scholarly journals Construction of a Self-Excisable Bacterial Artificial Chromosome Containing the Human Cytomegalovirus Genome and Mutagenesis of the Diploid TRL/IRL13 Gene

2002 ◽  
Vol 76 (5) ◽  
pp. 2316-2328 ◽  
Author(s):  
Dong Yu ◽  
Gregory A. Smith ◽  
Lynn W. Enquist ◽  
Thomas Shenk
Keyword(s):  
Bacterial Artificial Chromosome ◽  
Human Cytomegalovirus ◽  
Artificial Chromosome ◽  
Open Reading Frame ◽  
Wild Type Virus ◽  
Progeny Virus ◽  
Wild Type ◽  
Coding Region ◽  
Reading Frame ◽  
Strain Ad169

ABSTRACT The full-length genome of human cytomegalovirus strain AD169 was cloned as an infectious bacterial artificial chromosome (BAC) plasmid, pAD/Cre. The BAC vector, flanked by LoxP sites, was inserted immediately after the Us28 open reading frame without deletion of any viral sequences. The BAC vector contained the Cre recombinase-encoding gene disrupted by an intron under control of the simian virus 40 early promoter. When pAD/Cre was transfected into primary human foreskin fibroblast cells, Cre was expressed and mediated site-specific recombination between the two LoxP sites, excising the BAC DNA backbone. This gave rise to progeny virus that was wild type with the exception of an inserted 34-bp LoxP site. We performed site-directed mutagenesis on pAD/Cre to generate a series of viruses in which the TRL/IRL13 diploid genes were disrupted and subsequently repaired. The mutants reach the same titer as the wild-type virus, indicating that the TRL/IRL13 open reading frames are not required for virus growth in cell culture. The sequence of the TRL13 open reading frame in the low-passage Toledo strain of human cytomegalovirus is quite different from the corresponding region in the AD169 strain. One of multiple changes is a frameshift mutation. As a consequence, strain Toledo encodes a putative TRL13 protein whose C-terminal domain is larger (extending through the TRL14 coding region) and encodes in a reading frame different from that of strain AD169. We speculate that the strain AD169 coding region has drifted during passage in the laboratory. We propose that TRL13 has been truncated in strain AD169 and that the partially overlapping TRL14 open reading frame is not functional. This view is consistent with the presence of both TRL13 and -14 on all mRNAs that we have mapped from this region, an organization that would include the much longer strain Toledo TRL13 open reading frame on the mRNAs.

scholarly journals Deletion of Open Reading Frame UL26 from the Human Cytomegalovirus Genome Results in Reduced Viral Growth, Which Involves Impaired Stability of Viral Particles

2006 ◽  
Vol 80 (11) ◽  
pp. 5423-5434 ◽  
Author(s):  
Kerstin Lorz ◽  
Heike Hofmann ◽  
Anja Berndt ◽  
Nina Tavalai ◽  
Regina Mueller ◽  
...  
Keyword(s):  
Human Cytomegalovirus ◽  
Transcriptional Activation ◽  
Deletion Mutant ◽  
Artificial Chromosome ◽  
Open Reading Frame ◽  
Wild Type Virus ◽  
Wild Type ◽  
Reading Frame ◽  
Transcriptional Activation Domain ◽  
Viral Particles

ABSTRACT We previously showed that open reading frame (ORF) UL26 of human cytomegalovirus, a member of the US22 multigene family of betaherpesviruses, encodes a novel tegument protein, which is imported into cells in the course of viral infection. Moreover, we demonstrated that pUL26 contains a strong transcriptional activation domain and is capable of stimulating the major immediate-early (IE) enhancer-promoter. Since this suggested an important function of pUL26 during the initiation of the viral replicative cycle, we sought to ascertain the relevance of pUL26 by construction of a viral deletion mutant lacking the UL26 ORF using the bacterial artificial chromosome mutagenesis procedure. The resulting deletion virus was verified by PCR, enzyme restriction, and Southern blot analyses. After infection of human foreskin fibroblasts, the UL26 deletion mutant showed a small-plaque phenotype and replicated to significantly lower titers than wild-type or revertant virus. In particular, we noticed a striking decrease of infectious titers 7 days postinfection in a multistep growth experiment, whereas the release of viral DNA from infected cells was not impaired. A further investigation of this aspect revealed a significantly diminished stability of viral particles derived from the UL26 deletion mutant. Consistent with this, we observed that the tegument composition of the deletion mutant deviates from that of the wild-type virus. We therefore hypothesize that pUL26 plays a role not only in the onset of IE gene transcription but also in the assembly of the viral tegument layer in a stable and correct manner.

scholarly journals Human Cytomegalovirus UL47 Tegument Protein Functions after Entry and before Immediate-Early Gene Expression

2002 ◽  
Vol 76 (3) ◽  
pp. 1043-1050 ◽  
Author(s):  
Jill T. Bechtel ◽  
Thomas Shenk
Keyword(s):  
Human Cytomegalovirus ◽  
Human Fibroblasts ◽  
Open Reading Frame ◽  
Early Gene ◽  
Immediate Early ◽  
Wild Type Virus ◽  
Marker Genes ◽  
Type Virus ◽  
Wild Type ◽  
Reading Frame

ABSTRACT The human cytomegalovirus UL47 open reading frame encodes a 110-kDa protein that is a component of the virion tegument. We have constructed a cytomegalovirus mutant, ADsubUL47, in which the central portion of the UL47 open reading frame has been replaced by two marker genes. The mutant replicated to titers 100-fold lower than those for wild-type virus after infection at either a high or a low input multiplicity in primary human fibroblasts but was substantially complemented on cells expressing UL47 protein. A revertant virus in which the mutation was repaired, ADrevUL47, replicated with wild-type kinetics. Mutant virions lacked UL47 protein and contained reduced amounts of UL48 protein. The mutant was found to be less infectious than wild-type virus, and a defect very early in the replication cycle was observed. Transcription of the viral immediate-early 1 gene was delayed by 8 to 10 h. However, this delay was not the result of a defect in virus entry or of the inability of virion proteins to transactivate the major immediate-early promoter. We also show that the UL47 protein coprecipitated with the UL48 and UL69 tegument proteins and the UL86-encoded major capsid protein. We propose that a UL47-containing complex is involved in the release of viral DNA from the disassembling virus particle and that the loss of UL47 protein causes this process to be delayed.

scholarly journals Construction and Characterization of an Infectious Murine Gammaherpesivrus-68 Bacterial Artificial Chromosome

2011 ◽  
Vol 2011 ◽  
pp. 1-11 ◽  
Author(s):  
Ting-Ting Wu ◽  
Hsiang-I Liao ◽  
Leming Tong ◽  
Ronika Sitapara Leang ◽  
Greg Smith ◽  
...  
Keyword(s):  
Bacterial Artificial Chromosome ◽  
Cultured Cells ◽  
Artificial Chromosome ◽  
Productive Infection ◽  
Wild Type Virus ◽  
Wild Type ◽  
Bac Clone ◽  
Reading Frame ◽  
Murine Gammaherpesvirus ◽  
Gammaherpesvirus 68

Here we describe the cloning of a sequenced WUMS isolate of murine gammaherpesvirus-68 (MHV-68, γHV-68, also known as MuHV-4) as a bacterial artificial chromosome (BAC). We engineered the insertion of the BAC sequence flanked by loxP sites into the left end of the viral genome before the M1 open reading frame. The infectious viruses were reconstituted following transfection of the MHV-68 BAC DNA into cells. The MHV-68 BAC-derived virus replicated indistinguishably from the wild-type virus in cultured cells. Excision of the BAC insert was efficiently achieved by coexpressing the Cre recombinase. Although the BAC insertion did not significantly affect acute productive infection in the lung, it severely compromised the ability of MHV-68 to establish splenic latency. Removal of the BAC sequence restored the wild-type level of latency. Site-specific mutagenesis was carried out by RecA-mediated recombination to demonstrate that this infectious BAC clone can be used for genetic studies of MHV-68.

scholarly journals Identification of Glycoprotein gpTRL10 as a Structural Component of Human Cytomegalovirus

2002 ◽  
Vol 76 (3) ◽  
pp. 1450-1460 ◽  
Author(s):  
S. Spaderna ◽  
H. Blessing ◽  
E. Bogner ◽  
W. Britt ◽  
M. Mach
Keyword(s):  
Human Cytomegalovirus ◽  
Structural Component ◽  
Laboratory Strain ◽  
Immunoblot Analysis ◽  
Protein Product ◽  
Clinical Isolates ◽  
Coding Region ◽  
Reading Frame ◽  
Infected Cells ◽  
Strain Ad169

ABSTRACT Human cytomegalovirus (HCMV) has a coding capacity for glycoproteins which far exceeds that of other herpesviruses. Few of these proteins have been characterized. We have investigated the gene product(s) of reading frame 10, which is present in both the internal and terminal repeat regions of HCMV strain AD169 and only once in clinical isolates. The putative protein product is a 171-amino-acid glycoprotein with a theoretical mass of 20.5 kDa. We characterized the protein encoded by this reading frame in the laboratory strain AD169 and a recent isolate, TB40E. The results from both strains were comparable. Northern blot analyses showed that the gene was transcribed with early/late kinetics. Two proteins of 22 and 23.5-kDa were detected in virus-infected cells and in cells transiently expressing recombinant TRL10. Both forms contained only high-mannose-linked carbohydrate modifications. In addition, virus-infected cells expressed small amounts of the protein modified with complex N-linked sugars. Image analysis localized transiently expressed TRL10 to the endoplasmic reticulum. Immunoblot analyses as well as immunoelectron microscopy of purified virions demonstrated that TRL10 represents a structural component of the virus particle. Immunoblot analysis in the absence of reducing agents indicated that TRL10, like the other HCMV envelope glycoproteins, is present in a disulfide-linked complex. Sequence analysis of the TRL10 coding region in nine low-passage clinical isolates revealed strain-specific variation. In summary, the protein product of the TRL10 open reading frame represents a novel structural glycoprotein of HCMV and was termed gpTRL10.

scholarly journals Improved Detection of Mutated Human Cytomegalovirus UL97 by Pyrosequencing

2010 ◽  
Vol 54 (12) ◽  
pp. 5234-5241 ◽  
Author(s):  
Birgit Schindele ◽  
Luise Apelt ◽  
Jörg Hofmann ◽  
Andreas Nitsche ◽  
Detlef Michel ◽  
...  
Keyword(s):  
Human Cytomegalovirus ◽  
Artificial Chromosome ◽  
Prolonged Treatment ◽  
Solid Organ ◽  
Immune Functions ◽  
Wild Type ◽  
Hematopoietic Stem ◽  
Reading Frame ◽  
Mutant Sequence ◽  
Cell Transplants

ABSTRACT Ganciclovir (GCV) resistance frequently occurs upon prolonged treatment of ongoing active human cytomegalovirus (HCMV) infection in individuals with immature or compromised immune functions (e.g., recipients of solid-organ and hematopoietic stem cell transplants). Using pyrosequencing (PSQ), we established fast and sensitive detection of GCV resistance-associated mutations occurring in the HCMV open reading frame UL97. These mutations have been repeatedly associated with clinical treatment failure. We designed four PSQ assays and evaluated them by analyzing mixtures of plasmids or bacterial artificial chromosome-derived viruses containing UL97 wild-type and mutant sequences. A minimum level of 6% mutant sequence variants could be detected in these mixtures. In order to further evaluate the novel PSQ assays, we tested clinical specimens from patients with active HCMV infections. The results were compared with those obtained by conventional dideoxy chain terminator sequencing. As the PSQ method was more sensitive in detecting minor HCMV mutant fractions in a wild-type population, it is suggested that pyrosequencing is a useful tool for the early detection of emerging GCV-resistant HCMV in GCV-treated patients.

scholarly journals Cleavage Specificity of the UL48 Deubiquitinating Protease Activity of Human Cytomegalovirus and the Growth of an Active-Site Mutant Virus in Cultured Cells

2009 ◽  
Vol 83 (23) ◽  
pp. 12046-12056 ◽  
Author(s):  
Eui Tae Kim ◽  
Se Eun Oh ◽  
Yun-Ok Lee ◽  
Wade Gibson ◽  
Jin-Hyun Ahn
Keyword(s):  
Human Cytomegalovirus ◽  
Active Site ◽  
Protease Activity ◽  
Cultured Cells ◽  
Mutant Virus ◽  
Wild Type Virus ◽  
Progeny Virus ◽  
Type Virus ◽  
Wild Type ◽  
Cleavage Specificity

ABSTRACT The human cytomegalovirus (HCMV) open reading frame UL48 encodes a 253-kDa tegument protein that is closely associated with the capsid and was recently shown to have ubiquitin-specific protease activity (J. Wang, A. N. Loveland, L. M. Kattenhorn, H. L. Ploegh, and W. Gibson, J. Virol. 80:6003-6012, 2006). Here, we examined the cleavage specificity of this deubiquitinase (DUB) and replication characteristics of an active-site mutant virus. The purified catalytic domain of the UL48 DUB (1 to 359 amino acids), corresponding to the herpes simplex virus UL36USP DUB (L. M. Kattenhorn, G. A. Korbel, B. M. Kessler, E. Spooner, and H. L. Ploegh, Mol. Cell 19:547-557, 2005), efficiently released ubiquitin but not ubiquitin-like modifications from a hemagglutinin peptide substrate. Mutating the active-site residues Cys24 or His162 (C24S and H162A, respectively) abolished this activity. The HCMV UL48 and HSV UL36USP DUBs cleaved both Lys48- and Lys63-linked ubiquitin dimers and oligomers, showing more activity toward Lys63 linkages. The DUB activity of the full-length UL48 protein immunoprecipitated from virus-infected cells also showed a better cleavage of Lys63-linked ubiquitinated substrates. An HCMV (Towne) mutant virus in which the UL48 DUB activity was destroyed [UL48(C24S)] produced 10-fold less progeny virus and reduced amounts of viral proteins compared to wild-type virus at a low multiplicity of infection. The mutant virus also produced perceptibly less overall deubiquitination than the wild-type virus. Our findings demonstrate that the HCMV UL48 DUB contains both a ubiquitin-specific carboxy-terminal hydrolase activity and an isopeptidase activity that favors ubiquitin Lys63 linkages and that these activities can influence virus replication in cultured cells.

scholarly journals Murine Cytomegalovirus Open Reading Frame M27 Plays an Important Role in Growth and Virulence in Mice

2001 ◽  
Vol 75 (4) ◽  
pp. 1697-1707 ◽  
Author(s):  
Gerardo Abenes ◽  
Manfred Lee ◽  
Erik Haghjoo ◽  
Tuong Tong ◽  
Xiaoyan Zhan ◽  
...  
Keyword(s):  
Scid Mice ◽  
Open Reading Frame ◽  
Murine Cytomegalovirus ◽  
Wild Type Virus ◽  
Wild Type ◽  
Reading Frame ◽  
Viral Virulence ◽  
Carboxyl Terminal

ABSTRACT Using a Tn3-based transposon mutagenesis approach, we have generated a pool of murine cytomegalovirus (MCMV) mutants. In this study, one of the mutants, RvM27, which contained the transposon sequence at open reading frame M27, was characterized both in tissue culture and in immunocompetent BALB/c mice and immunodeficient SCID mice. Our results suggest that the M27 carboxyl-terminal sequence is dispensable for viral replication in vitro. Compared to the wild-type strain and a rescued virus that restored the M27 region, RvM27 was attenuated in growth in both BALB/c and SCID mice that were intraperitoneally infected with the viruses. Specifically, the titers of RvM27 in the salivary glands, lungs, spleens, livers, and kidneys of the infected SCID mice at 21 days postinfection were 50- to 500-fold lower than those of the wild-type virus and the rescued virus. Moreover, the virulence of the mutant virus appeared to be attenuated, because no deaths occurred among SCID mice infected with RvM27 for up to 37 days postinfection, while all the animals infected with the wild-type and rescued viruses died within 27 days postinfection. Our observations provide the first direct evidence to suggest that a disruption of M27 expression results in reduced viral growth and attenuated viral virulence in vivo in infected animals. Moreover, these results suggest that M27 is a viral determinant required for optimal MCMV growth and virulence in vivo and provide insight into the functions of the M27 homologues found in other animal and human CMVs as well as in other betaherpesviruses.

scholarly journals The Herpes Simplex Virus Type 1 UL17 Gene Encodes Virion Tegument Proteins That Are Required for Cleavage and Packaging of Viral DNA

1998 ◽  
Vol 72 (5) ◽  
pp. 3779-3788 ◽  
Author(s):  
Brandy Salmon ◽  
Charles Cunningham ◽  
Andrew J. Davison ◽  
Wendy J. Harris ◽  
Joel D. Baines
Keyword(s):  
Vero Cells ◽  
Open Reading Frame ◽  
Wild Type Virus ◽  
Type Virus ◽  
Wild Type ◽  
Viral Dna ◽  
Reading Frame ◽  
Simplex Virus ◽  
Hsv 1

ABSTRACT Previous studies have suggested that the UL17 gene of herpes simplex virus type 1 (HSV-1) is essential for virus replication. In this study, viral mutants incorporating either a lacZexpression cassette in place of 1,490 bp of the 2,109-bp UL17 open reading frame [HSV-1(ΔUL17)] or a DNA oligomer containing an in-frame stop codon inserted 778 bp from the 5′ end of the UL17 open reading frame [HSV-1(UL17-stop)] were plaque purified on engineered cell lines containing the UL17 gene. A virus derived from HSV-1(UL17-stop) but containing a restored UL17 gene was also constructed and was designated HSV-1(UL17-restored). The latter virus formed plaques and cleaved genomic viral DNA in a manner indistinguishable from wild-type virus. Neither HSV-1(ΔUL17) nor HSV-1(UL17-stop) formed plaques or produced infectious progeny when propagated on noncomplementing Vero cells. Furthermore, genomic end-specific restriction fragments were not detected in DNA purified from noncomplementing cells infected with HSV-1(ΔUL17) or HSV-1(UL17-stop), whereas end-specific fragments were readily detected when the viruses were propagated on complementing cells. Electron micrographs of thin sections of cells infected with HSV-1(ΔUL17) or HSV-1(UL17-stop) illustrated that empty capsids accumulated in the nuclei of Vero cells, whereas DNA-containing capsids accumulated in the nuclei of complementing cells and enveloped virions were found in the cytoplasm and extracellular space. Additionally, protein profiles of capsids purified from cells infected with HSV-1(ΔUL17) compared to wild-type virus show no detectable differences. These data indicate that the UL17 gene is essential for virus replication and is required for cleavage and packaging of viral DNA. To characterize the UL17 gene product, an anti-UL17 rabbit polyclonal antiserum was produced. The antiserum reacted strongly with a major protein of apparent M r 77,000 and weakly with a protein of apparent M r 72,000 in wild-type infected cell lysates and in virions. Bands of similar sizes were also detected in electrophoretically separated tegument fractions of virions and light particles and yielded tryptic peptides of masses characteristic of the predicted UL17 protein. We therefore conclude that the UL17 gene products are associated with the virion tegument and note that they are the first tegument-associated proteins shown to be required for cleavage and packaging of viral DNA.

scholarly journals Resistance of Human Cytomegalovirus to Benzimidazole Ribonucleosides Maps to Two Open Reading Frames: UL89 and UL56

1998 ◽  
Vol 72 (6) ◽  
pp. 4721-4728 ◽  
Author(s):  
Paula M. Krosky ◽  
Mark R. Underwood ◽  
Steven R. Turk ◽  
Kathryne W.-H. Feng ◽  
Rajeev K. Jain ◽  
...  
Keyword(s):  
Human Cytomegalovirus ◽  
Dna Cleavage ◽  
Drug Targets ◽  
Antiviral Drug ◽  
Nuclease Activity ◽  
Open Reading Frames ◽  
Wild Type Virus ◽  
Wild Type ◽  
Viral Dna ◽  
Reading Frame

ABSTRACT 2,5,6-Trichloro-1-β-d-ribofuranosyl benzimidazole (TCRB) is a potent and selective inhibitor of human cytomegalovirus (HCMV) replication. TCRB acts via a novel mechanism involving inhibition of viral DNA processing and packaging. Resistance to the 2-bromo analog (BDCRB) has been mapped to the UL89 open reading frame (ORF), and this gene product was proposed as the viral target of the benzimidazole nucleosides. In this study, we report the independent isolation of virus that is 20- to 30-fold resistant to TCRB (isolate C4) and the characterization of the virus. The six ORFs known to be essential for viral DNA cleavage and packaging (UL51, UL52, UL56, UL77, UL89, and UL104) were sequenced from wild-type HCMV, strain Towne, and from isolate C4. Mutations were identified in UL89 (D344E) and in UL56 (Q204R). The mutation in UL89 was identical to that previously reported for virus resistant to BDCRB, but the mutation in UL56 is novel. Marker transfer analysis demonstrated that each of these mutations individually caused ∼10-fold resistance to the benzimidazoles and that the combination of both mutations caused ∼30-fold resistance. The rate and extent of replication of the mutants was the same as for wild-type virus, but the viruses were less sensitive to inhibition of DNA cleavage by TCRB. Mapping of resistance to UL56 supports and extends recent work showing that UL56 codes for a packaging motif binding protein which also has specific nuclease activity (E. Bogner et al., J. Virol. 72:2259–2264, 1998). Resistance which maps to two different genes suggests that their putative proteins interact and/or that either or both have a benzimidazole ribonucleoside binding site. The results also suggest that the gene products of UL89 and UL56 may be antiviral drug targets.

scholarly journals Human Cytomegalovirus Latency-Associated Protein pORF94 Is Dispensable for Productive and Latent Infection

2000 ◽  
Vol 74 (19) ◽  
pp. 9333-9337 ◽  
Author(s):  
Kirsten Lofgren White ◽  
Barry Slobedman ◽  
Edward S. Mocarski
Keyword(s):  
Human Cytomegalovirus ◽  
Latent Infection ◽  
Cultured Cells ◽  
Wild Type Virus ◽  
Wild Type ◽  
Reading Frame ◽  
Latently Infected ◽  
Infected Cells ◽  
Latent Phase

ABSTRACT Human cytomegalovirus latency in bone marrow-derived myeloid progenitors is characterized by the presence of latency-associated transcripts encoded in the ie1/ie2 region of the viral genome. To assess the role of ORF94 (UL126a), a conserved open reading frame on these transcripts, a recombinant virus (RC2710) unable to express this gene was constructed. This virus replicated at wild-type levels and expressed productive as well as latency-associatedie1/ie2 region transcripts. During latency in granulocyte-macrophage progenitors, RC2710 DNA was detected at levels indistinguishable from wild-type virus, latent-phase transcription was present, and RC2710 reactivated when latently infected cells were cocultured with permissive fibroblasts. These data suggest pORF94 is not required for either productive or latent infection as assayed in cultured cells despite being the only known nuclear latency-associated protein.

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