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.

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 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.

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.

Mating type control in Saccharomyces cerevisiae: a frameshift mutation at the common DNA sequence, X, of the HML alpha locus

1984 ◽  
Vol 4 (1) ◽  
pp. 203-211
Author(s):  
K Tanaka ◽  
T Oshima ◽  
H Araki ◽  
S Harashima ◽  
Y Oshima
Keyword(s):  
Mating Type ◽  
Base Pair ◽  
Reading Frame ◽  
Base Pair Deletion ◽  
A Cell ◽  
Alpha Gene ◽  
The Common ◽  
Type Switch ◽  
Diploid Cells ◽  
Mat Locus

A mutation defective in the homothallic switching of mating type alleles, designated hml alpha-2, has previously been characterized. The mutation occurred in a cell having the HO MATa HML alpha HMRa genotype, and the mutant culture consisted of ca. 10% a mating type cells, 90% nonmater cells of haploid cell size, and 0.1% sporogenous diploid cells. Genetic analyses revealed that nonmater haploid cells have a defect in the alpha 2 cistron at the MAT locus. This defect was probably caused by transposition of a cassette originating from the hml alpha-2 allele by the process of the homothallic mating type switch. That the MAT locus of the nonmater cells is occupied by a DNA fragment indistinguishable from the Y alpha sequence in electrophoretic mobility was demonstrated by Southern hybridization of the EcoRI-HindIII fragment encoding the MAT locus with a cloned HML alpha gene as the probe. The hml alpha-2 mutation was revealed to be a one-base-pair deletion at the ninth base pair in the X region from the X and Y boundary of the HML locus. This mutation gave rise to a shift in the open reading frame of the alpha 2 cistron. A molecular mechanism for the mating type switch associated with the occurrence of sporogenous diploid cells in the mutant culture is discussed.

scholarly journals Involvement of the N-Terminal Deubiquitinating Protease Domain of Human Cytomegalovirus UL48 Tegument Protein in Autoubiquitination, Virion Stability, and Virus Entry

2016 ◽  
Vol 90 (6) ◽  
pp. 3229-3242 ◽  
Author(s):  
Young-Eui Kim ◽  
Se Eun Oh ◽  
Ki Mun Kwon ◽  
Chan Hee Lee ◽  
Jin-Hyun Ahn
Keyword(s):  
Human Cytomegalovirus ◽  
Virus Entry ◽  
Terminal Region ◽  
Mutant Virus ◽  
Wild Type Virus ◽  
Type Virus ◽  
Wild Type ◽  
Viral Growth ◽  
Internal Region ◽  
Deubiquitinating Protease

ABSTRACTHuman cytomegalovirus (HCMV) protein pUL48 is closely associated with the capsid and has a deubiquitinating protease (DUB) activity in its N-terminal region. Although this DUB activity moderately increases virus replication in cultured fibroblast cells, the requirements of the N-terminal region of pUL48 in the viral replication cycle are not fully understood. In this study, we characterized the recombinant viruses encoding UL48(ΔDUB/NLS), which lacks the DUB domain and the adjacent nuclear localization signal (NLS), UL48(ΔDUB), which lacks only the DUB, and UL48(Δ360–1200), which lacks the internal region (amino acids 360 to 1200) downstream of the DUB/NLS. While ΔDUB/NLS and Δ360–1200 mutant viruses did not grow in fibroblasts, the ΔDUB virus replicated to titers 100-fold lower than those for wild-type virus and showed substantially reduced viral gene expression at low multiplicities of infection. The DUB domain contained ubiquitination sites, and DUB activity reduced its own proteasomal degradation intrans. Deletion of the DUB domain did not affect the nuclear and cytoplasmic localization of pUL48, whereas the internal region (360–1200) was necessary for cytoplasmic distribution. In coimmunoprecipitation assays, pUL48 interacted with three tegument proteins (pUL47, pUL45, and pUL88) and two capsid proteins (pUL77 and pUL85) but the DUB domain contributed to only pUL85 binding. Furthermore, we found that the ΔDUB virus showed reduced virion stability and less efficiently delivered its genome into the cell than the wild-type virus. Collectively, our results demonstrate that the N-terminal DUB domain of pUL48 contributes to efficient viral growth by regulating its own stability and promoting virion stabilization and virus entry.IMPORTANCEHCMV pUL48 and its herpesvirus homologs play key roles in virus entry, regulation of immune signaling pathways, and virion assembly. The N terminus of pUL48 contains the DUB domain, which is well conserved among all herpesviruses. Although studies using the active-site mutant viruses revealed that the DUB activity promotes viral growth, the exact role of this region in the viral life cycle is not fully understood. In this study, using the mutant virus lacking the entire DUB domain, we demonstrate that the DUB domain of pUL48 contributes to viral growth by regulating its own stability via autodeubiquitination and promoting virion stability and virus entry. This report is the first to demonstrate the characteristics of the mutant virus with the entire DUB domain deleted, which, along with information on the functions of this region, is useful in dissecting the functions associated with pUL48.

scholarly journals Detection of novel HIV-1 drug resistance mutations by support vector analysis of deep sequence data and experimental validation

2019 ◽  
Author(s):  
Mariano Avino ◽  
Emmanuel Ndashimye ◽  
Daniel J. Lizotte ◽  
Abayomi S. Olabode ◽  
Richard M. Gibson ◽  
...  
Keyword(s):  
Drug Resistance ◽  
Drug Treatment ◽  
Treatment Outcomes ◽  
Sequence Data ◽  
Wild Type Virus ◽  
Support Vector ◽  
Wild Type ◽  
Subtype B ◽  
The World ◽  
Hiv 1

AbstractThe global HIV-1 pandemic comprises many genetically divergent subtypes. Most of our understanding of drug resistance in HIV-1 derives from subtype B, which predominates in North America and western Europe. However, about 90% of the pandemic represents non-subtype B infections. Here, we use deep sequencing to analyze HIV-1 from infected individuals in Uganda who were either treatment-naïve or who experienced virologic failure on ART without the expected patterns of drug resistance. Our objective was to detect potentially novel associations between mutations in HIV-1 integrase and treatment outcomes in Uganda, where most infections are subtypes A or D. We retrieved a total of 380 archived plasma samples from patients at the Joint Clinical Research Centre (Kampala), of which 328 were integrase inhibitor-naïve and 52 were raltegravir (RAL)-based treatment failures. Next, we developed a bioinformatic pipeline for alignment and variant calling of the deep sequence data obtained from these samples from a MiSeq platform (Illumina). To detect associations between within-patient polymorphisms and treatment outcomes, we used a support vector machine (SVM) for feature selection with multiple imputation to account for partial reads and low quality base calls. Candidate point mutations of interest were experimentally introduced into the HIV-1 subtype B NL4-3 backbone to determine susceptibility to RAL in U87.CD4.CXCR4 cells. Finally, we carried out replication capacity experiments with wild-type and mutant viruses in TZM-bl cells in the presence and absence of RAL. Our analyses not only identified the known major mutation N155H and accessory mutations G163R and V151I, but also novel mutations I203M and I208L as most highly associated with RAL failure. The I203M and I208L mutations resulted in significantly decreased susceptibility to RAL (44.0-fold and 54.9-fold, respectively) compared to wild-type virus (EC50=0.32 nM), and may represent novel pathways of HIV-1 resistance to modern treatments.Author summaryThere are many different types of HIV-1 around the world. Most of the research on how HIV-1 can become resistant to drug treatment has focused on the type (B) that is the most common in high-income countries. However, about 90% of infections around the world are caused by a type other than B. We used next-generation sequencing to analyze samples of HIV-1 from patients in Uganda (mostly infected by types A and D) for whom drug treatment failed to work, and whose infections did not fit the classic pattern of adaptation based on B. Next, we used machine learning to detect mutations in these virus populations that could explain the treatment outcomes. Finally, we experimentally added two candidate mutations identified by our analysis to a laboratory strain of HIV-1 and confirmed that they conferred drug resistance to the virus. Our study reveals new pathways that other types of HIV-1 may use to evolve resistance to drugs that make up the current recommended treatment for newly diagnosed individuals.

scholarly journals The AC4 Protein of a Cassava Geminivirus Is Required for Virus Infection

2019 ◽  
Vol 32 (7) ◽  
pp. 865-875 ◽  
Author(s):  
Kegui Chen ◽  
Behnam Khatabi ◽  
Vincent N. Fondong
Keyword(s):  
Plant Viruses ◽  
Wild Type Virus ◽  
Virus Infectivity ◽  
Type Virus ◽  
Wild Type ◽  
Knockout Mutant ◽  
East African ◽  
Reading Frame ◽  
Cell Membrane Binding

Geminiviruses (family Geminiviridae) are among the most devastating plant viruses worldwide, causing severe damage in crops of economic and subsistence importance. These viruses have very compact genomes and many of the encoded proteins are multifunctional. Here, we investigated the role of the East African cassava mosaic Cameroon virus (EACMCV) AC4 on virus infectivity in Nicotiana benthamiana. Results showed that plants inoculated with EACMCV containing a knockout mutation in an AC4 open reading frame displayed symptoms 2 to 3 days later than plants inoculated with wild-type virus, and these plants recovered from infection, whereas plants inoculated with the wild-type virus did not. Curiously, when an additional mutation was made in the knockout mutant, the resulting double mutant virus completely failed to cause any apparent symptoms. Interestingly, the role of AC4 on virus infectivity appeared to be dependent on an encoded N-myristoylation motif that mediates cell membrane binding. We previously showed that EACMCV containing the AC4T38I mutant produced virus progeny characterized by second-site mutations and reversion to wild-type virus. These results were confirmed in this study using additional mutations. Together, these results show involvement of EACMCV AC4 in virus infectivity; they also suggest a role for the combined action of mutation and selection, under prevailing environmental conditions, on begomovirus genetic variation and diversity.

scholarly journals Isolation and Characterization of HIV-1 Isolates Resistant to Oxathiin Carboxanilide Analogues: Evaluation of Variables in the Drug Resistance Selection Process

1996 ◽  
Vol 7 (5) ◽  
pp. 261-269 ◽  
Author(s):  
T.L. Kinjerski ◽  
L.A. Pallansch ◽  
R.W. Buckheit
Keyword(s):  
Drug Resistance ◽  
Amino Acid ◽  
Reverse Transcriptase ◽  
Selection Process ◽  
Antiviral Agents ◽  
Wild Type Virus ◽  
Wild Type ◽  
Resistance Selection ◽  
Isolation And Characterization ◽  
Virus Isolates

Virus isolates resistant to a variety of structural analogues of oxathiin carboxanilide (UC84) were selected in cell culture and phenotypically and mo!ecularly characterized in order to evaluate the effects of variables in the resistance selection process. The rate of appearance of the resistant viruses and the net loss of sensitivity of the virus isolates to the selecting compound were not dependent on the selective pressure employed against wild-type virus, but were associated with structural features of the compound. Although each of the compounds rapidly selected for resistant viruses, the isolates obtained varied in their overall level of resistance, in their cross-resistance to other non-nucleoside reverse transcriptase inhibitors (NNRTIs) and in the amino acid changes present in the reverse transcriptase (RT). The mutation Y181C was most commonly observed, often with a second mutation, such as K101E or V-106A. The amino acid change L100I was also observed upon selection. In order to determine the reproducibility of the in-vitro resistance selection process, 10 isolates resistant to UC84 were independently selected in parallel cultures. Mutations detected in the RT of these isolates were varied but included the commonly reported Y181C and V106A amino acid changes, as well as unique changes K101I, K101N, K103Q, G190A, T139I and A98S. These results demonstrate that from the heterogeneous wild-type population, antiviral agents, such as the UC compounds, may select a wide variety of virus isolates with resistance-engendering amino acid changes in the RT. In addition, the results also suggest that antiviral agents may select for resistant viruses at different rates and to different extents, offering the possibility that compounds might be identified which do not readily result in high level drug resistance.

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