scholarly journals Detection of human cytomegalovirus DNA replication in non-permissive Vero and 293 cells

2003 ◽  
Vol 84 (3) ◽  
pp. 639-645 ◽  
Author(s):  
Victoria Ellsmore ◽  
G. Gordon Reid ◽  
Nigel D. Stow
Keyword(s):  
Dna Replication ◽  
Dna Synthesis ◽  
Human Cytomegalovirus ◽  
Human Fibroblasts ◽  
Cell Types ◽  
Vero Cells ◽  
Progeny Virus ◽  
Viral Dna ◽  
Viral Origin ◽  
293 Cells

Human cytomegalovirus (HCMV) displays an exceptionally restricted host range in tissue culture with human fibroblasts being the principal fully permissive system. Nevertheless, immediate early (IE) proteins are expressed following infection of many non-permissive cell types of human, simian and murine origin, and viral origin-dependent DNA synthesis has been reconstituted by transfection of plasmids into Vero cells, a non-permissive line from African green monkey. We have examined the accumulation of HCMV strain AD169 DNA, and the replication of transfected HCMV origin-containing plasmids, in infected Vero and human embryonic kidney 293 cells, which were previously reported to express the major IE protein in a small proportion of infected cells but to be non-permissive for viral DNA synthesis. In Vero cells accumulation of origin-containing plasmid but not viral DNA occurred, whilst in 293 cells both DNAs accumulated. Immunofluorescence experiments indicated that following infection with 3 p.f.u. per cell, a small fraction of both cell types expressed the UL44 DNA replication protein. Neither cell line, however, supported the generation of infectious progeny virus. These results suggest that IE proteins expressed in Vero and 293 cells can induce the synthesis of early proteins capable of functioning in viral DNA replication, but there is a failure in later events on the pathway to infectious virus production. This provides further support for transfected Vero cells being a valid system in which to study HCMV DNA synthesis, and suggests that 293 cells may also prove useful in similar experiments.

scholarly journals Histone H3 Lysine 4 Methylation Marks Postreplicative Human Cytomegalovirus Chromatin

2012 ◽  
Vol 86 (18) ◽  
pp. 9817-9827 ◽  
Author(s):  
Alexandra Nitzsche ◽  
Charlotte Steinhäußer ◽  
Katrin Mücke ◽  
Christina Paulus ◽  
Michael Nevels
Keyword(s):  
Dna Replication ◽  
Dna Synthesis ◽  
Histone Modification ◽  
Dna Polymerase ◽  
Human Cytomegalovirus ◽  
Viral Genome ◽  
Histone H3 ◽  
Viral Dna ◽  
The Impact ◽  
Preferential Incorporation

In the nuclei of permissive cells, human cytomegalovirus genomes form nucleosomal structures initially resembling heterochromatin but gradually switching to a euchromatin-like state. This switch is characterized by a decrease in histone H3 K9 methylation and a marked increase in H3 tail acetylation and H3 K4 methylation across the viral genome. We used ganciclovir and a mutant virus encoding a reversibly destabilized DNA polymerase to examine the impact of DNA replication on histone modification dynamics at the viral chromatin. The changes in H3 tail acetylation and H3 K9 methylation proceeded in a DNA replication-independent fashion. In contrast, the increase in H3 K4 methylation proved to depend widely on viral DNA synthesis. Consistently, labeling of nascent DNA using “click chemistry” revealed preferential incorporation of methylated H3 K4 into viral (but not cellular) chromatin during or following DNA replication. This study demonstrates largely selective epigenetic tagging of postreplicative human cytomegalovirus chromatin.

scholarly journals Proteasome Subunits Relocalize during Human Cytomegalovirus Infection, and Proteasome Activity Is Necessary for Efficient Viral Gene Transcription

2009 ◽  
Vol 84 (6) ◽  
pp. 3079-3093 ◽  
Author(s):  
Karen Tran ◽  
Jeffrey A. Mahr ◽  
Deborah H. Spector
Keyword(s):  
Dna Replication ◽  
Dna Synthesis ◽  
Human Cytomegalovirus ◽  
Hcmv Infection ◽  
Proteasome Activity ◽  
Late Gene ◽  
Viral Dna ◽  
Rna Transcription ◽  
Viral Dna Replication ◽  
Early Protein

ABSTRACT We have continued studies to further understand the role of the ubiquitin-proteasome system (UPS) in human cytomegalovirus (HCMV) infection. With specific inhibitors of the proteasome, we show that ongoing proteasome activity is necessary for facilitating the various stages of the infection. Immediate-early protein 2 expression is modestly reduced with addition of proteasome inhibitors at the onset of infection; however, both early and late gene expression are significantly delayed, even if the inhibitor is removed at 12 h postinfection. Adding the inhibitor at later times during the infection blocks the further accumulation of viral early and late gene products, the severity of which is dependent on when the proteasome is inhibited. This can be attributed primarily to a block in viral RNA transcription, although DNA synthesis is also partially inhibited. Proteasome activity and expression increase as the infection progresses, and this coincides with the relocalization of active proteasomes to the periphery of the viral DNA replication center, where there is active RNA transcription. Interestingly, one 19S subunit, Rpn2, is specifically recruited into the viral DNA replication center. The relocalization of the subunits requires viral DNA replication, but their maintenance around or within the replication center is not dependent on continued viral DNA synthesis or the proteolytic activity of the proteasome. These studies highlight the importance of the UPS at all stages of the HCMV infection and support further studies into this pathway as a potential antiviral target.

scholarly journals Human Cytomegalovirus UL84 Insertion Mutant Defective for Viral DNA Synthesis and Growth

2004 ◽  
Vol 78 (19) ◽  
pp. 10360-10369 ◽  
Author(s):  
Yiyang Xu ◽  
Sylvia A. Cei ◽  
Alicia Rodriguez Huete ◽  
Gregory S. Pari
Keyword(s):  
Dna Replication ◽  
Dna Synthesis ◽  
Real Time ◽  
Human Cytomegalovirus ◽  
Viral Genome ◽  
Infectious Virus ◽  
Regulatory Protein ◽  
Insertion Mutant ◽  
Viral Dna ◽  
Transfected Cells

ABSTRACT Human cytomegalovirus (HCMV) UL84 is required for oriLyt-dependent DNA replication, and evidence from transient transfection assays suggests that UL84 directly participates in DNA synthesis. In addition, because of its apparent interaction with IE2, UL84 is implicated as a possible regulatory protein. To address the role of UL84 in the context of the viral genome, we generated a recombinant HCMV bacterial artificial chromosome (BAC) construct that did not express the UL84 gene product. This construct, BAC-IN84/Ep, displayed a null phenotype in that it failed to produce infectious virus after transfection into human fibroblast cells, whereas a revertant virus readily produced viral plaques and, subsequently, infectious virus. Real-time quantitative PCR showed that BAC-IN84/Ep was defective for DNA synthesis in that no increase in the accumulation of viral DNA was observed in transfected cells. We were unable to complement BAC-IN84/Ep in trans; however, oriLyt-dependent DNA replication was observed by the cotransfection of UL84 and BAC-IN84/Ep. An analysis of viral mRNA by real-time PCR indicated that, even in the absence of DNA synthesis, all representative kinetic classes of genes were expressed in cells transfected with BAC-IN84/Ep. The detection of UL44 and IE2 by immunofluorescence in BAC-IN84/Ep-transfected cells showed that these proteins failed to partition into replication compartments, indicating that UL84 expression is essential for the formation of these proteins into replication centers within the context of the viral genome. These results show that UL84 provides an essential DNA replication function and influences the subcellular localization of other viral proteins.

scholarly journals Differential Requirement of Human Cytomegalovirus UL112-113 Protein Isoforms for Viral Replication

2017 ◽  
Vol 91 (17) ◽  
Author(s):  
Tim Schommartz ◽  
Jiajia Tang ◽  
Rebekka Brost ◽  
Wolfram Brune
Keyword(s):  
Endothelial Cells ◽  
Dna Replication ◽  
Viral Replication ◽  
Dna Polymerase ◽  
Human Cytomegalovirus ◽  
Cell Nucleus ◽  
Human Fibroblasts ◽  
Gene Products ◽  
Viral Dna ◽  
Viral Dna Replication

ABSTRACT The UL112-113 gene is one of the few alternatively spliced genes of human cytomegalovirus (HCMV). It codes for four phosphoproteins, p34, p43, p50, and p84, all of which are expressed with early kinetics and accumulate at sites of viral DNA replication within the host cell nucleus. Although these proteins are known to play important, possibly essential, roles in the viral replication cycle, little is known about the contribution of individual UL112-113 protein products. Here we used splice site mutagenesis, intron deletion and substitution, and nonsense mutagenesis to prevent the individual expression of each UL112-113 protein isoform and to investigate the importance of each isoform for viral replication. We show that HCMV mutants lacking p34 or p50 expression replicated to high titers in human fibroblasts and endothelial cells, indicating that these proteins are nonessential for viral replication, while mutant viruses carrying a stop mutation within the p84 coding sequence were severely growth impaired. Viral replication could not be detected upon the inactivation of p43 expression, indicating that this UL112-113 protein is essential for viral replication. We also analyzed the ability of UL112-113 proteins to recruit other viral proteins to intranuclear prereplication compartments. While UL112-113 expression was sufficient to recruit the UL44-encoded viral DNA polymerase processivity factor, it was not sufficient for the recruitment of the viral UL84 and UL117 proteins. Remarkably, both the p43 and p84 isoforms were required for the efficient recruitment of pUL44, which is consistent with their critical role in the viral life cycle. IMPORTANCE Human cytomegalovirus requires gene products from 11 genetic loci for the lytic replication of its genome. One of these loci, UL112-113, encodes four proteins with common N termini by alternative splicing. In this study, we inactivated the expression of each of the four UL112-113 proteins individually and determined their requirement for HCMV replication. We found that two of the UL112-113 gene products were dispensable for viral replication in human fibroblasts and endothelial cells. In contrast, viral replication was severely reduced or absent when one of the other two gene products was inactivated, indicating that they are of crucial importance for the viral replication cycle. We further showed that the latter two gene products are involved in the recruitment of pUL44, an essential cofactor of the viral DNA polymerase, to specific sites within the cell nucleus that are thought to serve as starting points for viral DNA replication.

scholarly journals Human Cytomegalovirus Can Procure Deoxyribonucleotides for Viral DNA Replication in the Absence of Retinoblastoma Protein Phosphorylation

2016 ◽  
Vol 90 (19) ◽  
pp. 8634-8643 ◽  
Author(s):  
Chad V. Kuny ◽  
Robert F. Kalejta
Keyword(s):  
Dna Replication ◽  
Dna Synthesis ◽  
Human Cytomegalovirus ◽  
De Novo ◽  
Dna Virus ◽  
Viral Dna ◽  
Viral Oncoproteins ◽  
Viral Dna Replication ◽  
Rb Phosphorylation

ABSTRACTViral DNA replication requires deoxyribonucleotide triphosphates (dNTPs). These molecules, which are found at low levels in noncycling cells, are generated either by salvage pathways or throughde novosynthesis. Nucleotide synthesis utilizes the activity of a series of nucleotide-biosynthetic enzymes (NBEs) whose expression is repressed in noncycling cells by complexes between the E2F transcription factors and the retinoblastoma (Rb) tumor suppressor. Rb-E2F complexes are dissociated and NBE expression is activated during cell cycle transit by cyclin-dependent kinase (Cdk)-mediated Rb phosphorylation. The DNA virus human cytomegalovirus (HCMV) encodes a viral Cdk (v-Cdk) (the UL97 protein) that phosphorylates Rb, induces the expression of cellular NBEs, and is required for efficient viral DNA synthesis. A long-held hypothesis proposed that viral proteins with Rb-inactivating activities functionally similar to those of UL97 facilitated viral DNA replication in part by inducing thede novoproduction of dNTPs. However, we found that dNTPs were limiting even in cells infected with wild-type HCMV in which UL97 is expressed and Rb is phosphorylated. Furthermore, we revealed that bothde novoand salvage pathway enzymes contribute to viral DNA replication during HCMV infection and that Rb phosphorylation by cellular Cdks does not correct the viral DNA replication defect observed in cells infected with a UL97-deficient virus. We conclude that HCMV can obtain dNTPs in the absence of Rb phosphorylation and that UL97 can contribute to the efficiency of DNA replication in an Rb phosphorylation-independent manner.IMPORTANCETransforming viral oncoproteins, such as adenovirus E1A and papillomavirus E7, inactivate Rb. The standard hypothesis for how Rb inactivation facilitates infection with these viruses is that it is through an increase in the enzymes required for DNA synthesis, which include nucleotide-biosynthetic enzymes. However, HCMV UL97, which functionally mimics these viral oncoproteins through phosphorylation of Rb, fails to induce the production of nonlimiting amounts of dNTPs. This finding challenges the paradigm of the role of Rb inactivation during DNA virus infection and uncovers the existence of an alternative mechanism by which UL97 contributes to HCMV DNA synthesis. The ineffectiveness of the UL97 inhibitor maribavir in clinical trials might be better explained with a fuller understanding of the role of UL97 during infection. Furthermore, as the nucleoside analog ganciclovir is the current drug of choice for treating HCMV, knowing the provenance of the dNTPs incorporated into viral DNA may help inform antiviral therapeutic regimens.

scholarly journals Defective Growth Correlates with Reduced Accumulation of a Viral DNA Replication Protein after Low-Multiplicity Infection by a Human Cytomegalovirus ie1 Mutant

1998 ◽  
Vol 72 (1) ◽  
pp. 366-379 ◽  
Author(s):  
Richard F. Greaves ◽  
Edward S. Mocarski
Keyword(s):  
Dna Replication ◽  
Human Cytomegalovirus ◽  
Human Fibroblasts ◽  
Lytic Replication ◽  
Early Gene ◽  
Similar Proportion ◽  
Viral Dna ◽  
Viral Dna Replication ◽  
Primary Fibroblasts ◽  
Infected Cells

ABSTRACT To investigate the importance of the IE1 p72 regulatory protein during human cytomegalovirus replication, a recombinant virus unable to synthesize IE1 p72 was constructed. The Towne strain mutant CR208 lacked exon 4 of the major immediate-early gene and was isolated and complemented in an IE1-expressing immortalized human fibroblast line (ihfie1.3). Replication of CR208 in primary human fibroblasts was completed after an input multiplicity of 10 PFU/cell but was severely impaired at 0.1 PFU/cell. CR208 formed plaques with lower efficiency on primary fibroblasts than on ihfie1.3 cells, and the relationship between the CR208 inoculum size and the resulting number of undersized plaques was nonlinear, indicating that multiple particles of CR208 were required to initiate lytic replication in a single primary fibroblast. After infection of primary fibroblasts with CR208 at 5 PFU/cell, a normal pattern of viral antigens was detected, although IE1 p72 was absent. During lower-multiplicity infections, IE2 protein was consistently detected at similar levels in a similar proportion of CR208-infected cells relative to the case for a Towne infection, but many fewer CR208-infected cells contained the ppUL44 polymerase accessory protein when evaluated at 24 or 48 h after infection. Furthermore, fibroblasts infected with CR208 at a low multiplicity failed to form viral DNA replication compartments, despite having expressed IE2 p86. These low-multiplicity growth and expression defects were corrected in two rescued derivatives of CR208 able to synthesize IE1 p72. One rescued virus (CR249) carried a deletion removing the large intron between exons 1 and 2 of the ie1-ie2 locus, revealing that this intron was dispensable for growth in cell culture.

scholarly journals Inhibition of Human Cytomegalovirus DNA Replication with a Phosphorothioate Cholesteryl-Modified Oligonucleotide is Mediated by Rapid Cellular Association and Virus-Facilitated Nuclear Localization

1997 ◽  
Vol 8 (3) ◽  
pp. 255-264 ◽  
Author(s):  
Z Zhang ◽  
JA Smith ◽  
AP Smyth ◽  
J-Y Tang ◽  
W Eisenberg ◽  
...  
Keyword(s):  
Dna Replication ◽  
Human Cytomegalovirus ◽  
Nuclear Localization ◽  
Antimicrobial Agents ◽  
Human Fibroblasts ◽  
Viral Dna ◽  
Viral Dna Replication ◽  
Nuclease Resistance ◽  
Infected Cells ◽  
Modified Oligonucleotide

We have previously shown that an antisense phosphorothioate (PS) oligodeoxynucleotide has potent anti-human cytomegalovirus (HCMV) activity (GS Pari, AK Field & JA Smith, Antimicrobial Agents and Chemotherapy 1995, 39: 1157–1161). We have now used a modified PS oligonucleotide having three 2′-O-methyl nucleotides at the 3′ end and four 2′-O-methyl nucleotides at the 5′ end, containing a cholesteryl moiety linked to the 3′ end by a novel thiono-triester linkage. This compound, UL36ANTI-M, is superior to the PS (UL36ANTI) version with respect to antiviral potency, melting temperature and nuclease resistance. Also, we show that cellular association for this oligonucleotide is rapid, occurring within 15 min after treatment and is about 12-fold higher when compared to UL36ANTI. This increased rate of cellular association also correlates with antiviral properties in that a 15 min incubation with UL36ANTI-M was sufficient to achieve 75% inhibition of viral DNA replication and complete inhibition was achieved after only a 1 h pretreatment. In addition confocal microscopic examination showed a change in subcellular distribution from perinuclear to nuclear for oligonucleotides in HCMV-infected human fibroblasts. However, the total amount of cell-associated oligonucleotide was unchanged in infected cells.

Identification of temperature-sensitive DNA- mutants of Chinese hamster cells affected in cellular and viral DNA synthesis

1986 ◽  
Vol 6 (12) ◽  
pp. 4594-4601
Author(s):  
J J Dermody ◽  
B E Wojcik ◽  
H Du ◽  
H L Ozer
Keyword(s):  
Dna Replication ◽  
Dna Synthesis ◽  
Chinese Hamster ◽  
Human Adenovirus ◽  
Temperature Sensitive ◽  
Restrictive Temperature ◽  
Permissive Temperature ◽  
Dependent Manner ◽  
Viral Dna ◽  
Cell Functions

We described a strategy which facilitates the identification of cell mutants which are restricted in DNA synthesis in a temperature-dependent manner. A collection of over 200 cell mutants temperature-sensitive for growth was isolated in established Chinese hamster cell lines (CHO and V79) by a variety of selective and nonselective techniques. Approximately 10% of these mutants were identified as ts DNA- based on differential inhibition of macromolecular synthesis at the restrictive temperature (39 degrees C) as assessed by incorporation of [3H]thymidine and [35S]methionine. Nine such mutants, selected for further study, demonstrated rapid shutoff of DNA replication at 39 degrees C. Infections with two classes of DNA viruses extensively dependent on host-cell functions for their replication were used to distinguish defects in DNA synthesis itself from those predominantly affecting other aspects of DNA replication. All cell mutants supported human adenovirus type 2 (Ad2) and mouse polyomavirus DNA synthesis at the permissive temperature. Five of the nine mutants (JB3-B, JB3-O, JB7-K, JB8-D, and JB11-J) restricted polyomavirus DNA replication upon transfection with viral sequences at 33 degrees C and subsequent shift to 39 degrees C either before or after the onset of viral DNA synthesis. Only one of these mutants (JB3-B) also restricted Ad2 DNA synthesis after virion infection under comparable conditions. No mutant was both restrictive for Ad2 and permissive for polyomavirus DNA synthesis at 39 degrees C. The differential effect of these cell mutants on viral DNA synthesis is expected to assist subsequent definition of the biochemical defect responsible.

scholarly journals Adenovirus E1B 55-Kilodalton Protein Is Required for both Regulation of mRNA Export and Efficient Entry into the Late Phase of Infection in Normal Human Fibroblasts

2006 ◽  
Vol 80 (2) ◽  
pp. 964-974 ◽  
Author(s):  
Ramon Gonzalez ◽  
Wenying Huang ◽  
Renee Finnen ◽  
Courtney Bragg ◽  
S. J. Flint
Keyword(s):  
Dna Synthesis ◽  
Hela Cells ◽  
Human Fibroblasts ◽  
Late Phase ◽  
Mrna Export ◽  
Human Cells ◽  
Insertion Mutation ◽  
Viral Dna ◽  
Normal Human ◽  
Normal Human Cells

ABSTRACT The human adenovirus type 5 (Ad5) E1B 55-kDa protein is required for selective nuclear export of viral late mRNAs from the nucleus and concomitant inhibition of export of cellular mRNAs in HeLa cells and some other human cell lines, but its contributions(s) to replication in normal human cells is not well understood. We have therefore examined the phenotypes exhibited by viruses carrying mutations in the E1B 55-kDa protein coding sequence in normal human fibroblast (HFFs). Ad5 replicated significantly more slowly in HFFs than it does in tumor cells, a difference that is the result of delayed entry into the late phase of infection. The A143 mutation, which specifically impaired export of viral late mRNAs from the nucleus in infected HeLa cells (R. A. Gonzalez and S. J. Flint, J. Virol. 76:4507-4519, 2002), induced a more severe defect in viral mRNA export in HFFs. This observation indicates that the E1B 55-kDa protein regulates mRNA export during the late phase of infection of normal human cells. Other mutants exhibited phenotypes not observed in HeLa cells. In HFFs infected by the null mutant Hr6, synthesis of viral late mRNAs and proteins was severely impaired. Such defects in late gene expression were the result of inefficient progression into the late phase of infection, for viral DNA synthesis was 10-fold less efficient in Hr6-infected HFFs than in cells infected by Ad5. Similar, but less severe, defects in viral DNA synthesis were induced by the insertion mutation H224, which has been reported to inhibit binding of the E1B 55-kDa protein to p53 (C. C. Kao, P. R. Yew, and A. J. Berk, Virology 179:806-814, 1990).

Hairpin transfer-independent Parvovirus DNA Replication Produces Infectious Virus

2021 ◽  
Author(s):  
Weiran Shen ◽  
Zekun Wang ◽  
Kang Ning ◽  
Fang Cheng ◽  
John F. Engelhardt ◽  
...  
Keyword(s):  
Dna Replication ◽  
Hek293 Cells ◽  
Progeny Virus ◽  
Human Bocavirus ◽  
Viral Dna ◽  
Airway Epithelia ◽  
Viral Dna Replication ◽  
Human Airway ◽  
The Right ◽  
Human Airway Epithelia

Parvoviruses package a linear single-stranded DNA genome with hairpin structures at both ends. It has been thought that terminal hairpin sequences are indispensable for viral DNA replication. Here, we provide evidence that the hairpin-deleted duplex genomes of human bocavirus 1 (HBoV1) replicate in human embryonic kidney (HEK) 293 cells. We propose an alternative model for HBoV1 DNA replication in which the leading strand can initiate strand-displacement without “hairpin-transfer.” The transfection of the HBoV1 duplex genomes that retain a minimal replication origin at the right-end ( OriR ), but with extensive deletions in the right-end hairpin (REH), generated viruses in HEK293 cells at a level 10-20 times lower than the wild-type (WT) duplex genome. Importantly, these viruses that have a genome with various deletions after the OriR , but not the one retaining only the OriR , replicated in polarized human airway epithelia. We discovered that the 18-nt sequence (nt 5,403-5,420) beyond the OriR was sufficient to confer virus replication in polarized human airway epithelia, although its progeny virus production was ∼5 times lower than that of the WT virus. Thus, our study demonstrates that hairpin transfer-independent productive parvovirus DNA replication can occur. Importance Hairpin transfer-independent parvovirus replication was modeled with human bocavirus 1 (HBoV1) duplex genomes whose 5’ hairpin structure was ablated by various deletions. In HEK293 cells, these duplex viral genomes with ablated 5’/hairpin sequence replicated efficiently and generated viruses that productively infected polarized human airway epithelium. Thus, for the first time, we reveal a previously unknown phenomenon that the productive parvovirus DNA replication does not depend on the hairpin sequence at REH to initiate “rolling hairpin” DNA replication. Notably, the intermediates of viral DNA replication, as revealed two-dimensional electrophoresis, from transfections of hairpin sequence-deleted duplex genome and full-length genome in HEK293 cells, as well as from virus infection of polarized human airway epithelia are similar. Thus, the establishment of the hairpin transfer-independent parvoviral DNA replication deepens our understanding in viral DNA replication and may have implications in development of parvovirus-based viral vectors with alternative properties.

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