scholarly journals Exon 3 of the Human Cytomegalovirus Major Immediate-Early Region Is Required for Efficient Viral Gene Expression and for Cellular Cyclin Modulation

2005 ◽  
Vol 79 (12) ◽  
pp. 7438-7452 ◽  
Author(s):  
Elizabeth A. White ◽  
Deborah H. Spector
Keyword(s):  
Human Cytomegalovirus ◽  
Cyclin B1 ◽  
Mutant Virus ◽  
Immediate Early ◽  
Wild Type Virus ◽  
Viral Gene ◽  
Protein Accumulation ◽  
Wild Type ◽  
Altered Expression ◽  
Infected Cells

ABSTRACT The human cytomegalovirus (HCMV) major immediate-early (IE) proteins share an 85-amino-acid N-terminal domain specified by exons 2 and 3 of the major IE region, UL122-123. We have constructed IE Δ30-77, a recombinant virus that lacks the majority of IE exon 3 and consequently expresses smaller forms of both IE1 72- and IE2 86-kDa proteins. The mutant virus is viable but growth impaired at both high and low multiplicities of infection and exhibits a kinetic defect that is not rescued by growth in fibroblasts expressing IE1 72-kDa protein. The kinetics of mutant IE2 protein accumulation in IE Δ30-77 virus-infected cells are approximately normal compared to wild-type virus-infected cells, but the IE Δ30-77 virus is delayed in expression of early viral genes, including UL112-113 and UL44, and does not sustain expression of mutant IE1 protein as the infection progresses. Additionally, cells infected with IE Δ30-77 exhibit altered expression of cellular proteins compared to wild-type HCMV-infected cells. PML is not dispersed but is retained at ND10 sites following infection with IE Δ30-77 mutant virus. While the deletion mutant retains the ability to mediate the stabilization of cyclin B1, cdc6, and geminin in infected cells, its capacity to upregulate the expression of cyclin E has been reduced. These data indicate that the activity of one or both of the HCMV major IE proteins is required in vivo for the modulation of cell cycle proteins observed in cells infected with wild-type HCMV.

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 Mutant Human Cytomegalovirus Lacking the Immediate-Early TRS1 Coding Region Exhibits a Late Defect

2002 ◽  
Vol 76 (23) ◽  
pp. 12290-12299 ◽  
Author(s):  
Catherine A. Blankenship ◽  
Thomas Shenk
Keyword(s):  
Human Cytomegalovirus ◽  
Infectious Virus ◽  
Human Fibroblasts ◽  
Reporter Genes ◽  
Immediate Early ◽  
Viral Gene ◽  
Wild Type ◽  
Viral Mrnas ◽  
Virus Particles ◽  
Infected Cells

ABSTRACT The human cytomegalovirus IRS1 and TRS1 open reading frames encode immediate-early proteins with identical N-terminal domains and divergent C-terminal regions. Both proteins have been shown previously to activate reporter genes in transfection assays in cooperation with other viral gene products. We have constructed two viruses carrying substitution mutations within either the IRS1 or TRS1 open reading frame. ADsubIRS1 failed to produce the related IRS1 and IRS1263 proteins, but it replicated with normal kinetics to produce a wild-type yield in human fibroblasts. The addition in trans of the IRS1263 protein, which antagonizes the ability of IRS1 and TRS1 proteins to activate reporter genes, did not inhibit the growth of the mutant virus. ADsubTRS1 failed to produce the TRS1 protein, and it generated an ∼200-fold-reduced yield of infectious virus in comparison to its wild-type parent. Viral DNA accumulated normally, as did a set of viral mRNAs that were monitored in ADsubTRS1-infected cells. However, two tegument proteins were partially mislocalized and infectious virus particles did not accumulate to normal levels within ADsubTRS1-infected cells. Further, infectious ADsubTRS1 particles sedimented abnormally in a glycerol-tartrate gradient, indicating that the structure of the mutant particles is aberrant. Our analysis of the ADsubTRS1 phenotype indicates that the TRS1 protein is required, either directly or indirectly, for efficient assembly of virus particles.

scholarly journals Diverse Roles for E4orf3 at Late Times of Infection Revealed in an E1B 55-Kilodalton Protein Mutant Background

2004 ◽  
Vol 78 (18) ◽  
pp. 9924-9935 ◽  
Author(s):  
Robin N. Shepard ◽  
David A. Ornelles
Keyword(s):  
Gene Expression ◽  
Double Mutant ◽  
Mutant Virus ◽  
Wild Type Virus ◽  
Viral Gene ◽  
Type Virus ◽  
Wild Type ◽  
Viral Dna ◽  
Early Region ◽  
Infected Cells

ABSTRACT Species C human adenovirus mutants that fail to express open reading frame 3 of early region 4 (E4orf3) are phenotypically indistinguishable from the wild-type virus when evaluated in cells cultured in vitro. However, E4orf3 gene function has been productively studied in the context of additional viral mutations. This study identifies diverse roles for the E4orf3 protein that are evident in the absence of early region 1B 55-kDa protein (E1B-55K) function. In an E1B-55K-deficient background, the E4orf3 protein promotes viral replication by increasing both the burst size and the probability that an infected cell will produce virus. Early viral gene expression is not impaired in E1B-55K/E4orf3 double mutant virus-infected cells. Cells infected with the double mutant virus accumulated concatemers of viral DNA. However, the E1B-55K/E4orf3 double mutant virus did not replicate any better in MO59J cells, in which viral DNA concatemers did not accumulate, than in MO59K cells, in which viral DNA concatemers were produced, suggesting that viral DNA concatenation is not the primary growth defect of the E1B-55K/E4orf3 double mutant virus. Accumulation of viral mRNA in the nucleus and cytoplasm of E1B-55K/E4orf3 double mutant virus-infected cells was severely reduced compared to that on wild-type virus-infected cells. Thus, in an E1B-55K mutant background, the E4orf3 protein promotes the accumulation of late viral RNA and enhances late gene expression. Finally, within the context of an E1B-55K mutant virus, the E4orf3 protein acts to suppress host cell translation and preserve the viability of cells at moderately late times of infection.

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 Herpes Simplex Virus 1 Induces Cytoplasmic Accumulation of TIA-1/TIAR and both Synthesis and Cytoplasmic Accumulation of Tristetraprolin, Two Cellular Proteins That Bind and Destabilize AU-Rich RNAs

2004 ◽  
Vol 78 (16) ◽  
pp. 8582-8592 ◽  
Author(s):  
Audrey Esclatine ◽  
Brunella Taddeo ◽  
Bernard Roizman
Keyword(s):  
Herpes Simplex Virus ◽  
Herpes Simplex ◽  
Mutant Virus ◽  
Wild Type Virus ◽  
Type Virus ◽  
Wild Type ◽  
Herpes Simplex Virus 1 ◽  
Infected Cells ◽  
Cytoplasmic Accumulation ◽  
Simplex Virus

ABSTRACT Herpes simplex virus 1 causes a shutoff of cellular protein synthesis through the degradation of RNA that is mediated by the virion host shutoff (Vhs) protein encoded by the UL41 gene. We reported elsewhere that the Vhs-dependent degradation of RNA is selective, and we identified RNAs containing AU-rich elements (AREs) that were upregulated after infection but degraded by deadenylation and progressive 3′-to-5′ degradation. We also identified upregulated RNAs that were not subject to Vhs-dependent degradation (A. Esclatine, B. Taddeo, L. Evans, and B. Roizman, Proc. Natl. Acad. Sci. USA 101:3603-3608, 2004). Among the latter was the RNA encoding tristetraprolin, a protein that binds AREs and is known to be associated with the degradation of RNAs containing AREs. Prompted by this observation, we examined the status of the ARE binding proteins tristetraprolin and TIA-1/TIAR in infected cells. We report that tristetraprolin was made and accumulated in the cytoplasm of wild-type virus-infected human foreskin fibroblasts as early as 2 h and in HEp-2 cells as early as 6 h after infection. The amounts of tristetraprolin that accumulated in the cytoplasm of cells infected with a mutant virus lacking UL41 were significantly lower than those in wild-type virus-infected cells. The localization of tristetraprolin was not modified in cells infected with a mutant lacking the gene encoding infected cell protein 4 (ICP4). TIA-1 and TIAR are two other proteins that are associated with the regulation of ARE-containing RNAs and that normally reside in nuclei. In infected cells, they started to accumulate in the cytoplasm after 6 h of infection. In cells infected with the mutant virus lacking UL41, TIA-1/TIAR accumulated in the cytoplasm in granular structures reminiscent of stress granules in a significant percentage of the cells. In addition, an antibody to tristetraprolin coprecipitated the Vhs protein from lysates of cells late in infection. The results indicate that the Vhs-dependent degradation of ARE-containing RNAs correlates with the transactivation, cytoplasmic accumulation, and persistence of tristetraprolin in infected cells.

scholarly journals The chromatin-tethering domain of human cytomegalovirus immediate-early (IE) 1 mediates associations of IE1, PML and STAT2 with mitotic chromosomes, but is not essential for viral replication

2012 ◽  
Vol 93 (4) ◽  
pp. 716-721 ◽  
Author(s):  
Hye Jin Shin ◽  
Young-Eui Kim ◽  
Eui Tae Kim ◽  
Jin-Hyun Ahn
Keyword(s):  
Viral Replication ◽  
Human Cytomegalovirus ◽  
Immediate Early ◽  
Wild Type Virus ◽  
Type Virus ◽  
Virus Infections ◽  
Wild Type ◽  
Mitotic Chromosomes ◽  
Carboxyl Terminal ◽  
Chromosome Tethering

Human cytomegalovirus (HCMV) immediate-early (IE) 1 protein associates with chromosomes in mitotic cells using its carboxyl-terminal 16 aa region. However, the role of this IE1 activity in viral growth has not been evaluated in the context of mutant virus infection. We produced a recombinant HCMV encoding mutant IE1 with the carboxyl-terminal chromosome-tethering domain (CTD) deleted. This IE1(ΔCTD) virus grew like the wild-type virus in fibroblasts, indicating that the CTD is not essential for viral replication in permissive cells. Unlike wild-type virus infections, PML and STAT2, which interact with IE1, did not accumulate at mitotic chromosomes in IE1(ΔCTD) virus-infected fibroblasts, demonstrating that their associations with chromosomes are IE1 CTD-dependent. IE1 SUMOylation did not affect IE1 association with chromosomes. Our results provide genetic evidence that the CTD is required for the associations of IE1, PML and STAT2 with mitotic chromosomes, but that these IE1-related activities are not essential for viral replication in fibroblasts.

scholarly journals Role of Herpes Simplex Virus ICP27 in the Degradation of mRNA by Virion Host Shutoff RNase

2010 ◽  
Vol 84 (19) ◽  
pp. 10182-10190 ◽  
Author(s):  
Brunella Taddeo ◽  
Weiran Zhang ◽  
Bernard Roizman
Keyword(s):  
Mutant Virus ◽  
Structural Proteins ◽  
Wild Type Virus ◽  
Tegument Protein ◽  
Wild Type ◽  
Virion Host Shutoff ◽  
Infected Cells ◽  
Host Shutoff ◽  
Simplex Virus

ABSTRACT The virion host shutoff (VHS) RNase tegument protein released into cells by infecting virus has two effects. Preexisting stable mRNAs (e.g., GAPDH [glyceraldehyde-3-phosphate dehydrogenase]) are rapidly degraded. Stress response RNAs containing AU-rich elements (AREs) in the 3′ untranslated region (3′UTR) are deadenylated and cleaved, but the cleavage products persist for hours, in contrast to the short half-lives of ARE-containing mRNAs in uninfected cells. At late times, the VHS RNase is neutralized by the viral structural proteins VP16 and VP22. A recent study (J. A. Corcoran, W. L. Hsu, and J. R. Smiley, J. Virol. 80:9720-9729, 2006) reported that, at relatively late times after infection, ARE RNAs are rapidly degraded in cells infected with ΔICP27 mutant virus and concluded that ICP27 “stabilizes” ARE mRNAs. We report the following. (i) The rates of degradation of ARE mRNA at early times (3 h) after infection with the wild type or the ΔICP27 mutant virus are virtually identical, and hence ICP27 plays no role in this process. (ii) In noncomplementing cells, VHS RNase or VP22 is not synthesized. Therefore, the only VHS that is active is brought into cells by the ΔICP27 mutant. (ii) The VHS RNase brought into the cells by the ΔICP27 virus is reduced in potency relative to that of wild-type virus. Hence the rapid degradation of ARE mRNAs noted in ΔICP27 mutant-infected cells at late times is similar to that taking place in mock-infected or in ΔVHS RNase mutant-virus-infected cells and does not by itself support the hypothesis that ICP27 stabilizes ARE mRNAs. (iii) Concurrently, we present the first evidence that VHS RNase interacts with ICP27 most likely when bound to cap- and poly(A)-binding proteins, respectively.

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 UL26-Deficient Human Cytomegalovirus Produces Virions with Hypophosphorylated pp28 Tegument Protein That Is Unstable within Newly Infected Cells

2006 ◽  
Vol 80 (7) ◽  
pp. 3541-3548 ◽  
Author(s):  
Joshua Munger ◽  
Dong Yu ◽  
Thomas Shenk
Keyword(s):  
Human Cytomegalovirus ◽  
Deletion Mutant ◽  
Open Reading Frame ◽  
Early Gene ◽  
Immediate Early ◽  
Tegument Protein ◽  
Protein Accumulation ◽  
Reading Frame ◽  
Tegument Proteins ◽  
Infected Cells

ABSTRACT The human cytomegalovirus UL26 open reading frame encodes proteins of 21 and 27 kDa that result from the use of two different in-frame initiation codons. The UL26 protein is a constituent of the virion and thus is delivered to cells upon viral entry. We have characterized a mutant of human cytomegalovirus in which the UL26 open reading frame has been deleted. The UL26 deletion mutant has a profound growth defect, the magnitude of which is dependent on the multiplicity of infection. Two very early defects were discovered. First, even though they were present in normal amounts within mutant virions, the UL99-coded pp28 and UL83-coded pp65 tegument proteins were present in reduced amounts at the earliest times assayed within newly infected cells; second, there was a delay in immediate-early mRNA and protein accumulation. Further analysis revealed that although wild-type levels of the pp28 tegument protein were present in UL26 deletion mutant virions, the protein was hypophosphorylated. We conclude that the UL26 protein influences the normal phosphorylation of at least pp28 in virions and possibly additional tegument proteins. We propose that the hypophosphorylation of tegument proteins causes their destabilization within newly infected cells, perhaps disrupting the normal detegumentation process and leading to a delay in the onset of immediate-early gene expression.

scholarly journals Poliovirus 3A Protein Limits Interleukin-6 (IL-6), IL-8, and Beta Interferon Secretion during Viral Infection

2001 ◽  
Vol 75 (17) ◽  
pp. 8158-8165 ◽  
Author(s):  
Dana A. Dodd ◽  
Thomas H. Giddings ◽  
Karla Kirkegaard
Keyword(s):  
Protein Secretion ◽  
Interleukin 6 ◽  
Secretory Pathway ◽  
Mutant Virus ◽  
Cytokine Secretion ◽  
Wild Type Virus ◽  
General Mechanism ◽  
Wild Type ◽  
Beta Interferon ◽  
Infected Cells

ABSTRACT During viral infections, the host secretory pathway is crucial for both innate and acquired immune responses. For example, the export of most proinflammatory and antiviral cytokines, which recruit lymphocytes and initiate antiviral defenses, requires traffic through the host secretory pathway. To investigate potential effects of the known inhibition of cellular protein secretion during poliovirus infection on pathogenesis, cytokine secretion from cells infected with wild-type virus and with 3A-2, a mutant virus carrying an insertion in viral protein 3A which renders the virus defective in the inhibition of protein secretion, was tested. We show here that cells infected with 3A-2 mutant virus secrete greater amounts of cytokines interleukin-6 (IL-6), IL-8, and beta interferon than cells infected with wild-type poliovirus. Increased cytokine secretion from the mutant-infected cells can be attributed to the reduced inhibition of host protein secretion, because no significant differences between 3A-2- and wild-type-infected cells were observed in the inhibition of viral growth, host cell translation, or the ability of wild-type- or 3A-2-infected cells to support the transcriptional induction of beta interferon mRNA. We surmise that the wild-type function of 3A in inhibiting ER-to-Golgi traffic is not required for viral replication in tissue culture but, by altering the amount of secreted cytokines, could have substantial effects on pathogenesis within an infected host. The global inhibition of protein secretion by poliovirus may reflect a general mechanism by which pathogens that do not require a functional protein secretory apparatus can reduce the native immune response and inflammation associated with infection.

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