNPC and FSE RNA and inhibiting coronavirus replication by decreasing -1 PRF efficiency. Collectively, our study identifies hnRNPC as a key host cofactor for coronaviruses and provides a novel target for broad-spectrum antiviral drug development.

RNA, Viral↗

PubMed1984

Independent mutations in Ad2ts111 cause degradation of cellular DNA and defective viral DNA replication.

An adenovirus mutant, Ad2ts111, has previously been shown to be temperature sensitive for viral DNA replication in vivo and also to induce degradation of cellular DNA. Soluble nuclear extracts prepared from Ad2ts111-infected HeLa cells grown at either the permissive (32 degrees C) or the nonpermissive (39.5 degrees C) temperature are thermolabile for elongation but not for initiation of DNA replication in vitro. Adenovirus single-stranded-DNA-binding protein purified from wild-type-infected cells can complement these extracts at the restrictive temperature in vitro. The DNA-binding protein synthesized in Ad2ts111-infected cells is stable at the nonpermissive temperature and is phosphorylated, as is the wild-type protein. In contrast, the mutant DNA-binding protein synthesized in Ad5ts125-infected cells is unstable. Ad2ts111 and Ad5ts125 do not complement each other for virus growth in vivo. These results suggest that Ad2ts111 contains a mutation in the DNA-binding protein that affects viral DNA synthesis. Finally, we demonstrated that, unlike viral DNA synthesis, the induction of cellular DNA degradation in Ad2ts111-infected cells is not temperature sensitive and that this phenotype is a result of a mutation in early region 1 on the virus genome. Thus, the two phenotypes displayed in Ad2ts111-infected cells, namely, the temperature-sensitive replication of viral DNA and the degradation of cell DNA, are the result of two separate mutations.

Adenoviruses, Human↗

PubMed1995

Expression of human cytomegalovirus UL36 and UL37 genes is required for viral DNA replication.

It was previously reported that the region encoding human cytomegalovirus (HCMV) genes UL36 to UL38 was required for origin-dependent DNA replication. These genes encode transactivators that upregulate viral and cellular transcription. However, their requirement for viral DNA replication has not been demonstrated. We have now used an antisense phosphorothioate oligonucleotide complementary to the intron-exon boundary of the UL36 and UL37 unspliced RNA to show that these gene products are required for HCMV DNA replication. Southern analysis showed that this oligonucleotide almost completely inhibits HCMV DNA replication when used at concentrations as low as 0.08 microM. The ability of this oligonucleotide to inhibit DNA replication was not the result of an inhibition of virus adsorption. Southern blots showed no impairment of viral adsorption or internalization in the presence of either specific or nonspecific phosphorothioate oligonucleotides. In addition, Northern (RNA) blots confirm that this antisense compound specifically reduced UL36 mRNA in treated cells to undetectable levels while the steady-state levels of immediate-early transcripts IE1 and IE2 were unaffected. These results demonstrate that the UL36 and UL37 gene products provide an essential function in initiation of HCMV DNA replication.

Amino Acid Sequence↗

PubMed1990

[Mechanism mediating HIV replication in infected individuals].

A long asymptomatic period is one of the characteristics of human immunodeficiency virus (HIV). How actively HIV replicates in vivo and what controls viral replication during this period remains to be clarified. Proviral DNA integrated into the cellular DNA of peripheral blood lymphocytes (PBL) is not completely silent, but the viral antigens in PBL are extremely low. Several mechanisms have been proposed to explain the low levels of viral replication: i) the regulatory genes encoded in HIV might control the expression of viral DNA, ii) the majority of the proviruses might be defective, iii) host immune systems such as T cell-mediated HIV-specific cytotoxicity and antibody-dependent cellular cytotoxicity might eliminate infected cells, and iv) CD8+ lymphocytes might suppress spontaneous viral replication in PBL.

Acquired Immunodeficiency Syndrome↗

PubMed1999

Protein 2A of grapevine fanleaf nepovirus is implicated in RNA2 replication and colocalizes to the replication site.

RNA2 of grapevine fanleaf virus is replicated in trans by the RNA1-encoded replication machinery. Full processing of the RNA2-encoded polyprotein P2 yields protein 2A of unknown function, the movement protein 2B(MP), and the coat protein 2C(CP). Analysis of a set of deletion mutants in the P2-coding sequence revealed that protein 2A is necessary but not sufficient for RNA2 replication. In addition to the 5' and 3' noncoding sequences and the 2A-coding sequence, an additional sequence coding for 2B(MP) and/or 2C(CP) or the green fluorescent protein (GFP) is necessary for RNA2 replication. When 2A fused to GFP (2AGFP) was transiently expressed in uninfected T-BY2 protoplasts, 2AGFP appeared as punctate structures evenly distributed in the cytoplasm. However, in cells cotransfected with grapevine fanleaf virus RNAs and the 2AGFP construct, 2AGFP was predominantly found in a juxtanuclear location along with 1D(pro) and 1C(VPg), two RNA1-encoded proteins involved in RNA replication. Viral RNA replication as traced by 5-bromouridine 5' triphosphate (BrUTP) incorporation into newly synthesized RNA occurred at the same location. This colocalization is consistent with the hypothesis that 2A enables RNA2 replication through its association with the replication complex assembled from RNA1-encoded proteins.

Base Sequence↗