†The statements on this Web site have not been evaluated by the Food and Drug Administration (FDA). En effet, du fait de leur simplicité extrême, les virus ne peuvent pas se multiplier, du moins se multiplier par eux-mêmes. Until some years ago, cyclins were presumed to be encoded exclusively by eukaryotic cells. As a result, these viruses profoundly alter the biochemical pathways that normally control cellular growth, and may thus promote uncontrolled cell proliferation. Even though all known herpesviruses utilize very similar mechanisms for amplification of their genomes, the nuclear import of the replication complex components appears to be a heterogeneous and highly regulated process to ensure the correct spatiotemporal localization of each protein. Viruses can tailor the cellular response by suppressing downstream signalling from DNA-damage sensors, as exemplified by EBV. Combination treatment studies indicated complementary activities between YC15-PE38 and the herpesviral DNA replication inhibitor ganciclovir.
The identity of other components remains obscure. Therefore we concluded that deletion of gG in EHV-1 seems to lead to an exacerbation of respiratory disease in the mouse. On the basis of very recent findings, it is becoming increasingly clear that pUL97 is a component of a multiprotein nuclear egress complex (NEC). Immunological assays are an essential part of studies aimed at determining the kinetics of expression and the cellular location of DEV pUL38 in vitro. In this study, we obtained rabbit anti-pUL38 polyclonal sera, which were shown to be functional in immunofluorescence and western blotting assays. The DEV CHv strain used throughout this study was grown in duck embryo fibroblast (DEF) cells. Several of the proteins we identified had previously unknown functions or were structural components of the virion.
In a previous study, we had amplified the ORF of pUL38 (1398 bp) from the DEV genome . The amplified product was cloned between the Bam HI and Xho I sites of a pET32(+) plasmid, and a pET32-pUL38 plasmid construct was created. Escherichia coli BL21(DE3) was transformed with the recombinant construct, and protein expression was induced with 1 mM IPTG at 37°C for 4 h. The bacterial proteins were analyzed by 12% SDS-PAGE under denaturing conditions. Protein bands were visualized after staining with 0.1% Coomassie blue R250, and the protein concentration was determined using the software program BandScan 5.0 [ 17 ]. The recombinant pUL38 was successfully expressed in the transformed cells (Fig. 1 ).
Figure 1 Expression and purification of the DEV pUL38. SDS-PAGE of the expressed peptide in E. coli BL21 (DE3) is shown. M Marker; 1 the total cell proteins uninduced with IPTG; 2 the total cell proteins induced with IPTG; 3 the insoluble fraction after purification with IMAC. The black arrow points to the recombinant pUL38 (approximately 70 kDa). The expressed recombinant pUL38, however, was trapped in inclusion bodies. The HCMV serostatus of 99% (89/90) was also higher in our cohort than reported by Chiu et al.
The cells were later lysed by using lysozyme (0.1 mg/mL) at 4°C for 1 h and sonicated on ice for 5 min at an amplitude of 30% with a 30-s pulse frequency. The lysate was centrifuged at 10,000 × g for 20 min at 4°C. This complex formation is important for proper positioning of both proteins at the inner nuclear membrane (28, 36, 45). The suspension was centrifuged at 10,000 × g for 20 min at 4°C, and then the resulting precipitate was resuspended in regeneration buffer containing 6 M urea, 0.5 M NaCl, 20 mM Tris-HCl (pH 7.9) and incubated at room temperature (25°C) for 30 min. The incubated mixture was then centrifuged at 10,000 × g for 20 min. The two viruses cross-react serologically, but some unique proteins exist for each type. The recombinant His-tagged proteins were purified from the above supernatant by immobilized metal affinity chromatography (IMAC) on a Ni-NTA affinity resin (Bio-Rad, California, USA) according to the protocol of Cai et al.
. Finally, homogeneity of the proteins was verified by an SDS-PAGE assay (Fig. These various findings demonstrate important roles for BPLF1 and potentially its homologs in viral replication and infectivity. ClustalW2 multiple sequence alignment program was used to align amino acid sequences of glycoprotein B ectodomain between the reference strains of members of the Herpesviridae family. Furthermore, Poxviridae have been suggested to promote selective and augmented translation of viral mRNAs in in vitro studies (26). Farina A, Feederle R, Raffa S, Gonnella R, Santarelli R, Frati L, Angeloni A, Torrisi MR, Faggioni A, Delecluse HJ: BFRF1 of Epstein-Barr virus is essential for efficient primary viral envelopment and egress. Two weeks after the last immunization, the antiserum was harvested from the carotid artery.
To determine the kinetics of pUL38 expression, DEF cells were infected with DEV. Cell lysates were prepared at 2 h, 4 h, 8 h, 12 h, 24 h, and 48 h post-infection (h.p.i). Les protéines précoces b sont des enzymes impliquées dans la réplication de l’ADN viral. As seen in Fig. 2A , DEV pUL38 (molecular mass, approximately 51 kDa), was detectable in DEF cells as early as 8 h.p.i. Once in the cytoplasm, the action of RanGAP catalyzes the hydrolysis of GTP to GDP, resulting in the dissociation of RanGDP from the IMPs which are then available for a new round of nuclear import, while the newly generated RanGDP can be transported into the nucleus by NTF2 . As a consequence of the elevated abundance of E2F1 protein and activation of Chk2, expression of p73 is upregulated (Refs 18, 19), leading to the induction of apoptosis (Refs 19, 20).
Immediate-early (α) transcripts are expressed first, and the proteins encoded by these mRNA species are required for the subsequent expression of all other kinetic classes of viral genes. Delayed-early (β) genes, many of which encode proteins involved in the replication of the viral genome, are maximally expressed before or at the peak of DNA replication and are then switched off. Late transcripts (γ) are maximally expressed only after the onset of viral DNA replication and encode proteins involved in virion assembly. As reported in previous studies, 2 identified immediate-early products, namely, protein kinase pUS3 and dUTPase, were first detected at 2 h.p.i. and 4 h p.i. respectively [ 9 , 10 ]. In contrast, 2 identified late products–tegument protein pUL31 and pUL51–were first detected at 6 h.p.i.
and 8 h.p.i., respectively [ 5 , 8 ]. Hence, we concluded that pUL38 may be a late gene product and may be a part of virion architecture. Figure 2 Kinetics of expression and immunolocalization of the DEV pUL38 in infected DEF cells. A Western blot of lysates from mock-infected or DEV-infected DEF cells with polyclonal antibodies specific to pUL38 protein, showing that pUL38 is expressed as a 51 kDa protein from 8 h onward following infection. B Immunofluorescence detection of pUL38 in mock-infected (a) or DEV-infected DEF cells at 8(c), 18(d), 30(e) and 56 h(f) post-infection. Cells were incubated with preimmune serum(b) pUL38-specific antibody and subsequently stained with fluorescein isothiocyanate (FITC)-conjugated secondary antibody. Nuclei were counterstained with DAPI (blue).
To confirm the intracellular distribution of pUL38, DEF cells were plated on coverslips and infected with DEV at an MOI of 5. The cells were processed at 8 h, 18 h, 30 h, and 56 h.p.i., and pUL38 was detected using pUL38-specific antibody and fluorescein isothiocyanate (FITC)-conjugated secondary antibody. As can be seen in Fig. 2B, the pUL38 distribution pattern appeared to change over the course of DEV infection. At 8 h.p.i., pUL38 was expressed diffusely throughout the cytoplasm of cells. At 18 h.p.i., it was detected close to the nucleus and showed a fine speckled pattern. At later times following infection (30 h), the pUL38 protein was localized in very fine punctate forms dispersed throughout the nucleus of infected cells.
These results suggest a putative change in the intracellular localization of pUL38 during the course of DEV infection. Since pUL38 is localized to the nucleus, we investigated the possibility of this protein being incorporated into DEV virions by probing the western blots of highly purified virions. The extracellular virions were collected from culture media harvested at 48 h.p.i. For construction of a HSV-1 UL25 deletion mutant, pcDNA-UL25(HSV-1) was digested with SbfI and AleI, thereby removing codons 55 to 516 of the 580-codon open reading frame (Fig. The purified DEV virions were separated by SDS-PAGE, and western blots were performed with rabbit antisera against the pUL38 protein. A protein band corresponding to the molecular weight of 51 kDa was clearly seen in the blots (Fig. 3 ).
This result suggests that pUL38 is a component of DEV virions. Figure 3 The association of DEV pUL38 with purified virions. Virus particles were collected from culture medium harvested at 48 h.p.i. H1299 cells were either transfected with pEYFP-N1-Rad18 or cotransfected with pEYFP-N1-Rad18 and the first 246 N-terminal amino acids of BPLF1, FLAG tagged (FLAG-BPLF1 1-246). Purified virions were lysed in SDS sample buffer, separated by SDS-PAGE, stained with Coomassie brilliant blue (lane 1), and then analyzed by western blotting with the UL38 antiserum (lane 2). Upon internalization of virus particles, viral cores are transported to the MTOC by cellular dynein/dynactin motors, and viral genomes are delivered into the nucleus by disassembly of the viral capsid at the nuclear pore (reviewed in references 5, 58, 59, and 60). In most herpesviruses, after assembly of the capsid and packaging of the viral genome–a process that occurs in the nucleus–the nucleocapsid is translocated to the cytoplasm .
For final maturation within the cytoplasmic tegument, components associate with the translocated nucleocapsid, with themselves, and with the future envelope; this results in the formation of an infectious herpes virion. However, there are 2 assembly pathways in DEV infection in both the cytoplasm and the nucleus . The majority of nucleocapsids acquire teguments in the nucleus, which are enveloped by the inner nuclear membrane, after which mature viruses are released into the cytoplasm. Une fois entré dans la cellule, l’ARN viral va être rétrotranscrit dans le cytoplasme en ADN par la transcriptase inverse virale (TI). At later times following infection, pUL38 localized in the nucleus of infected cells and was not detectable in the cytoplasm. The results suggested that pUL38 may be an internal component of the DEV nucleocapsid and may be involved in stabilizing the capsid.