Cell & Bioscience

Cell & Bioscience

The US11 protein of herpes simplex virus type 1 (HSV-1) is a small, highly basic phosphoprotein expressed at late times during infection. Periodically, the virus can reactivate and the progeny viruses spread back to the epithelium. P., Sakai, D. However, whether the VP19C NES is required for efficient HSV-1 production is unknown. While the function of this complex interferon-induced proteome is not completely understood, several key double-stranded RNA (dsRNA)-responsive effectors, notably the dsRNA-inducible protein kinase PKR and the 2′-5′ oligoadenylate synthetase (OAS), act to control protein synthesis and RNA stability in virus-infected cells. The virus binds to proteoliposomes containing APOE or APOA1 and also to CR1, and both clusterin and PICALM are related to a mannose 6-phosphate receptor used by the virus for cellular entry and intracellular transport. The Herpesviridae is a large family of DNA viruses, including eight known types of human herpesviruses.

Nuclear export is mediated via disruption of the nuclear lamina and binding to LMNA. These results illustrate that protein phosphorylation events mediated by viral protein kinases serve to coordinate the roles of VP11/12 as a virion component and intracellular signaling molecule. A previous study revealed a leucine-rich sequcnce “ 342 LERLFGRLRI 351 ” in VP19C functioning as a nuclear export signal (NES) was responsible for its nucleocytoplasmic shuttling [ 2 ]. Herpes simplex infection in mice also induces neuronal degeneration in the entorhinal cortex and hippocampus, as well as memory deficits, all features of Alzheimer’s disease [2] and C. Cloning of the HSV-1 genome into infectious bacterial artificial chromosomes (BACs) has facilitated genetic manipulation of the HSV-1 genome in Escherichia coli (E. coli) . To investigate whether abrogation of VP19C nuclear export has an effect on HSV-1 lytic replication, a VP19C NES-mutated recombinant virus was constructed.

Sery was one of the pioneers in developing an antiviral eye drop to use on eye ulcers caused by a herpes virus, said William Tasman, former ophthalmologist in chief at Wills Eye. coli GS1783 strain using a HSV-1 17-37BAC [ 3 ]. The scheme of constructing a VP19C NES-mutated BAC was shown in Figure 1 . Primer sequences used for BAC mutagenesis are listed in Table 1 . Construction of the recombinant BAC pVP19C NESm/17-37BAC was performed as described previously [ 4 ]. The clones with the desired recombination were selected on kanamycin- and L-arabinose-containing plates and then subjected to PCR and restricted fragment length polymorphisms (RFLP) analysis. By binding to TAR, the HIV Tat protein prevents activation of both PKR and OAS (28).

Possession of the human APOE4 allele in mice also favours cerebral infection by the herpes virus [16] . A series of 2D tilt images is recorded at various tilting angles, aligned and combined to obtain the 3D structure. (A) Schematic representation of the HSV-1 genome containing terminal long (TRL) and short (TRS) and internal long (IRL) and short (IRS) repeats flanking the unique long (UL) and short (US) regions. (B) The NES mutations were introduced by using two-step red recombination. The sequences corresponding to the first (red and wathet) and second (blue) recombination events are shown in the indicated colors. This forms a channel that inserts into pathogen cell membranes, killing them by lysis. coli containing the HSV-1 17-37BAC.

After the first recombination, the PCR product was inserted into the homologous location within the UL38 gene. After confirming that the PCR product was inserted into the correct locus by using RFLP and Southern blot hybridizations, the second recombination was performed in which the Kan R gene plus one of the complimentary homologous sequences were removed. State officials said the outbreak, only the second in the state in four years, should not lower the grade of New Jersey’s 24,000 hogs and should not wreak havoc on the $1.2 million market. Figure 2 Identification of the role of VP19C NES in HSV-1 life cycle. (A) RFLP analysis of BAC recombinant variants. Parental p17-37BAC, pVP19C NESm/Kan/17-37BAC and pVP19C NESm/17-37BAC were digested with Bam HI. The sizes of the molecular markers are shown on the left.

(B) VP19C NESm/17-37BAC recombinant virus reconstitution. The n12 ICP4 mutant (8) was obtained from N. This is primarily mediated via lipoprotein receptors. The major events of the viral life cycle and the cellular compartments where they took place were shown in . Vero cells were infected with wild-type HSV-1, parental 17-37BAC or VP19C NESm/17-37BAC virus at an MOI of 1, and virus was harvested at the indicated time points and titrated on a Vero monolayer. The data plotted show the mean of three independent experiments. (E) Individual plaque area assays of wild-type, parental and recombinant VP19C NESm/17-37BAC virus are shown.

The diameter of 80 plaques was measured for each virus, and the means ± standard deviations of the diameters were calculated and are shown. (F) Immunofluorescence assay for detecting the subcellular localization of VP19C in wild-type HSV-1 and VP19C NESm/17-37BAC virus-infected cells. Vero cells were infected with wild-type HSV-1 or VP19C NESm/17-37BAC virus at an MOI of 3 for 12 h and then probed with antibody against VP19C. (G) Western blot analysis of virion lysates was performed. Players have been divided on the proposal, ratified unanimously by owners in New York and touted by union leadership as containing significant gains in salaries and a quicker route to free agency. Expression was determined in cell lysates by Western blot analysis with antibodies against VP19C, VP22 and VP23. (H) The relative expression of virion proteins in the lysates was analyzed using Quantity One Imaging Software (Bio-Rad).
Cell & Bioscience

To investigate the effect of the NES mutation of VP19C on the life cycle of HSV-1, 1–2 μg of recombinant pVP19C NESm/17-37BAC DNA was electroporated into Vero cells to reconstitute the VP19C NESm/17-37BAC virus (Figure 2B). Sequence analysis confirmed that the NES mutation in VP19C was not repaired during viral replication (Figure 2C), which indicated that the NES is not required for virus growth in vitro. To compare the in vitro growth properties of VP19C NESm/17-37BAC virus with those of the parental virus and wild-type HSV-1, single step growth kinetics were determined as described previously [4]. The gel was prerun at 2,000 V for 30 min. It is bound to chondroitin-4,6 sulphate [56] , a receptor for HSV-1 [57] , raising the intriguing possibility that this form of APP  could be used as a viral receptor. De-enveloped capsids were first observed in cells fixed at 20 min p.i. Immunofluorescence staining was applied to visualize the subcellular localization of VP19C in wild-type HSV-1 and VP19C NESm/17-37BAC virus-infected cells.

As shown in Figure 2F, VP19C displayed a nuclear localization in VP19C NESm/17-37BAC virus-infected cells as it did in wild-type HSV-1-infected cells, indicating that the NES mutation of VP19C did not affect its nuclear localization in infection background. Western blot was also performed to detect the expression of virion proteins VP19C, VP23 and VP22. As it is shown in Figure 2G and H, the expression of VP19C, VP23 and VP22 was unchanged. These results collectively indicated that abrogation of VP19C nuclear export does not affect subcellular localization of VP19C and viral gene expression. The HSV-1 capsid is the most morphologically regular component of the virus structure. VP19C is an integral capsid protein that interacts with VP23 to form the triplex, which is the unit that forms the capsid. Capsid assembly of HSV-1 is known to take place in the nuclei of infected cells.

It is reported that correct transport of the protein components to the site of capsid assembly is an important function of VP19C [5]. Because of its nuclear localization signal (NLS) and NES, VP19C is able to shuttle between the nucleus and the cytoplasm as part of a chromosome region maintenance 1 (CRM1) dependent pathway as well as through the atypical Ran- and importin β-dependent, but not importin α5, mechanisms [4]. These lines of evidence suggest that VP19C and VP23 may interact in the cytoplasm, and then, VP23 is translocated into the nucleus because of the NLS on VP19C to participate in capsid assembly. The hydrophobic residues in the NES are important for nuclear export of VP19C, and VP19C is exported from the nucleus through the NES via a CRM1-dependent pathway. However, CRM1-mediated transport is highly subjected to various regulation processes, including the masking of NES, phosphorylation and heterodimerization of the protein and formation of disulfide bonds by an oxidative process [6]. The efficacy of the heat inactivation step was tested in a standard OAS in vitro reaction mixture using 10 μg heat-inactivated extract and 10 μg/ml poly(I)(C). The relationships between key Alzheimer’s disease susceptibility gene products and herpes simplex.

In three 1 nm-thick digital slices from different positions in z-direction, filaments emanating from the NPC were visible () and the viral DNA releasing from the vertex through the NPC was resolved (). The mechanism by which subcellular localization is regulated and whether it is regulated over the course of HSV-1 infection will be interesting to determine. BAC recombineering allows for the rapid and efficient modification of viral genomes in E. coli, thus providing an invaluable tool for the genetic manipulation of herpesvirus genomes and the production of altered forms of viruses to decipher the specific function of a gene in the virus life cycle and pathogenesis [7]. In the present study, we constructed a VP19C NES-mutated recombinant virus using BAC recombineering technology. The recombinant virus with the NES mutation in VP19C did not impair virus replication, and viral gene expression was also unaffected. Our previous study identified a non-classical NLS of VP19C, and the nuclear import of VP19C is required for efficient HSV-1 production [4].

These lines of evidence together suggest that the role of nuclear import and export may function differently in HSV-1 lytic replication. HSV-1 ICP27 was shown to shuttle between the nucleus and cytoplasm through a leucine-rich NES in a CRM1-dependent pathway [8]. However, ICP27 shuttling is not involved in viral DNA replication, but affects the expression of HSV-1 late genes [9]. Li et al. also identified a functional NLS and NES in ORF45 of Kaposi’s sarcoma-associated herpesvirus 8, and demonstrated that the NES is important for ORF45 nucleocytoplasmic shuttling. However, only the NLS-mutated virus had impaired virus production, and the NES-mutated virus did not affect viral replication [10]. Additionally, because VP19C could interact with other cellular proteins (such as nuclear pore complexs) or viral proteins (such as UL25) [4], it cannot be excluded that VP19C may export from the nucleus through other pathways in the context of infection.

The samples were boiled for 5 min, resolved by SDS-polyacrylamide gel electrophoresis (PAGE), and immunoblotted following standard procedures. 41 (2009) 1088-1093. Based on the amount of DNA and the presence or absence of scaffolding protein inside the capsids, we sorted the six groups of capsids and proposed two possible DNA encapsidation mechanisms: DNA encapsidation could take place in both capsids without scaffolding protein density () and capsids with deformed scaffolding protein (). Acknowledgements This work was supported by funding from the National Nature Science Foundation of China (grant numbers 31171219, 81271213, 81070878, 81271214, 81300929 and 81261120404). We thank Dr. Frazer J. Rixon, Dr.

C. Sweet, Dr. David Leib, Dr. Gregory Smith and Dr. Nikolaus Osterrieder for the generous gift of the antibodies against VP19C and VP23, the antibody against VP22, 17-37BAC, E. coli GS1783 and pEPkan-S, respectively. LY and MDW performed the experiments, and drafted the manuscript.

CLL, MGD and TH carried out the virus titration. ZHH and LWD analyzed the data. ). Virol.

You may also like