Biomedical subjects

Chengjun Mo

Publications and source records attributed to Chengjun Mo.

5 recordsLinked to original sources

PubMed2005

Human cytomegalovirus uracil DNA glycosylase associates with ppUL44 and accelerates the accumulation of viral DNA.

BACKGROUND: Human cytomegalovirus UL114 encodes a uracil-DNA glycosylase homolog that is highly conserved in all characterized herpesviruses that infect mammals. Previous studies demonstrated that the deletion of this nonessential gene delays significantly the onset of viral DNA synthesis and results in a prolonged replication cycle. The gene product, pUL114, also appears to be important in late phase DNA synthesis presumably by introducing single stranded breaks. RESULTS: A series of experiments was performed to formally assign the observed phenotype to pUL114 and to characterize the function of the protein in viral replication. A cell line expressing pUL114 complemented the observed phenotype of a UL114 deletion virus in trans, confirming that the observed defects were the result of a deficiency in this gene product. Stocks of recombinant viruses without elevated levels of uracil were produced in the complementing cells; however they retained the phenotype of poor growth in normal fibroblasts suggesting that poor replication was unrelated to uracil content of input genomes. Recombinant viruses expressing epitope tagged versions of this gene demonstrated that pUL114 was expressed at early times and that it localized to viral replication compartments. This protein also coprecipitated with the DNA polymerase processivity factor, ppUL44 suggesting that these proteins associate in infected cells. This apparent interaction did not appear to require other viral proteins since ppUL44 could recruit pUL114 to the nucleus in uninfected cells. An analysis of DNA replication kinetics revealed that the initial rate of DNA synthesis and the accumulation of progeny viral genomes were significantly reduced compared to the parent virus. CONCLUSION: These data suggest that pUL114 associates with ppUL44 and that it functions as part of the viral DNA replication complex to increase the efficiency of both early and late phase viral DNA synthesis.

Cells, Cultured↗

PubMed2004

Development of an efficient fluorescence-based microneutralization assay using recombinant human cytomegalovirus strains expressing green fluorescent protein.

MedImmune Vaccines has created four, live, attenuated human cytomegalovirus (HCMV) vaccine candidates, each derived from defined portions of the parental strains, Towne and Toledo. To determine each candidate's ability to induce HCMV specific immunity, a fluorescence-based microneutralization assay was developed using recombinants of Toledo and Towne which express enhanced green fluorescent protein (EGFP). Replication of the EGFP recombinants in cell culture was the same as the respective parental strains. Using the EGFP recombinants, this fluorescence-based microneutralization assay was compared with the traditional plaque reduction assay. Serum samples were analyzed by both the fluorescence microneutralization and plaque reduction assays and regression analysis showed a correlation of R2 > or = 0.90 between the two assays. As an alternative to measuring fluorescence, infected cells were examined microscopically and the number of green fluorescent cells was counted automatically. Regression lines between fluorescent cell counting and fluorescence in the well also showed a high correlation (R2 > or = 0.92). An excellent linear concordance in titers was observed between the two assays. Using the plaque reduction assay, serum samples were identified that preferentially neutralized the Toledo strain compared to the Towne strain. The same preferences were observed with the fluorescence-based microneutralization assay. This new assay is adaptable to rapid, automated collection of neutralization data and would therefore be suitable for the examination of large numbers of clinical serum samples.

Cell Line↗

PubMed2004

Functions of the C-terminal domain of varicella-zoster virus glycoprotein E in viral replication in vitro and skin and T-cell tropism in vivo.

Varicella-zoster virus (VZV) glycoprotein E (gE) is essential for VZV replication. To further analyze the functions of gE in VZV replication, a full deletion and point mutations were made in the 62-amino-acid (aa) C-terminal domain. Targeted mutations were introduced in YAGL (aa 582 to 585), which mediates gE endocytosis, AYRV (aa 568 to 571), which targets gE to the trans-Golgi network (TGN), and SSTT, an "acid cluster" comprising a phosphorylation motif (aa 588 to 601). Substitutions Y582G in YAGL, Y569A in AYRV, and S593A, S595A, T596A, and T598A in SSTT were introduced into the viral genome by using VZV cosmids. These experiments demonstrated a hierarchy in the contributions of these C-terminal motifs to VZV replication and virulence. Deletion of the gE C terminus and mutation of YAGL were lethal for VZV replication in vitro. Mutations of AYRV and SSTT were compatible with recovery of VZV, but the AYRV mutation resulted in rapid virus spread in vitro and the SSTT mutation resulted in higher virus titers than were observed for the parental rOka strain. When the rOka-gE-AYRV and rOka-gE-SSTT mutants were evaluated in skin and T-cell xenografts in SCIDhu mice, interference with TGN targeting was associated with substantial attenuation, especially in skin, whereas the SSTT mutation did not alter VZV infectivity in vivo. These results provide the first information about how targeted mutations of this essential VZV glycoprotein affect viral replication in vitro and VZV virulence in dermal and epidermal cells and T cells within intact tissue microenvironments in vivo.

Animals↗

PubMed2003

Varicella-zoster virus infection facilitates VZV glycoprotein E trafficking to the membrane surface of melanoma cells.

Varicella-zoster virus glycoprotein E (gE) is the most abundant VZV glycoprotein on the surface of virus-infected cells. VZV gE has targeting sequences for the trans-Golgi network (TGN) and is transported from the ER to the TGN in infected and gE-transfected cells. In this study, VZV gE expressing melanoma cell lines were generated. gE is expressed under the control of the reverse Tet repressor (Tet-On). gE induced by Tet-On is retained at the ER as well as in the cis Golgi by immunofluorescence confocal microscopy. To test whether other viral protein(s) may facilitate gE trafficking and surface localization, MSPgE-vOka virus that contains MSPgE in place of wt gE was made. MAb 3B3 anti-gE does not bind to MSPgE. This MAb was used to track the localization of gE in Met-gE cells post MSPgE-vOka infection. gE became detectable mostly at the TGN and on the cell surface after viral infection. These data indicate that viral proteins facilitate the trafficking and cell surface expression of gE.

Biological Transport, Active↗

PubMed2002

The requirement of varicella zoster virus glycoprotein E (gE) for viral replication and effects of glycoprotein I on gE in melanoma cells.

The glycoprotein E (gE) of varicella zoster virus (VZV), encoded by ORF68, is the most abundant viral glycoprotein. In the current experiments, we demonstrated that ORF68 deletion was incompatible with recovery of infectious virus from VZV cosmids. Replacing ORF68 at a nonnative AvrII site in the genome restored infectivity. Further, we found that VZV gE could be expressed under the control of the Tet-On promoter in stably transfected melanoma cell lines (Met-gE cells) without evidence of toxicity. In these Met-gE cells, gE colocalized with gamma-adaptin, a trans Golgi network marker, in perinuclear sites, but did not reach plasma membranes. In order to investigate how infection altered gE localization, we made a recombinant virus, vOka-MSPgE, with ORF68 from the VZV MSP strain. VZV MSP encodes a mutant gE protein (D150N) that lacks the mAb epitope, 3B3 (Santos et al., Virology 275, 306-317, 2000), whereas Met-gE protein binds mAb 3B3. Within 48 h after Met-gE cells were infected with vOka-MSPgE, the steady-state distribution of Met-gE protein extended beyond the perinuclear areas to other cytoplasmic sites and to plasma membranes. A second recombinant, vOka-MSPgE without gI (vOka-MSPgEdeltagI), was constructed to investigate Met-gE protein distribution in the absence of gI. The redistribution of Met-gE protein which was observed by 48 h after vOka-MSPgE infection did not occur until 5 days (140 h) within vOka-MSPgEdeltagI infected cells. After vOka-MSPgE infection of Met-gE cells, most Met-gE protein was in the final 94K mature form by 72 h. However, progression to predominance of mature gE was delayed in Met-gE cells infected with vOka-MSPgEdeltagI. These observations confirm our hypothesis that VZV gE is essential, based upon the demonstration of restored infectivity after replacing ORF68 in a nonnative site in the genome, and provide further evidence of the role of gI in facilitating the maturation and intracellular distribution of this critical VZV glycoprotein.

Doxycycline↗