• Journal of Microbiology
• /
• 제38권1호
• /
• pp.24-30
• /
• 2000

US3 gene is a member of the human cytomegalovirus (HCMV) immediate early gene. Although the precise functions of the US3 gene in HCMV replication and pathogenesis are not known, it has been reported to play a role in inhibiting major histocompatibility class I antigen presentation. For further knowledge of US3 gene expression, rabbit polyclonal antiserum of the US3 gene product was used for indirect immunofluorescence assay. In permissive human foreskin fibroblast (HFF) cells, US3 gene expression was detectable as crescent or half-moon shape in the perinuclear region at immediate early times after virus infection. HFF cells infected with mutant HCMV lacking US3 open reading frames were negative for US3 immunofluorescence assay. Double immunofluorescence assay using monoclonal antibody to gamma adaptin (specific for the Golgi complex) and rabbit anti-US3 antiserum revealed that US3 gene product could be localized to the Golgi complex. At later time after HCMV infection, US3 gene products were detected as globular aggregates in the cytosol. These aggregates were positive for gamma adaptin and stained with preimmune serum, suggesting a nonspecific reaction to the Golgi complex. Northern blot analysis revealed that transcription of US3 was observed only during immediate early times after virus infection (until 6 h postinfection). Therefore US3 gene expression appears to be confined to immediate early time and its gene products are localized to the Golgi complex as crescent shaped forms in the perinuclear cytoplasm.

• Molecules and Cells
• /
• 제38권2호
• /
• pp.122-129
• /
• 2015

DBM-2198, a six-membered azasugar nucleotide (6-AZN)-containing phosphorothioate (P = S) oligonucleotide (AZPSON), was described in our previous publication [Lee et al. (2005)] with regard to its antiviral activity against a broad spectrum of HIV-1 variants. This report describes the mechanisms underlying the anti-HIV-1 properties of DBM-2198. The LTR-mediated reporter assay indicated that the anti-HIV-1 activity of DBM-2198 is attributed to an extracellular mode of action rather than intracellular sequence-specific antisense activity. Nevertheless, the antiviral properties of DBM-2198 and other AZPSONs were highly restricted to HIV-1. Unlike other P = S oligonucleotides, DBM-2198 caused no host cell activation upon administration to cultures. HIV-1 that was pre-incubated with DBM-2198 did not show any infectivity towards host cells whereas host cells pre-incubated with DBM-2198 remained susceptible to HIV-1 infection, suggesting that DBM-2198 acts on the virus particle rather than cell surface molecules in the inhibition of HIV-1 infection. Competition assays for binding to HIV-1 envelope protein with anti-gp120 and anti-V3 antibodies revealed that DBM-2198 acts on the viral attachment site of HIV-1 gp120, but not on the V3 region. This report provides a better understanding of the antiviral mechanism of DBM-2198 and may contribute to the development of a potential therapeutic drug against a broad spectrum of HIV-1 variants.

• 한국잠사학회:학술대회논문집
• /
• 한국잠사학회 1997년도 Progress and Future Development of Sericultural Science and Technology 40th Anniversary Commemoration Symposium
• /
• pp.73-101
• /
• 1997

Baculoviruses are characterized by large double-stranded circular DNA genomes and rod-shaped enveloped virions. Bombyx mori nucleopolyhedrovirus(BmNPV) is a major pathogen, which causes severe damage in sericulture. Currently, BmNPV is recogtnized as an improtant tool in molecular biology, especially for expression of useful genes in B.mori cells and silkworm larvae. Our laboratories have focused on the studies of the molecular mechanisms of BmNPV replication and the application of BmNPV to agriculture and medicine. The entire nucleotide sequence of the BmNPV genome has recently determined. The BmNPV genome possessed 135 putative genes and 7 homologous repeated sequence (hrs) regions. Relatively little space, a few to a few hundred base-pairs, was observed between the open reading frames and hrs. Termination codons often overlapped. These results showed a compactly packde BmNPV genome. Based on comparative sequence analyses, we speculated that the ancestor of BmNPV was a baculovirus similar to Autographa californica NPV(AcNPV). The function of the BmNPV genes were characterized by gene deletion analysis; p35 was found to be involved in blocking apoptosis and cysteine proteinase was found to be involved in horizontal virus transmission by degrading viral-infected larval host. By AcNPV and BmNPV coinfection experiments, we identified a BmNPV gene involved in expanding host specificity of AcNPV. The identified gene was likely encoded a DNA helicase based on the amino acid sequence analysis; a few amino acid substitutions in the putative DNA helicase gene resulted in the expansion of host range of AcNPV. These findings indicate that BmNPV evolved within a short period from an AcNPV-like ancestral virus due to rapid evolution including specific amino acid substitutions and gene deletions/insertions.

• 한국어병학회지
• /
• 제35권2호
• /
• pp.141-155
• /
• 2022

Rock bream iridovirus (RBIV) causes high mortality and economic losses in the rock bream (Oplegnathus fasciatus) aquaculture industry in Korea. Viral open reading frames (ORFs) expression profiling at different RBIV infection stages was investigated using microarray approaches. Rock bream were exposed to the virus and held for 7 days at 23 ℃ before the water temperature was reduced to 17 ℃. Herein, 28% mortality was observed from 24 to 35 days post infection (dpi), after which no mortality was observed until 70 dpi (end of the experiment). A total of 27 ORFs were significantly up- or down-regulated after RBIV infection. In RBIV-infected rock bream, four viral genes were expressed after 2 dpi. Most RBIV ORFs (26 genes, 96.2%) were significantly elevated between 7 and 20 dpi. Among them, 12 ORF (44.4%) transcripts reached their peak expression intensity at 15 dpi, and 14 ORFs (51.8%) were at peak expression intensity at 20 dpi. Expression levels began to decrease after 25 dpi, and 92.6% of ORFs (25 genes) were expressed below 1-fold at 70 dpi. From the microarray data, in addition to the viral infection, viral gene expression profiles were categorized into three infection stages, namely, early (2 dpi), middle (7 to 20 dpi), and recovery (25 and 70 dpi). RBIV ORFs 009R, 023R, 032L, 049L, and 056L were remarkably expressed during RBIV infection. Furthermore, six ORFs (001L, 013R, 052L, 053L, 058L, and 061L) were significantly expressed only at 20 dpi. To verify the cDNA microarray data, we performed quantitative real-time PCR, and the results were similar to that of the microarray. Our results provide novel observations on broader RBIV gene expression at different stages of infection and the development of control strategies against RBIV infection.

• 식물병연구
• /
• 제12권3호
• /
• pp.298-302
• /
• 2006

전형적인 줄무늬 모자이크 증상을 나타낸 칸나로부터 Cucumber mosaic virus(CMV)의 한 계통(Can-CMV)을 분리하고, 특성을 조사하였다. Can-CMV는 대부분의 전신감염 기주에서 병징이 발현되지 않았고, 이들 기주의 접종엽 및 상엽에서 RT-PCR로 바이러스의 감염여부를 조사한 결과, N. benthamiana와 N. glutinosa에서만 바이러스가 검출되었으며, 다른 기주로부터는 확인되지 않았다. 한편 Chenopodium amaranticolor의 접종엽에 발현된 국부 괴사병반은 대조로 접종한 Fny-CMV나 LS-CMV에 의해서 형성된 병반보다 매우 작은 크기의 반점을 형성하는 특성을 보였다. 또한 Vigna unguiculata의 접종엽에는 Fny-CMV나 LS-CMV가 접종 2-3일 후에 뚜렷한 괴사병반을 형성하는데 반해서, Can-CMV는 접종 4-5일 후에 윤곽이 확실치 않은 퇴록병반을 형성하였다. Can-CMV에 감염된 N. benthamiana로부터 추출한 dsRNA는 4종의 밴드가 검출되었으며, 이들 분자의 크기 및 종수는 Fny-CMV나 LS-CMV와 차이를 나타내지 않았다. Can-CMV의 항원은 Fny-CMV의 항혈청에 대해서 한 종의 뚜렷한 침강선을 형성하였으며, Fny-CMV의 항원에 의해서 형성된 침강선과 융합하였고, LS-CMV의 침강선과는 분지선을 형성함으로서, 혈청학적으로 서브그룹 I에 속하는 계통으로 판단되었다. 또한 Can-CMV에 감염된 N. benthamiana로부터 RNA를 추출하여 CMV-specific 프라이머를 이용한 CMV-RNA3의 외피단백질 유전자를 포함하는 3'영역에 대해서 RT-PCR을 실시하였다. Can-CMV는 Fny-CMV나 LS-CMV와 마찬가지로 예상되었던 약 950bp크기의 cDNA가 증폭되었으며, 이 cDNA를 EcoRI으로 처리하였을 때에는 절단되지 않았고, MspI에서는 595bp 및 350bp의 절편으로 절단되었다. 이와 같은 결과는 Fny-CMV의 패턴과 일치하였으며, 이러한 결과로부터 Can-CMV가 서브그룹 IA에 속하는 계통으로 확인되었다. 이상과 같은 결과들로부터, Can-CMV는 앞으로 바이러스와 기주의 다양한 상호관계를 이해하는데 있어서 중요한 병원학적 성질을 가지고 있는 것으로 생각되었다.

• BMB Reports
• /
• 제40권5호
• /
• pp.814-824
• /
• 2007

Liver cirrhosis (LC) is defined as comprising diffuse fibrosis and regenerating nodules of the liver. The biochemical and anatomical dysfunction in LC results from both reduced liver cell number and portal vascular derangement. Although several studies have investigated dysregulated genes in cirrhotic nodules, little is known about the genes implicated in the pathophysiologic change of LC or about their relationship with the degree of decompensation. Here, we applied cDNA microarray analysis using 38 HBsAg-positive LC specimens to identify the genes dysregulated in HBV-associated LC and to evaluate their relation to disease severity. Among 1063 known cancer- and apoptosis-related genes, we identified 104 genes that were significantly up- (44) or down- (60) regulated in LC. Interestingly, this subset of 104 genes was characteristically correlated with the degree of decompensation, called the Pugh-Child classification (20 Pugh-Child A, 10 Pugh-Child B, and 8 Pugh-Child C). Patient samples from Pugh-Child C exhibited a distinct pattern of gene expression relative to those of Pugh-Child A and B. Especially in Pugh-Child C, genes encoding hepatic proteins and metabolizing enzymes were significantly down-regulated, while genes encoding various molecules related to cell replication were up-regulated. Our results suggest that subsets of genes in liver cells correspond to the pathophysiologic change of LC according to disease severity and possibly to hepatocarcinogenesis.

• BMB Reports
• /
• 제38권1호
• /
• pp.34-40
• /
• 2005

GLPG (Ganoderma lucidum proteoglycan) was a bioactive fraction obtained by the liquid fermentation of the mycelia of Ganoderma lucidum, EtOH precipitation, and DEAE-cellulose column chromatography. GLPG was a proteoglycan with a carbohydrate: protein ratio of 10.4: 1. Its antiviral activities against herpes simplex virus type 1 (HSV-1) and type 2 (HSV-2) were investigated using a cytopathic inhibition assay. GLPG inhibited cell death in a dose-dependent manner in HSV-infected cells. In addition, it had no cytotoxic effect even at 2 mg/ml. In order to study the mode of action of the antiviral activity of GLPG, cells were treated with GLPG before, during, and after infection, and viral titer in the supernatant of cell culture 48 h post-infection was determined using a $TCID_{50}$ assay. The antiviral effects of GLPG were more remarkable before viral treatment than after treatment. Although the precise mechanism has yet to be defined, our work suggests that GLPG inhibits viral replication by interfering with the early events of viral adsorption and entry into target cells. Thus, this proteoglycan appears to be a candidate anti-HSV agent.

• BMB Reports
• /
• 제48권2호
• /
• pp.121-126
• /
• 2015

Here we report a new chemical inhibitor against HIV-1 with a novel structure and mode of action. The inhibitor, designated as A1836, inhibited HIV-1 replication and virus production with a 50% inhibitory concentration ($IC_{50}$) of $2.0{\mu}M$ in an MT-4 cell-based and cytopathic protection antiviral assay, while its 50% cytotoxic concentration ($CC_{50}$) was much higher than $50{\mu}M$. Examination of the effect of A1836 on in vitro HIV-1 reverse transcriptase (RT) and integrase showed that neither were molecular targets of A1836. The characterization and re-infection assay of the HIV-1 virions generated in the presence of A1836 showed that the synthesis of early RT products in the cells infected with the virions was inhibited dose-dependently, due in part to abnormal protein formation within the virions, thus resulting in an impaired infectivity. These results suggest that A1836 might be a novel candidate for the development of a new type of HIV-1 inhibitor.

• Journal of Microbiology and Biotechnology
• /
• 제18권2호
• /
• pp.328-333
• /
• 2008

A series of pep tides binding to the HCV NS5B polymerase was selected from phage display peptide libraries. A conserved motif of Ser-Arg-X-Arg/Leu was identified among the selected peptides, and Pep2 (Trp-Ser-Arg-Pro-Arg-Ser-Leu) was chosen for further characterization. The binding of Pep2 to HCV NS5B in vivo was shown by a yeast two-hybrid assay and by subcellular colocalization analysis using immunofluorescence confocal microscopy. The in vitro interaction was also confirmed by GST pulldown assay. The replication of the HCV 1b subgenomic replicon was efficiently inhibited by the presence of the peptide. By using a subtractive biopanning against Pep2, the binding site of the peptide was mapped at the pocket of Pro388 to Pro391 in the thumb subdomain of the polymerase. A yeast two-hybrid analysis using Pro388Ala and Pro391Ala mutants of NS5B confirmed the binding.

• Journal of Microbiology
• /
• 제35권2호
• /
• pp.152-157
• /
• 1997

Infection of human fibroblast (HF) cells with human cytomegalovirus (HCMV) result in changes in the intracellular level of second messengers. Since nitric oxide (NO) production has been known to be related with other second messengers, it is probable that HCMV infection of HF cells may involve NO. To test this possibility, the amount of NO was measured following ogenous addition of NO generators such as sodium nitroprusside (SNP) or S-nitroso-N-a-cetylpenicillamine (SNAP) immediately after HCMV infection, however, inhibited virus multiplication. Furthermore, immunoblot experiment using monoclonal antibody to HCMV major immediate early (MIE) proteins or CAT assay using pCMVIE/CAT (plasmid containing CAT gene driven by HCMV MIE promoter) revealed that SNP or SNAP blocked the MIE gene expression. SNP was more effective than SNAP in hibiting HCMV multiplication or MIE gene expression. SNP produced more NO than SNAP in inhibiting HCMV multiplication or MIE gene expression. SNP produced more NO than SNAP. Although the mechanism for the inhibition of HCMV multiplication and MIE gene expression by NO is still elusive some correlation with NO-mediated inhibition of HCMV-induced increase in cytosolic free Ca$\^$2+/ concentration ([Ca$\^$2+/]) was observed. The increase of [Ca$\^$2+/] following HCMV infection was inhibited by SNP, and less effectively by SNAP. Raising [Ca$\^$2+/ with bromo-A23187 partially reversed the SNP block of MIE gene expression. Thus, there appear to e some relationships among NO. [Ca$\^$2+/], and HCMV MIE gene expression.