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PubMed1995

Recombinational rescue of the stalled DNA replication fork: a model based on analysis of an Escherichia coli strain with a chromosome region difficult to replicate.

To examine the physiological effects of DNA replication arrest at the terminus (Ter), we constructed a replication-blocked Escherichia coli strain so that both bidirectional replication forks would be impeded at two flanking Ter sites, one artificial and the other natural. While the blocked strain grew slightly more slowly than a control strain, it had abnormal phenotypes similar to those of E. coli dam mutants, i.e., hyper-Rec phenotype, recA(+)- and recB+ (C+)-dependent growth, and constitutive SOS induction. The observation that these two apparently unrelated mutants cause similar phenotypes led us to design a model. We propose that the following sequential events may occur in both strains. A double-strand (ds) break occurs at the blocked replication fork in the blocked strain and at the ongoing fork in the dam mutant, through which RecBCD enzyme enters and degrades the ds DNA molecule, and the degradation product serves as the signal molecule for SOS induction. When RecBCD enzyme meets an appropriately oriented Chi sequence, its DNase activity is converted to recombinase enzyme, which is able to repair the ds end, recombinationally. this model (i) explains the puzzling phenotype of recA and recB (C) mutants and the SOS-inducing phenotype of polA, lig, and dna mutants under restrictive conditions, (ii) provides an interpretation for the role of the Chi sequence, and (iii) suggests a possible key role for homologous recombination with regard to cell survival following the arrest of DNA replication.

Chromosomes, Bacterial↗

PubMed1979

Replication of bacteriophage phiK duplex replicative-form DNA in dnaB and dnaC mutants of Escherichia coli.

We have directly tested the effects of host cell DNA synthesis mutations on bacteriophage phiK replicative-form (RF) DNA replication in vivo. We observed that phiK RF DNA replication continued at normal rates in both dnaB and dnaC mutant hosts under conditions in which the activities of the dnaB and dnaC gene products were shown to be markedly reduced. This suggests that these two host proteins are not essential for normal phiK RF DNA replication. In control experiments we observed markedly reduced rates of phiK RF DNA replication in temperature-sensitive dnaG and dnaE host mutants, indicating that the products of these genes are essential. Thus, the mechanism of DNA chain initiation in vivo on the duplex RF DNA templates of isometric phages such as phiK apparently is different from that on the similar templates of isometric phages such as phiX174. The implications of this difference are discussed in the text.

Bacterial Proteins↗

PubMed1988

Restricted replication of hepatitis A virus in cell culture: encapsidation of viral RNA depletes the pool of RNA available for replication.

The replication of hepatitis A virus (HAV) in BS-C-1 cells was examined under single-cycle growth conditions by using strand-specific probes for detection of viral RNA species. No measurable lag phase was demonstrated between accumulation of positive-strand HAV RNA and production of infectious virions, indicating that replication of virion RNA is rate limiting for the production of infectious virus. Intracellular viral RNA was further analyzed by using 2 M LiCl to fractionate the insoluble nonvirion 35S RNA and replicative intermediates (RI) from the soluble virions and double-stranded replicative forms, in conjunction with sucrose density gradient ultracentrifugation to separate the different forms of viral RNA. Throughout the productive phase of HAV infection, 95 to 97% of positive-strand HAV RNA was soluble in 2 M LiCl and was shown to be contained in mature virions. Of the LiCl-insoluble HAV RNA, more than 99% was positive-stranded 35S RNA, whereas 0.4% was negative stranded and had the sedimentation and partial RNase resistance characteristics of RI. The pattern of RNA accumulation in HAV-infected cells is thus very different from that seen in poliovirus-infected cells, where large pools of RI and mRNA are produced before RNA is sequestered into mature virions. The results of this study suggest that encapsidation of positive-strand HAV RNA inhibits transcription at all times during the growth cycle, thereby reducing the pool of replicating RNA and the final yield of infectious HAV.

Capsid↗

PubMed1997

Effects of mutations in the Exo III motif of the herpes simplex virus DNA polymerase gene on enzyme activities, viral replication, and replication fidelity.

The herpes simplex virus DNA polymerase catalytic subunit, which has intrinsic polymerase and 3'-5' exonuclease activities, contains sequence motifs that are homologous to those important for 3'-5' exonuclease activity in other polymerases. The role of one such motif, Exo III, was examined in this study. Mutated polymerases containing either a single tyrosine-to-histidine change at residue 577 or this change plus an aspartic acid-to-alanine at residue 581 in the Exo III motif exhibited defective or undetectable exonuclease activity, respectively, yet retained substantial polymerase activity. Despite the defects in exonuclease activity, the mutant polymerases were able to support viral replication in transient complementation assays, albeit inefficiently. Viruses replicated via the action of these mutant polymerases exhibited substantially increased frequencies of mutants resistant to ganciclovir. Furthermore, when the Exo III mutations were incorporated into the viral genome, the resulting mutant viruses displayed only modestly defect in replication in Vero cells and exhibited substantially increased mutation frequencies. The results suggest that herpes simplex virus can replicate despite severely impaired exonuclease activity and that the 3'-5' exonuclease contributes substantially to the fidelity of viral DNA replication.

Amino Acid Sequence↗

PubMed2002

Sequence requirements for viral RNA replication and VPg uridylylation directed by the internal cis-acting replication element (cre) of human rhinovirus type 14.

Until recently, the cis-acting signals required for replication of picornaviral RNAs were believed to be restricted to the 5' and 3' noncoding regions of the genome. However, an RNA stem-loop in the VP1-coding sequence of human rhinovirus type 14 (HRV-14) is essential for viral minus-strand RNA synthesis (K. L. McKnight and S. M. Lemon, RNA 4:1569-1584, 1998). The nucleotide sequence of the apical loop of this internal cis-acting replication element (cre) was critical for RNA synthesis, while secondary RNA structure, but not primary sequence, was shown to be important within the duplex stem. Similar cres have since been identified in other picornaviral genomes. These RNA segments appear to serve as template for the uridylylation of the genome-linked protein, VPg, providing the VPg-pUpU primer required for viral RNA transcription (A. V. Paul et al., J. Virol. 74:10359-10370, 2000). Here, we show that the minimal functional HRV-14 cre resides within a 33-nucleotide (nt) RNA segment that is predicted to form a simple stem-loop with a 14-nt loop sequence. An extensive mutational analysis involving every possible base substitution at each position within the loop segment defined the sequence that is required within this loop for efficient replication of subgenomic HRV-14 replicon RNAs. These results indicate that three consecutive adenosine residues (nt 2367 to 2369) within the 5' half of this loop are critically important for cre function and suggest that a common RNNNAARNNNNNNR loop motif exists among the cre sequences of enteroviruses and rhinoviruses. We found a direct, positive correlation between the capacity of mutated cres to support RNA replication and their ability to function as template in an in vitro VPg uridylylation reaction, suggesting that these functions are intimately linked. These data thus define more precisely the sequence and structural requirements of the HRV-14 cre and provide additional support for a model in which the role of the cre in RNA replication is to act as template for VPg uridylylation.

Base Sequence↗

PubMed2004

Nef stimulates human immunodeficiency virus type 1 replication in primary T cells by enhancing virion-associated gp120 levels: coreceptor-dependent requirement for Nef in viral replication.

The Nef protein enhances human immunodeficiency virus type 1 (HIV-1) replication through an unknown mechanism. We and others have previously reported that efficient HIV-1 replication in activated primary CD4(+) T cells depends on the ability of Nef to downregulate CD4 from the cell surface. Here we demonstrate that Nef greatly enhances the infectivity of HIV-1 particles produced in primary T cells. Nef-defective HIV-1 particles contained significantly reduced quantities of gp120 on their surface; however, Nef did not affect the levels of virion-associated gp41, indicating that Nef indirectly stabilizes the association of gp120 with gp41. Surprisingly, Nef was not required for efficient replication of viruses that use CCR5 for entry, nor did Nef influence the infectivity or gp120 content of these virions. Nef also inhibited the incorporation of CD4 into HIV-1 particles released from primary T cells. We propose that Nef, by downregulating cell surface CD4, enhances HIV-1 replication by inhibiting CD4-induced dissociation of gp120 from gp41. The preferential requirement for Nef in the replication of X4-tropic HIV-1 suggests that the ability of Nef to downregulate CD4 may be most important at later stages of disease when X4-tropic viruses emerge.

CD4 Antigens↗

PubMed2004

A cis-acting replication element in the sequence encoding the NS5B RNA-dependent RNA polymerase is required for hepatitis C virus RNA replication.

RNA structures play key roles in the replication of RNA viruses. Sequence alignment software, thermodynamic RNA folding programs, and classical comparative phylogenetic analysis were used to build models of six RNA elements in the coding region of the hepatitis C virus (HCV) RNA-dependent RNA polymerase, NS5B. The importance of five of these elements was evaluated by site-directed mutagenesis of a subgenomic HCV replicon. Mutations disrupting one of the predicted stem-loop structures, designated 5BSL3.2, blocked RNA replication, implicating it as an essential cis-acting replication element (CRE). 5BSL3.2 is about 50 bases in length and is part of a larger predicted cruciform structure (5BSL3). As confirmed by RNA structure probing, 5BSL3.2 consists of an 8-bp lower helix, a 6-bp upper helix, a 12-base terminal loop, and an 8-base internal loop. Mutational analysis and structure probing were used to explore the importance of these features. Primary sequences in the loops were shown to be important for HCV RNA replication, and the upper helix appears to serve as an essential scaffold that helps maintain the overall RNA structure. Unlike certain picornavirus CREs, whose function is position independent, 5BSL3.2 function appears to be context dependent. Understanding the role of 5BSL3.2 and determining how this new CRE functions in the context of previously identified elements at the 5' and 3' ends of the RNA genome should provide new insights into HCV RNA replication.

Base Sequence↗

PubMed1995

A murine replication protein accumulates temporarily in the heterochromatic regions of nuclei prior to initiation of DNA replication.

We have analyzed the expression of the murine P1 gene, the mammalian homologue of the yeast MCM3 protein, during the mitotic cell cycle. The MCM3 protein has previously been shown to be of importance for initiation of DNA replication in Saccharomyces cerevisiae. We found that the murine P1 protein was present in the nuclei of mammalian cells throughout interphase of the cell cycle. This is in contrast to the MCM3 protein, which is located in the nuclei of yeast cells only between the M and the S phase of the cell cycle. Detailed analysis of the intranuclear localization of the P1 protein during the cell cycle revealed that it accumulates transiently in the heterochromatic regions towards the end of G1. The accumulation of the P1 protein in the heterochromatic regions prior to activation of DNA replication suggests that the mammalian P1 protein is also of importance for initiation of DNA replication. The MCM2-3.5 proteins have been suggested to represent yeast equivalents of a hypothetical replication licensing factor initially described in Xenopus. Our data support this model and indicate that the murine P1 protein could function as replication licensing factor. The chromosomal localization of the P1 gene was determined by fluorescence in situ hybridization to region 6p12 in human metaphase chromosomes.

3T3 Cells↗

PubMed2003

Toll-like receptor 2 (TLR2) and TLR9 signaling results in HIV-long terminal repeat trans-activation and HIV replication in HIV-1 transgenic mouse spleen cells: implications of simultaneous activation of TLRs on HIV replication.

Opportunistic infections are common in HIV-infected patients; they activate HIV replication and contribute to disease progression. In the present study we examined the role of Toll-like receptor 2 (TLR2) and TLR9 in HIV-long terminal repeat (HIV-LTR) trans-activation and assessed whether TLR4 synergized with TLR2 or TLR9 to induce HIV replication. Soluble Mycobacterium tuberculosis factor (STF) and phenol-soluble modulin from Staphylococcus epidermidis induced HIV-LTR trans-activation in human microvessel endothelial cells cotransfected with TLR2 cDNA. Stimulation of ex vivo spleen cells from HIV-1 transgenic mice with TLR4, TLR2, and TLR9 ligands (LPS, STF, and CpG DNA, respectively) induced p24 Ag production in a dose-dependent manner. Costimulation of HIV-1 transgenic mice spleen cells with LPS and STF or CpG DNA induced TNF-alpha and IFN-gamma production in a synergistic manner and p24 production in an additive fashion. In the THP-1 human monocytic cell line stably expressing the HIV-LTR-luciferase construct, LPS and STF also induced HIV-LTR trans-activation in an additive manner. This is the first time that TLR2 and TLR9 and costimulation of TLRs have been shown to induce HIV replication. Together these results underscore the importance of TLRs in bacterial Ag- and CpG DNA-induced HIV-LTR trans-activation and HIV replication. These observations may be important in understanding the role of the innate immune system and the molecular mechanisms involved in the increased HIV replication and HIV disease progression associated with multiple opportunistic infections.

Adaptor Proteins, Signal Transducing↗

PubMed2002

[High-level mIL-12 expression and replication of replicating adenovirus carrying mIL12 gene in naso-pharyngeal carcinoma cells].

OBJECTIVE: To enhance the therapeutic effects on nasopharyngeal carcinoma by combining treatment with selectively replicating adenovirus and IL12. METHODS: Replicating adenovirus with mouse IL12 gene insert (CNHK200-mIL12) was constructed to transfect nasopharyngeal carcinoma cell lines CNE3 and 915. Adenovirus hexon was detected by immunohistochemical staining and flow cytometry (FCM), and mIL12 expression examined by enzyme-linked immunosorbent assay (ELISA). The replication rates of CNHK200-mIL12 and dl1520 was determined by 50% tissue culture infectious dose (TCID50). RESULTS: Twenty-four hours after transfection with CNHK200-mIL12, most of cells were positive for adenovirus hexon and FCM demonstrated increased positivity rates of 39% and 4% among CNE3 and 915 cells respectively. It was observed that CNHK200-mIL12 replication increased by 1 000 folds with mIL12 expression level reaching as high as 84.5+/-4.6 ng in CNE3 cells and 75.6+/-3.4 ng in 915 cells as determined 72 h after transfection with 1x10(5) PFU CNHK200-mIL12 into 1x10(4) cells. CONCLUSION: CNHK200-mIL12 can replicate in vitro in nasopharyngeal carcinoma cells (dl1520 cells, for instance) with high mIL12 expression, which suggests that CNHK200-mIL12 may potentially be used to treat nasopharyngeal carcinoma.

Adenoviridae↗

PubMed1990

[The role of transposons in replication: Tn5 suppresses ts-mutation for plasmid RP1 replication in Escherichia coli cells].

Effect of restoration by transposon Tn5 of genetic damage in RP1 plasmid replication (named transposon suppression) was described. Hybrid plasmid, a derivative of RP1 and RP4, having ts mutation for replication--tsr12 and deletion in the aphA gene controlling kanamycin resistance, was constructed. Five of derivatives of this plasmid containing transposon Tn5 were made, and the strains containing both the Tn5 integrated into the chromosome and intact hybrid plasmid or the parental plasmid with the replication ts mutation, were constructed. It was shown that transposon Tn5 comprised within the hybrid plasmid or in the chromosome promotes maintenance of these replication defective plasmids in the bacterial culture at a non-permissive temperature and thus suppresses plasmid mutation tsr12. It was determined that the extent of suppression of plasmid replication ts mutation depends on the localization of transposon Tn5.

Conjugation, Genetic↗

PubMed1984

Protein-primed replication of plasmids containing the terminus of the adenovirus genome. I. Characterization of an in vitro DNA replication system dependent on adenoviral DNA sequences.

An in vitro system which replicates plasmid DNA containing the replication origin of adenovirus DNA has been established. Replication of plasmid pLA1 DNA, which contains the left-hand terminus (0-9.4 map units) of adenovirus serotype 5 DNA but which lacks the 55,000-dalton terminal protein, is initiated by a protein-primed mechanism in a manner similar to that found with adenovirus DNA. Initiation of DNA replication using plasmid pLA1 as a template requires (i) that the cloned adenovirus sequence be present at the terminus of a linearized (form III) DNA molecule ( Tamanoi , F., and Stillman , B. W. (1982) Proc. Natl. Acad. Sci. U. S. A., 79, 2221-2225; van Bergen, B. G. M., van der Ley , P. A., van Driel , W., van Mansfield , A. D. M., and van der Vliet , P. A. (1983) Nucleic Acid Res. 11, 1975-1979), and (ii) the presence of the 80,000-dalton precursor to the 55,000-dalton terminal protein and the adenovirus coded DNA-dependent DNA polymerase. In the presence of the four deoxy-nucleoside triphosphates, the preterminal protein, the adenovirus coded DNA binding protein, and an extract prepared from uninfected HeLa nuclei, the adenovirus DNA polymerase can elongate the preterminal-protein dCMP initiation complex formed on pLA1 DNA to full length (6.6 kilobase) DNA molecules. These results suggest that the 55,000-dalton terminal protein covalently linked to the 5' termini of adenovirus DNA is not essential for the replication of this DNA.

Adenoviridae↗

PubMed1999

Demethylation of the Epstein-barr virus origin of lytic replication and of the immediate early gene BZLF1 is DNA replication independent. Brief report.

Epstein-Barr virus (EBV) episomal DNA is extensively methylated in Burkitt lymphoma derived cell lines. In this study we examined whether lytic viral cycle reactivation is dependent on demethylation of critical viral genes. Viral replication was induced in the Burkitt's lymphoma cell line Daudi by the combination of 12-O-tetradecanoylphorbol-13-acetate (TPA) and sodium-butyrate. Two regions necessary for EBV replication, the BZLF1 immediate early region and the origin of lytic cycle replication (ori Lyt) were demethylated during the early phase of the lytic virus cycle. Demethylation was observed while production of new (unmethylated) viral DNA was blocked by phosphonoformic acid (PFA). This suggests that demethylation, which may be instrumental for the onset of the lytic cycle, is an active process independent of viral DNA replication

Antigens, Viral↗

PubMed2005

Regeneration of the replication-associated proteins tandem direct repeat recognition nucleotide sequence at the origin of DNA replication of porcine circovirus type 1.

Four copies of a hexanucleotide (H) sequence are located to the right of the palindrome at the origin of DNA replication of the porcine circovirus type 1 (PCV1) genome. These sequences are organized in two direct tandems, the proximal H1/H2 and the distal H3/H4 repeats, and they have been shown to be binding sites for the essential Rep and Rep' proteins. Previous work demonstrated that infectious PCV1 virion can accommodate a variable number of H sequences at the origin of DNA replication. In this work, mutational analysis was conducted to elucidate the critical core element within the hexanucleotide with respect to self-DNA replication and progeny virus synthesis. It was found that while a single H sequence abutting the palindrome is sufficient for PCV1 viability, a tandem repeat arrangement is the more stable and thus preferred configuration. Within the H sequence, selected nucleotides at specific positions are critical for Rep-associated protein recognition and for viral DNA replication.

Animals↗

PubMed1994

Anatomy of a DNA replication fork revealed by reconstitution of SV40 DNA replication in vitro.

Complete enzymatic replication of DNA from the simian virus 40 origin has been reconstituted with T antigen and highly purified cellular proteins. DNA polymerase-alpha/primase functions primarily to synthesize RNA-DNA primers for initiation of DNA replication at the origin and for priming each Okazaki fragment. A polymerase switching mechanism requiring replication factor C and the proliferating cell nuclear antigen allows two molecules of DNA polymerase-delta to replicate both strands of the double helix conjointly.

Animals↗

PubMed2004

Human cytomegalovirus UL84 oligomerization and heterodimerization domains act as transdominant inhibitors of oriLyt-dependent DNA replication: evidence that IE2-UL84 and UL84-UL84 interactions are required for lytic DNA replication.

Human cytomegalovirus (HCMV) UL84 encodes a 75-kDa protein required for oriLyt-dependent DNA replication and interacts with IE2 in infected and transfected cells. UL84 localizes to the nucleus of transfected and infected cells and is found in viral replication compartments. In transient assays it was shown that UL84 can interfere with the IE2-mediated transactivation of the UL112/113 promoter of HCMV. To determine whether UL84 protein-protein interactions are necessary for lytic DNA synthesis, we purified UL84 and used this protein to generate a monoclonal antibody. Using this antibody, we now show that UL84 forms a stable interaction with itself in vivo. The point of self-interaction maps to a region of the protein between amino acids 151 and 200, a domain that contains a series of highly charged amino acid residues. Coimmunoprecipitation assays determined that UL84 interacts with a protein domain present within the first 215 amino acids of IE2. We also show that an intact leucine zipper domain of UL84 is required for a stable interaction with IE2 and UL84 leucine zipper mutants fail to complement oriLyt-dependent DNA replication. UL84 leucine zipper mutants no longer interfere with IE2-mediated transactivation of the UL112/113 promoter, confirming that the leucine zipper is essential for a functional interaction with IE2. In addition, we demonstrate that both the leucine zipper and oligomerization domains of UL84 can act as transdominant-negative inhibitors of lytic replication in the transient assay, strongly suggesting that both an IE2-UL84 and a UL84-UL84 interaction are required for DNA synthesis.

Antibodies, Monoclonal↗

PubMed2000

Assembly of a complex containing Cdc45p, replication protein A, and Mcm2p at replication origins controlled by S-phase cyclin-dependent kinases and Cdc7p-Dbf4p kinase.

In Saccharomyces cerevisiae, replication origins are activated with characteristic timing during S phase. S-phase cyclin-dependent kinases (S-CDKs) and Cdc7p-Dbf4p kinase are required for origin activation throughout S phase. The activation of S-CDKs leads to association of Cdc45p with chromatin, raising the possibility that Cdc45p defines the assembly of a new complex at each origin. Here we show that both Cdc45p and replication protein A (RPA) bind to Mcm2p at the G(1)-S transition in an S-CDK-dependent manner. During S phase, Cdc45p associates with different replication origins at specific times. The origin associations of Cdc45p and RPA are mutually dependent, and both S-CDKs and Cdc7p-Dbf4p are required for efficient binding of Cdc45p to origins. These findings suggest that S-CDKs and Cdc7p-Dbf4p promote loading of Cdc45p and RPA onto a preformed prereplication complex at each origin with preprogrammed timing. The ARS1 association of Mcm2p, but not that of the origin recognition complex, is diminished by disruption of the B2 element of ARS1, a potential origin DNA-unwinding element. Cdc45p is required for recruiting DNA polymerase alpha onto chromatin, and it associates with Mcm2p, RPA, and DNA polymerase epsilon only during S phase. These results suggest that the complex containing Cdc45p, RPA, and MCMs is involved in origin unwinding and assembly of replication forks at each origin.

Carrier Proteins↗

PubMed2006

HMGB1 activates replication of latent HIV-1 in a monocytic cell-line, but inhibits HIV-1 replication in primary macrophages.

High mobility group box protein 1 (HMGB1) is an abundant component of mammalian cells that can be released into extracellular milieu actively or by cells that undergo necrosis. Exposure of inflammatory and endothelial cells to HMGB1 leads to the release of cytokines, including TNF-alpha and IL-6. To evaluate the impact of exogenous HMGB1 on viral replication in HIV-1 infected cells, we studied models of latent and acute infection. Extracellular HMGB1 dose dependently increased HIV-1 replication in the monocytic cells, U1, which is an established model for studying latent HIV-1 infection. Dexamethasone, a known inhibitor of NF-kappaB signaling in U1 cells, inhibited HMGB1-induced stimulation of the viral production. Addition of HMGB1 to primary monocytic cells with active HIV-1 infection elicited the opposite effect, due to suppression of the viral replication. The mechanism of this unexpected finding was explained by an HMGB1-mediated increased release of chemokines (RANTES, MIP-1alpha, and MIP-1beta) that are known to inhibit HIV-1 replication. The stimulatory effect of the HMGB1 was not present when latently infected T-cells (ACH-2) were used as target cells. Our data suggest that extracellular HMGB1 has a dichotomic effect on the HIV-1 infection in monocytes but not in lymphocytes. Both activation of latent HIV-1 infection and inhibition of active replication can thus be seen in vitro.

Cell Survival↗