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 The Human Cytomegalovirus UL97 Protein Kinase, an Antiviral Drug Target, Is Required at the Stage of Nuclear Egress

2003 ◽  
Vol 77 (2) ◽  
pp. 905-914 ◽  
Author(s):  
Paula M. Krosky ◽  
Moon-Chang Baek ◽  
Donald M. Coen
Keyword(s):  
Gene Expression ◽  
Protein Kinase ◽  
Virus Infection ◽  
Human Cytomegalovirus ◽  
Antiviral Drug ◽  
Virus Yield ◽  
Wild Type Virus ◽  
Wild Type ◽  
Viral Dna ◽  
Nuclear Egress

ABSTRACT Human cytomegalovirus encodes an unusual protein kinase, UL97, that activates the established antiviral drug ganciclovir and is specifically inhibited by a new antiviral drug, maribavir. We used maribavir and a UL97 null mutant, which is severely deficient in viral replication, to determine what stage of virus infection critically requires UL97. Compared with wild-type virus, there was little or no decrease in immediate-early gene expression, viral DNA synthesis, late gene expression, or packaging of viral DNA into nuclease-resistant structures in mutant-infected or maribavir-treated cells under conditions where the virus yield was severely impaired. Electron microscopy studies revealed similar proportions of various capsid forms, including DNA-containing capsids, in the nuclei of wild-type- and mutant-infected cells. However, capsids were rare in the cytoplasm of mutant-infected or maribavir-treated cells; the magnitudes of these decreases in cytoplasmic capsids were similar to those for virus yield. Thus, genetic and pharmacological evidence indicates that UL97 is required at the stage of infection when nucleocapsids exit from the nucleus (nuclear egress), and this poorly understood stage of virus infection can be targeted by antiviral drugs. Understanding UL97 function and maribavir action should help elucidate this interesting biological process and help identify new antiviral drug targets for an important pathogen in immunocompromised patients.

scholarly journals Human Cytomegalovirus Resistance to Deoxyribosylindole Nucleosides Maps to a Transversion Mutation in the Terminase Subunit-Encoding GeneUL89

2014 ◽  
Vol 59 (1) ◽  
pp. 226-232 ◽  
Author(s):  
Brian G. Gentry ◽  
Quang Phan ◽  
Ellie D. Hall ◽  
Julie M. Breitenbach ◽  
Katherine Z. Borysko ◽  
...  
Keyword(s):  
Human Cytomegalovirus ◽  
Structural Similarity ◽  
Open Reading Frames ◽  
Wild Type Virus ◽  
Wild Type ◽  
Term Administration ◽  
Transversion Mutation ◽  
Exon 1

ABSTRACTHuman cytomegalovirus (HCMV) infection can cause severe illnesses, including encephalopathy and mental retardation, in immunocompromised and immunologically immature patients. Current pharmacotherapies for treating systemic HCMV infections include ganciclovir, cidofovir, and foscarnet. However, long-term administration of these agents can result in serious adverse effects (myelosuppression and/or nephrotoxicity) and the development of viral strains with reduced susceptibility to drugs. The deoxyribosylindole (indole) nucleosides demonstrate a 20-fold greater activityin vitro(the drug concentration at which 50% of the number of plaques was reduced with the presence of drug compared to the number in the absence of drug [EC50] = 0.34 μM) than ganciclovir (EC50= 7.4 μM) without any observed increase in cytotoxicity. Based on structural similarity to the benzimidazole nucleosides, we hypothesize that the indole nucleosides target the HCMV terminase, an enzyme responsible for packaging viral DNA into capsids and cleaving the DNA into genome-length units. To test this hypothesis, an indole nucleoside-resistant HCMV strain was isolated, the open reading frames of the genes that encode the viral terminase were sequenced, and a G766C mutation in exon 1 ofUL89was identified; this mutation resulted in an E256Q change in the amino acid sequence of the corresponding protein. An HCMV wild-type strain, engineered with this mutation to confirm resistance, demonstrated an 18-fold decrease in susceptibility to the indole nucleosides (EC50= 3.1 ± 0.7 μM) compared to that of wild-type virus (EC50= 0.17 ± 0.04 μM). Interestingly, this mutation did not confer resistance to the benzimidazole nucleosides (EC50for wild-type HCMV = 0.25 ± 0.04 μM, EC50for HCMV pUL89 E256Q = 0.23 ± 0.04 μM). We conclude, therefore, that the G766C mutation that results in the E256Q substitution is unique for indole nucleoside resistance and distinct from previously discovered substitutions that confer both indole and benzimidazole nucleoside resistance (D344E and A355T).

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 pUS24 Is a Virion Protein That Functions Very Early in the Replication Cycle

2006 ◽  
Vol 80 (17) ◽  
pp. 8371-8378 ◽  
Author(s):  
Xuyan Feng ◽  
Jörg Schröer ◽  
Dong Yu ◽  
Thomas Shenk
Keyword(s):  
Human Cytomegalovirus ◽  
Mutant Virus ◽  
Replication Cycle ◽  
Immediate Early ◽  
Wild Type Virus ◽  
Type Virus ◽  
Wild Type ◽  
Virion Protein ◽  
Viral Dna ◽  
Infected Cells

ABSTRACT We have characterized the function of the human cytomegalovirus US24 gene, a US22 gene family member. Two US24-deficient mutants (BADinUS24 and BADsubUS24) exhibited a 20- to 30-fold growth defect, compared to their wild-type parent (BADwt), after infection at a relatively low (0.01 PFU/cell) or high (1 PFU/cell) input multiplicity. Representative virus-encoded proteins and viral DNA accumulated with normal kinetics to wild-type levels after infection with mutant virus when cells received equal numbers of mutant and wild-type infectious units. Further, the proteins were properly localized and no ultrastructural differences were found by electron microscopy in mutant-virus-infected cells compared to wild-type-virus-infected cells. However, virions produced by US24-deficient mutants had a 10-fold-higher genome-to-PFU ratio than wild-type virus. When infections were performed using equal numbers of input virus particles, the expression of immediate-early, early, and late viral proteins was substantially delayed and decreased in the absence of US24 protein. This delay is not due to inefficient virus entry, since two tegument proteins and viral DNA moved to the nucleus equally well in mutant- and wild-type-virus-infected cells. In summary, US24 is a virion protein and virions produced by US24-deficient viruses exhibit a block to the human cytomegalovirus replication cycle after viral DNA reaches the nucleus and before immediate-early mRNAs are transcribed.

scholarly journals Resistance of Human Cytomegalovirus to the Benzimidazole l-Ribonucleoside Maribavir Maps to UL27

2003 ◽  
Vol 77 (21) ◽  
pp. 11499-11506 ◽  
Author(s):  
Gloria Komazin ◽  
Roger G. Ptak ◽  
Brian T. Emmer ◽  
Leroy B. Townsend ◽  
John C. Drach
Keyword(s):  
Dna Synthesis ◽  
Human Cytomegalovirus ◽  
Resistance Mutation ◽  
Open Reading Frames ◽  
Cosmid Library ◽  
Wild Type Virus ◽  
Wild Type ◽  
Nuclear Egress ◽  
The One

ABSTRACT 1-(β-d-Ribofuranosyl)-2,5,6-trichlorobenzimidazole (TCRB) and its 2-bromo analog, BDCRB, are potent and selective inhibitors of human cytomegalovirus (HCMV) DNA processing and packaging. Since they are readily metabolized in vivo, analogs were synthesized to improve biostability. One of these, 1-(β-l-ribofuranosyl)-2-isopropylamino-5,6-dichlorobenzimidazole (1263W94; maribavir), inhibits viral DNA synthesis and nuclear egress. Resistance to maribavir was mapped to UL97, and this viral kinase was shown to be a direct target of maribavir. In the present study, an HCMV strain resistant to TCRB and BDCRB was passaged in increasing concentrations of maribavir, and resistant virus was isolated. This strain (G2) grew at the same rate as the wild-type virus and was resistant to both BDCRB and maribavir. Resistance to BDCRB was expected, because the parent strain from which G2 was isolated was resistant due to known mutations in UL56 and UL89. However, no mutations were found in UL97 or other relevant open reading frames that could explain resistance to maribavir. Because sequencing of selected HCMV genes did not identify the resistance mutation, a cosmid library was made from G2, and a series of recombinant G2 wild-type viruses were constructed. Testing the recombinants for sensitivity to maribavir narrowed the locus of resistance to genes UL26 to UL32. Sequencing identified a single coding mutation in ORF UL27 (Leu335Pro) as the one responsible for resistance to maribavir. These results establish that UL27 is either directly or indirectly involved in the mechanism of action of maribavir. They also suggest that UL27 could play a role in HCMV DNA synthesis or egress of HCMV particles from the nucleus.

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 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.

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 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 Resistance of Human Cytomegalovirus to Cyclopropavir Maps to a Base Pair Deletion in the Open Reading Frame ofUL97

2013 ◽  
Vol 57 (9) ◽  
pp. 4343-4348 ◽  
Author(s):  
Brian G. Gentry ◽  
Laura E. Vollmer ◽  
Ellie D. Hall ◽  
Katherine Z. Borysko ◽  
Jiri Zemlicka ◽  
...  
Keyword(s):  
Drug Resistance ◽  
Human Cytomegalovirus ◽  
Mechanism Of Action ◽  
Base Pair ◽  
Kinase Domain ◽  
Wild Type Virus ◽  
Resistant Virus ◽  
Wild Type ◽  
Reading Frame ◽  
Base Pair Deletion

ABSTRACTHuman cytomegalovirus (HCMV) is a widespread pathogen in the human population, affecting many immunologically immature and immunocompromised patients, and can result in severe complications, such as interstitial pneumonia and mental retardation. Current chemotherapies for the treatment of HCMV infections include ganciclovir (GCV), foscarnet, and cidofovir. However, the high incidences of adverse effects (neutropenia and nephrotoxicity) limit the use of these drugs. Cyclopropavir (CPV), a guanosine nucleoside analog, is 10-fold more active against HCMV than GCV (50% effective concentrations [EC50s] = 0.46 and 4.1 μM, respectively). We hypothesize that the mechanism of action of CPV is similar to that of GCV: phosphorylation to a monophosphate by viral pUL97 protein kinase with further phosphorylation to a triphosphate by endogenous kinases, resulting in inhibition of viral DNA synthesis. To test this hypothesis, we isolated a CPV-resistant virus, sequenced its genome, and discovered that bp 498 ofUL97was deleted. This mutation caused a frameshift inUL97resulting in a truncated protein that lacks a kinase domain. To determine if this base pair deletion was responsible for drug resistance, the mutation was engineered into the wild-type viral genome, which was then exposed to increasing concentrations of CPV. The results demonstrate that the engineered virus was approximately 72-fold more resistant to CPV (EC50= 25.8 ± 3.1 μM) than the wild-type virus (EC50= 0.36 ± 0.11 μM). We conclude, therefore, that this mutation is sufficient for drug resistance and that pUL97 is involved in the mechanism of action of CPV.

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