Prokaryotic expression of a large fragment of the most antigenic cytomegalovirus DNA-binding protein (ppUL44) and its reactivity with human antibodies

Ripalti, Alessandro; Monte, Paola Dal; Boccuni, M C; Campanini, Fabio; Lazzarotto, Tiziana; Campisi, B.; Ruan, Qiaoqiao; Landini, Maria Paola

doi:10.1016/0166-0934(94)90015-9

Cited by 21 publications

(17 citation statements)

References 18 publications

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“…HCMV UL44 is the polymerase accessory protein and was shown to interact with UL54, an HCMV-encoded polymerase (20)(21)(22)32). The evidence presented here suggests that UL44 may play a role in initiation of lytic DNA synthesis.…”

Section: Discussionmentioning

confidence: 78%

Identification of Human Cytomegalovirus UL84 Virus- and Cell-Encoded Binding Partners by Using Proteomics Analysis

Gao

Colletti

Pari

2008

J Virol

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Human cytomegalovirus (HCMV) UL84 is a phosphoprotein that shuttles from the nucleus to the cytoplasm and is required for oriLyt-dependent DNA replication and viral growth. UL84 was previously shown to interact with IE2 (IE86) in infected cells, and this interaction down-regulates IE2-mediated transcriptional activation in transient assays. UL84 and IE2 were also shown to cooperatively activate a promoter within HCMV oriLyt. UL84 alone can interact with an RNA stem-loop within oriLyt and is bound to this structure within the virion. In an effort to investigate the binding partners for UL84 in infected cells, we pulled down UL84 from protein lysates prepared from HCMV-infected human fibroblasts by using a UL84-specific antibody and resolved the immunoprecipitated protein complexes by two-dimensional gel electrophoresis. We subsequently identified individual proteins by matrix-assisted laser desorption ionization-tandem time of flight analysis. This analysis revealed that UL84 interacts with viral proteins UL44, pp65, and IE2. In addition, a number of cell-encoded proteins were identified, including ubiquitin-conjugating enzyme E2, casein kinase II (CKII), and the multifunctional protein p32. We also confirmed the interaction between UL84 and IE2 as well as the interaction of UL84 with importin ␣. UL44, pp65, and CKII interactions were confirmed to occur in infected and cotransfected cells by coimmunoprecipitation assays followed by Western blotting. Ubiquitination of UL84 occurred in the presence and absence of the proteasome activity inhibitor MG132 in infected cells. The identification of UL84 binding partners is a significant step toward the understanding of the function of this significant replication protein.Human cytomegalovirus (HCMV) lytic DNA replication requires the cis-acting oriLyt sequence, six core replication proteins, and, in human fibroblasts, the immediate-early protein IE2 and the multifunctional protein UL84 (4, 29, 30). Recent evidence indicates that UL84 may have several roles in the virus life cycle. Also, a number of activities have been associated with UL84. These include a shuttling function, inhibition of IE2-mediated transcriptional activation, RNA binding, and UTPase activity (9,12,17,43). The UL84-IE2 protein complex can activate a strong bidirectional promoter within oriLyt, presumably to initiate a transcription event that triggers initiation of DNA synthesis. Evidence that UL84 is the initiator protein for lytic DNA synthesis comes from recent data demonstrating that UL84 binds to a stem-loop RNA structure within oriLyt (7). In vitro binding assays suggest that UL84 can change the conformation of RNA stem-loop structures and possibly allow for the assembly of the replication complex. UL84 was shown to be a component of the virion (40) and is bound to viral DNA at the lytic origin within isolated virions (7). This suggests that UL84 can play a positive or a negative role in initiation of lytic DNA synthesis. Both the wide range of activities associated with UL84 and analysis of the...

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Section: Discussionmentioning

confidence: 78%

Identification of Human Cytomegalovirus UL84 Virus- and Cell-Encoded Binding Partners by Using Proteomics Analysis

Gao

Colletti

Pari

2008

J Virol

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“…The HCMV-coded proteins reactive with IgM antibody are VOL. 15,2002 DIAGNOSIS OF CONGENITAL HCMV INFECTION 685 both structural and nonstructural (39,144,151,213,223,224,280). Major structural proteins include pp150 (UL32), pp65 (UL83), and pp38 (UL80a), while nonstructural proteins include pp52 (UL44) and p130 (UL57).…”

Section: Serologymentioning

confidence: 99%

Diagnosis and Management of Human Cytomegalovirus Infection in the Mother, Fetus, and Newborn Infant

2002

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Human cytomegalovirus (HCMV) is the leading cause of congenital viral infection and mental retardation. HCMV infection, while causing asymptomatic infections in most immunocompetent subjects, can be transmitted during pregnancy from the mother with primary (and also recurrent) infection to the fetus. Hence, careful diagnosis of primary infection is required in the pregnant woman based on the most sensitive serologic assays (immunoglobulin M [IgM] and IgG avidity assays) and conventional virologic and molecular procedures for virus detection in blood. Maternal prognostic markers of fetal infection are still under investigation. If primary infection is diagnosed in a timely manner, prenatal diagnosis can be offered, including the search for virus and virus components in fetal blood and amniotic fluid, with fetal prognostic markers of HCMV disease still to be defined. However, the final step for definite diagnosis of congenital HCMV infection is detection of virus in the blood or urine in the first 1 to 2 weeks of life. To date, treatment of congenital infection with antiviral drugs is only palliative both prior to and after birth, whereas the only efficacious preventive measure seems to be the development of a safe and immunogenic vaccine, including recombinant, subunit, DNA, and peptide-based vaccines now under investigation. The following controversial issues are discussed in the light of the most recent advances in the field: the actual perception of the problem; universal serologic screening before pregnancy; the impact of correct counseling on decision making by the couple involved; the role of prenatal diagnosis in ascertaining transmission of virus to the fetus; the impact of preconceptional and periconceptional infections on the prevalence of congenital infection; and the prevalence of congenitally infected babies born to mothers who were immune prior to pregnancy compared to the number born to mothers undergoing primary infection during pregnancy

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“…Variations in the relative amounts of these antigens produced during growth and purification of the virus can result in a different stoichiometric composition of the viral antigens used in the various IgM tests, leading to the discordant interassay results observed. Antigenic materials composed of peptides or purified recombinant proteins produced through molecular biology offer an attractive alternative for the detection of CMV-specific IgM [16,18,24,25,[31][32][33][34][35]. We have shown that a cocktail of purified recombinant protein antigens containing portions of pUL32, pUL44, pUL83 and pUL80a is both necessary and sufficient to replace the virus in a microtiter ELISA for the detection of CMV-specific IgM [19,36].…”

Section: Serological Advancesmentioning

confidence: 99%

“…The key serological targets for detection of CMV-specific IgM include both structural (pUL32, pUL83, pUL80a) [25][26][27][28][29], and nonstructural (pUL57, pUL44) [24,[30][31][32] viral proteins. Variations in the relative amounts of these antigens produced during growth and purification of the virus can result in a different stoichiometric composition of the viral antigens used in the various IgM tests, leading to the discordant interassay results observed.…”

Section: Serological Advancesmentioning

confidence: 99%

New Advances in the Diagnosis of Congenital Cytomegalovirus Infection

Lazzarotto¹,

Varani²,

Gabrielli³

et al. 1999

Intervirology

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With the advances in anticytomegalovirus (anti-CMV) serology, the new recombinant IgM tests seem likely to become the screening tests for pregnant women whose prepregnancy serological status for CMV is unknown. When a woman is found to be IgM-positive, further diagnostic evaluation focused on determining whether this is due to a primary infection should be carried out. Maternal primary infections that were difficult to determine until a few years ago unless documented by seroconversion can now be readily diagnosed from the presence of low-avidity anti-CMV antibody which persists for approximately 20 weeks after primary infection. In primarily infected mothers prenatal diagnosis can be performed between 21 and 23 weeks of gestation, and the amniotic fluid (AF) represents the pathological material of choice to determine intrauterine virus transmission. In AF, the virus can be detected by culture and/or PCR. Both procedures differentiate uninfected from infected fetuses, but cannot predict fetal outcome. The determination of the viral load in AF carried out by quantitative PCR is more promising and could represent an important starting point for preemptive fetal therapy.

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Prokaryotic expression of a large fragment of the most antigenic cytomegalovirus DNA-binding protein (ppUL44) and its reactivity with human antibodies

Cited by 21 publications

References 18 publications

Identification of Human Cytomegalovirus UL84 Virus- and Cell-Encoded Binding Partners by Using Proteomics Analysis

Identification of Human Cytomegalovirus UL84 Virus- and Cell-Encoded Binding Partners by Using Proteomics Analysis

Diagnosis and Management of Human Cytomegalovirus Infection in the Mother, Fetus, and Newborn Infant

New Advances in the Diagnosis of Congenital Cytomegalovirus Infection

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