scholarly journals The Full-Length Protein Encoded by Human Cytomegalovirus Gene UL117 Is Required for the Proper Maturation of Viral Replication Compartments

2008 ◽  
Vol 82 (7) ◽  
pp. 3452-3465 ◽  
Author(s):  
Zhikang Qian ◽  
Baoqin Xuan ◽  
Te Tee Hong ◽  
Dong Yu
Keyword(s):  
Human Cytomegalovirus ◽  
Large Scale ◽  
Full Length ◽  
Wild Type ◽  
Virus Growth ◽  
Reading Frame ◽  
Early Proteins ◽  
Immediate Early Proteins ◽  
Novel Transcripts ◽  
Related Proteins

ABSTRACT Previously, two large-scale mutagenic analyses showed that mutations in the human cytomegalovirus (HCMV) gene UL117 resulted in a defect in virus growth in fibroblasts. Early transcriptional analyses have revealed several mRNAs from the UL119-UL115 region; however, specific transcripts encoding UL117-related proteins have not been identified. In this study, we identified two novel transcripts arising from the UL117 gene locus, and we reported that the UL117 open reading frame encoded the full-length protein pUL117 (45 kDa) and the shorter isoform pUL117.5 (35 kDa) as the result of translation initiation at alternative in-frame ATGs. Both proteins were expressed with early kinetics, but pUL117 accumulated at a lower abundance relative to that of pUL117.5. During HCMV infection, both proteins localized predominantly to the nucleus, and the major fraction of pUL117 localized in viral nuclear replication compartments. We constructed mutant HCMV viruses in which the entire UL117 coding sequence was deleted or the expression of pUL117 was specifically abrogated. The growth of mutant viruses was significantly attenuated, indicating that pUL117 was required for efficient virus infection in fibroblasts. Cells infected with the pUL117-deficient mutant virus accumulated representative viral immediate-early proteins and early proteins normally. In the absence of pUL117, the accumulation of replicating viral DNA was reduced by no more than twofold at early times and was indistinguishable from that of the wild type at 72 h postinfection. Strikingly, there was a 12- to 24-h delay in the development of nuclear replication compartments and a marked delay in the expression of late viral proteins. We conclude that pUL117 acts to promote the development of nuclear replication compartments to facilitate viral growth.

scholarly journals Human Cytomegalovirus Gene UL21a Encodes a Short-Lived Cytoplasmic Protein and Facilitates Virus Replication in Fibroblasts

2009 ◽  
Vol 84 (1) ◽  
pp. 291-302 ◽  
Author(s):  
Anthony R. Fehr ◽  
Dong Yu
Keyword(s):  
Human Cytomegalovirus ◽  
Large Scale ◽  
Hcmv Infection ◽  
Productive Infection ◽  
Lung Fibroblasts ◽  
Wild Type Virus ◽  
Growth Defect ◽  
Cytoplasmic Protein ◽  
Wild Type ◽  
Virus Growth

ABSTRACT The human cytomegalovirus (HCMV) gene UL21a was recently annotated by its conservation in chimpanzee cytomegalovirus. Two large-scale mutagenic analyses showed that mutations in overlapping UL21a/UL21 resulted in a severe defect of virus growth in fibroblasts. Here, we characterized UL21a and demonstrated its role in HCMV infection. We mapped a UL21a-specific transcript of ∼600 bp that was expressed with early kinetics. UL21a encoded pUL21a, a protein of ∼15 kDa, which was unstable and localized predominantly to the cytoplasm during HCMV infection or when expressed alone. Interestingly, pUL21a was drastically stabilized in the presence of proteasome inhibitor MG132, but its instability was independent of a functional ubiquitin-mediated pathway, suggesting that pUL21a underwent proteasome-dependent, ubiquitin-independent degradation. A UL21a deletion virus was attenuated in primary human newborn foreskin fibroblasts (HFFs) and embryonic lung fibroblasts (MRC-5), whereas a marker-rescued virus and mutant viruses lacking the neighboring or overlapping genes UL20, UL21, or UL21.5-UL23 replicated at wild-type levels. The growth defect of UL21a-deficient virus in MRC-5 cells was more pronounced than that in HFFs. At a high multiplicity of infection, the UL21a deletion virus synthesized viral proteins with wild-type kinetics but had a two- to threefold defect in viral DNA replication. More importantly, although pUL21a was not detected in the virion, progeny virions produced by the mutant virus were ∼10 times less infectious than wild-type virus, suggesting that UL21a is required for HCMV to establish efficient productive infection. We conclude that UL21a encodes a short-lived cytoplasmic protein and facilitates HCMV replication in fibroblasts.

scholarly journals Characterization of a Novel Golgi Apparatus-Localized Latency Determinant Encoded by Human Cytomegalovirus

2009 ◽  
Vol 83 (11) ◽  
pp. 5615-5629 ◽  
Author(s):  
Alex Petrucelli ◽  
Michael Rak ◽  
Lora Grainger ◽  
Felicia Goodrum
Keyword(s):  
Golgi Apparatus ◽  
Human Cytomegalovirus ◽  
Recombinant Virus ◽  
Latent Infection ◽  
Immediate Early ◽  
Productive Infection ◽  
Reading Frame ◽  
Early Proteins ◽  
Immediate Early Proteins

ABSTRACT Human cytomegalovirus (HCMV) exists indefinitely in infected individuals by a yet poorly characterized latent infection in hematopoietic cells. We previously demonstrated a requirement for the putative UL138 open reading frame (ORF) in promoting a latent infection in CD34+ hematopoietic progenitor cells (HPCs) infected in vitro. In our present study, we have identified two coterminal transcripts of 2.7 and 3.6 kb and a 21-kilodalton (kDa) protein (pUL138) that are derived from the UL138 locus with early-late gene kinetics during productive infection. The UL138 transcripts and protein are detected in both fibroblasts and HPCs. A recombinant virus, FIX-UL138STOP, that synthesizes the UL138 transcripts but not the protein exhibited a partial loss-of-latency phenotype in HPCs, similar to the phenotype observed for the UL138-null recombinant virus. This finding suggests that the UL138 protein is required for latency, but it does not exclude the possibility that the UL138 transcripts or other ORFs also contribute to latency. The mechanisms by which pUL138 contributes to latency remain unknown. While the 86- and 72-kDa immediate-early proteins were not detected in HPCs infected with HCMV in vitro, pUL138 did not function directly to suppress expression from the major immediate-early promoter in reporter assays. Interestingly, pUL138 localizes to the Golgi apparatus in infected cells but is not incorporated into virus particles. The localization of pUL138 to the Golgi apparatus suggests that pUL138 contributes to HCMV latency by a novel mechanism. pUL138 is the first HCMV protein demonstrated to promote an infection with the hallmarks of latency in CD34+ HPCs.

Human cytomegalovirus US3 and UL36-38 immediate-early proteins regulate gene expression.

1992 ◽  
Vol 66 (1) ◽  
pp. 95-105 ◽  
Author(s):  
A M Colberg-Poley ◽  
L D Santomenna ◽  
P P Harlow ◽  
P A Benfield ◽  
D J Tenney
Keyword(s):  
Gene Expression ◽  
Human Cytomegalovirus ◽  
Immediate Early ◽  
Regulate Gene Expression ◽  
Early Proteins ◽  
Immediate Early Proteins ◽  
Regulate Gene

scholarly journals Effects of Human Cytomegalovirus Immediate-Early Proteins on p53-mediated Apoptosis in Coronary Artery Smooth Muscle Cells

Circulation ◽  
1999 ◽  
Vol 99 (13) ◽  
pp. 1656-1659 ◽  
Author(s):  
Koichi Tanaka ◽  
Jian-Ping Zou ◽  
Kazuyo Takeda ◽  
Victor J. Ferrans ◽  
Gordon R. Sandford ◽  
...  
Keyword(s):  
Smooth Muscle ◽  
Coronary Artery ◽  
Smooth Muscle Cells ◽  
Human Cytomegalovirus ◽  
Muscle Cells ◽  
Immediate Early ◽  
Early Proteins ◽  
Immediate Early Proteins

scholarly journals The Use of Recombinant Baculoviruses for Sustained Expression of Human Cytomegalovirus Immediate Early Proteins in Fibroblasts

Virology ◽  
2001 ◽  
Vol 284 (2) ◽  
pp. 297-307 ◽  
Author(s):  
Roopashree S. Dwarakanath ◽  
Charles L. Clark ◽  
Anita K. McElroy ◽  
Deborah H. Spector
Keyword(s):  
Human Cytomegalovirus ◽  
Immediate Early ◽  
Recombinant Baculoviruses ◽  
Early Proteins ◽  
Immediate Early Proteins

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 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.

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