Maria Teresa Sciortino

Of the three tegument proteins that package mRNA in herpes simplex virions, one (VP22) transports the mRNA to uninfected cells for expression prior to viral infection

An earlier report has shown that herpes simplex virus 1 virions package RNA. Experiments designed to reveal the identity of the virion proteins capable of binding the RNA and to show whether the mRNA carried in the newly infected cells was expressed showed the following: (i) 32P-labeled riboprobe generated by in vitro transcription of the US8.5 ORF bound three proteins identified as the products of US11, UL47, and UL49 (VP22) genes. (ii) Viral RNA was bound to UL47 or US11 proteins immune precipitated from cells transduced with baculoviruses expressing UL47 or US11 and then superinfected with HSV-1 under conditions that blocked DNA synthesis and assembly of virions. (iii) Virions were purified from cells transduced with a baculovirus encoding a US8.5 protein fused to green fluorescent protein and superinfected with an HSV-1 mutant lacking the US8–12 genes. HEp-2 cells infected with these virions expressed the chimeric protein in ≈1% of infected cells. (iv) In mixed cultures, untreated Vero cells acquired the mRNA encoding the green fluorescent–US8.5 chimeric protein from HEp-2 cells doubly transduced with the genes encoding VP22 and the chimeric protein. The transfer was RNase sensitive and VP22 dependent, indicating that the RNA encoded by the chimeric gene was transferred to Vero cells as mRNA. We conclude that (i) three virion proteins are capable of binding RNA; (ii) the packaged RNA can be expressed in newly infected cells; and (iii) the UL47 protein was earlier reported to shuttle from nucleus to the cytoplasm and may transport RNA. VP22 thus appears to be a member of a new class of viral proteins whose major function is to bind and transport infected cell mRNA to uninfected cells to create the environment for effective initiation of infection.

The intracellular effects of non-ionic amphiphilic cyclodextrin nanoparticles in the delivery of anticancer drugs

The aim of this study was to develop nanoparticles made of the amphiphilic cyclodextrin heptakis (2-O-oligo(ethyleneoxide)-6-hexadecylthio-)-beta-CD (SC16OH) entrapping docetaxel (Doc) and establish their in vivo potential. Doc-loaded SC16OH nanoparticles were prepared by the emulsion-solvent evaporation technique and fully characterized for size, zeta potential, amount of entrapped drug, release rate and degradation rate. Spherical vesicular nanoparticles displaying a hydrodynamic radius of about 95 nm which did not change upon storage as an aqueous dispersion, a negative zeta potential and entrapment efficiency of Doc very close to 100% were produced. DSC study highlighted the crystalline nature of SC16OH, unloaded and Doc-loaded SC16OH nanoparticles which resulted in their very slow dissolution during release stage and well-modulated release of entrapped Doc for about 8 weeks. Doc-loaded SC16OH nanoparticles were not hemolytic toward red blood cells as compared to a commercial Doc formulation (Taxotere) which shows a dose-dependent toxicity. After exposure of HEp-2 cells to equivalent doses of free Doc and Doc-loaded SC16OH nanoparticles, superior cell killing and cell damage were observed for nanoparticles. Finally, cell damage was attributed to aberrant mitosis which was found to be significantly higher for HEp-2 cells treated with Doc-loaded SC16OH nanoparticles as compared to free Doc likely due to the ability of nanoparticles to slowly release the drug allowing prolonged cell arrest in mitosis. Taken together, these results highlights a great potential of nanoparticles based on SC16OH in solid tumors therapy.

Interaction of herpes simplex virus RNase with VP16 and VP22 is required for the accumulation of the protein but not for accumulation of mRNA

The virion host shutoff (vhs) protein encoded by the UL41 gene of herpes simplex virus 1 is an endoribonuclease. The enzyme is introduced into the cell during unpackaging of the virion upon entry and selectively degrades mRNA for several hours. The RNase activity ceases after the onset of synthesis of late (γ) viral proteins. Here we report that vhs protein does not accumulate in cells transiently transfected with only a plasmid encoding the UL41 gene. However, vhs does accumulate in cells cotransfected with plasmids expressing two other tegument proteins, VP16 and VP22. vhs does not directly interact with VP22 but, instead, binds VP22 only in the presence of VP16. In contrast to these findings, the amounts of vhs mRNA accumulating in the cells transfected solely with vhs are not significantly different from those detected in cells coexpressing vhs, VP16, and VP22. We conclude from these studies that the steady state of vhs mRNA, reflecting synthesis and turnover of mRNA, is not affected by the interaction of vhs protein with VP16 with VP22. A model is proposed in which the vhs protein may function to sequester mRNAs in compartments inaccessible to the cellular translational machinery and that VP16 and VP22 rescue the mRNAs by interacting with the vhs protein.

A Cyclodextrin‐Based Nanoassembly with Bimodal Photodynamic Action

We have developed a supramolecular nanoassembly capable of inducing remarkable levels of cancer cell mortality through a bimodal action based on the simultaneous photogeneration of nitric oxide (NO) and singlet oxygen ((1)O(2)). This was achieved through the appropriate incorporation of an anionic porphyrin (as (1)O(2) photosensitizer) and of a tailored NO photodonor in different compartments of biocompatible nanoparticles based on cationic amphiphilic cyclodextrins. The combination of steady-state and time-resolved spectroscopic techniques showed the absence of significant intra- and interchromophoric interaction between the two photoactive centers embedded in the nanoparticles, with consequent preservation of their photodynamic properties. Photodelivery of NO and (1)O(2) from the nanoassembly on visible light excitation was unambiguously demonstrated by direct and real-time monitoring of these transient species through amperometric and time-resolved infrared luminescence measurements, respectively. The typical red fluorescence of the porphyrin units was essentially unaffected in the bichromophoric nanoassembly, allowing its localization in living cells. The convergence of the dual therapeutic action and the imaging capacities in one single structure makes this supramolecular architecture an appealing, multifunctional candidate for applications in biomedical research.

Herpes simplex virus 2 causes apoptotic infection in monocytoid cells

Increasing evidence indicates that apoptosis can be associated with several viral infections. Here we demonstrate, that infection of monocytoid cells by Herpes simplex virus 2 (HSV-2) resulted, in time- and dose-dependent induction of apoptosis as an exclusive cytopathic effect. The phenomenon was confirmed using four different techniques. Conversely, apoptosis was not observed in the Vero cell line. Virus yield in monocytoid cells was delayed and reduced, although well detectable, in comparison with that observed in Vero cells. Nevertheless, released virions exhibited full infecting capability. Apoptosis induced by HSV-2 was not inhibited by cycloheximide and only partially by an UV-treatment which completely abrogated infectivity. Virus-induced apoptosis was partly inhibited by indomethacin and was associated with a down-regulation of Bcl-2. A similar, but less pronounced, apoptosis-inducing effect in monocytoid cells was also observed with HSV-1 infection. Depending on the target cells, therefore, HSV could complete a cycle of infection which is characterized by apoptosis of infected cells.

Replication-competent herpes simplex virus 1 isolates selected from cells transfected with a bacterial artificial chromosome DNA lacking only the UL49 gene vary with respect to the defect in the UL41 gene encoding host shutoff RNase.

ABSTRACT To generate a null UL49 gene mutant of herpes simplex virus 1 (HSV-1), we deleted from the viral DNA, encoded as a bacterial artificial chromosome (BAC), the UL49 open reading frame and, in a second step, restored it. Upon transfection into Vero cells, the BAC-ΔUL49 DNA yielded foci of degenerated cells that could not be expanded and a few replication-competent clones. The replication-competent viral clones derived from independent transfections yielded viruses that expressed genes with some delay, produced smaller plaques, and gave lower yields than wild-type virus. A key finding is that the independently derived replication-competent viruses lacked the virion host shutoff (vhs) activity expressed by the RNase encoded by the UL41 gene. One mutant virus expressed no vhs protein, whereas two others, derived from independent transfections, produced truncated vhs proteins consistent with the spontaneous in-frame deletion. In contrast, cells infected with the virus recovered upon transfection of the BAC-UL49R DNA (R-UL49) accumulated a full-length vhs protein, indicating that in the parental BAC-ΔUL49 DNA, the UL41 gene was intact. We conclude that expression of the vhs protein in the absence of UL49 protein is lethal, a conclusion bolstered by the evidence reported elsewhere that in transfected cells vhs requires both VP16 and VP22, the product of UL49, to be neutralized.

Nitric oxide photocaging platinum nanoparticles with anticancer potential

In this contribution we report the design, fabrication and properties of hydrosoluble platinum nanoparticles decorated with a nitric oxide (NO) caging compound. Direct monitoring of NO through an ultrasensitive NO electrode demonstrate that the nanoparticles are stable in the dark but supply NO at nanomolar levels exclusively upon light excitation. The biocompatibility of these nanohybrid systems and their potential in photoactivated anticancer therapy have been explored by in vitro experiments using tumor cell lines. Overall these nanoparticles meet a combination of ideal prerequisites in the context of biomedical applications. Indeed, they associate small sizes, good water solubility and thermal stability under physiological conditions with excellent biocompatibility and appreciable tumor cell mortality upon irradiation with visible light. All these features make our photoactivable nanoparticles very appealing point sources of NO from the viewpoint of practical application in the emerging field of nanomedicine.

Involvement of HVEM receptor in activation of nuclear factor κB by herpes simplex virus 1 glycoprotein D

The UV‐inactivated herpes simplex virus 1 (HSV‐1) and glycoprotein D (gD) of HSV‐1 have been shown to activate nuclear factor κB (NF‐κB) in U937 cells, but mechanisms involved in this activation have not been elucidated. Here we report that: (i) UV‐inactivated HSV‐1 induced an increased NF‐κB activation in cells expressing human HVEM (for herpesvirus entry mediator) at surface level, naturally or following stable transfection, but not in cells in which this receptor was not detected by flow cytometry analysis, (ii) treatment with soluble gD induced a dose‐dependent NF‐κB activation in THP‐1 cells naturally expressing HVEM, and a monoclonal antibody that prevents binding of gD to HVEM significantly reduced NF‐κB activation by soluble gD in the same cells, (iii) coculture with transfectants expressing wild‐type gD on their surface induced an approximately twofold increase in NF‐κB activation in cells naturally expressing HVEM, while coculture with transfectants expressing a mutated form of gD, lacking its capability to bind HVEM, did not induce a similar effect and (iv) treatment with soluble gD induced a dose‐dependent NF‐κB activation in CHO transfectants expressing HVEM, but not in control CHO transfectants lacking any functional gD receptor. Overall, these results establish that HVEM is involved in NF‐κB activation by HSV‐1 gD.

Involvement of gD/HVEM interaction in NF-kB-dependent inhibition of apoptosis by HSV-1 gD.

In the present paper, we aimed to verify whether the interaction of the glycoprotein D (gD) of herpes simplex 1 (HSV-1) with the HSV-1 receptor HVEM is involved in NF-kappaB-dependent protection against apoptosis by gD. To this purpose, first we utilized MAbs that interfere with gD/HVEM interaction and U937 cells that naturally express human HVEM on their surface. Pre-incubation with these MAbs, but not with a control antibody, partially reverted the protection of infectious HSV-1 towards anti-Fas induced apoptosis in U937 cells. Similarly, pre-incubation of UV-inactivated HSV-1 (UV-HSV-1) or recombinant gD with the same MAbs, significantly reduced the inhibition of Fas-mediated apoptosis by UV-HSV-1 or gD, respectively, in U937 cells. Moreover, coculture with stable transfectants expressing at surface level wild type gD protected U937 cells against Fas-induced apoptosis, while coculture with transfectants expressing a mutated form of gD, incapable to bind HVEM, did not protect. Finally, UV-HSV-1 protected against staurosporine-induced apoptosis in U937 cells as well as in the CHO transfectants expressing human HVEM on their surface, but not in the control CHO transfectants, which did not express HVEM. These results suggest that signaling triggered by binding of gD to HVEM could represent an additional mechanism of evasion from premature apoptotic death exerted by HSV-1-gD in HVEM-expressing cells, disclosing new opportunities of cell death manipulation by using gD preparations.

The Virion Host Shutoff RNase Plays a Key Role in Blocking the Activation of Protein Kinase R in Cells Infected with Herpes Simplex Virus 1

ABSTRACT Earlier studies have shown that active MEK blocks the activation of protein kinase R (PKR), a component of antiviral innate immune responses. In this report we show that the herpes simplex virus 1 virion host shutoff (VHS) RNase protein and MEK (mitogen-activated protein kinase kinase) act cooperatively in blocking the activation of PKR. This conclusion is based on the following. (i) In contrast to viral gene expression in the parental cell line or a cell line expressing a constitutively active MEK, the replication of a VHS mutant is particularly impaired in cells expressing dominant negative MEK. In this cell line PKR is activated by phosphorylation, and the accumulation of several viral proteins is delayed. (ii) In transfected cells, wild-type VHS blocked the activation of PKR, whereas PKR was activated in cells transfected with a mutant VHS or with plasmids encoding the VHS RNase and VP16 and VP22, the two viral proteins that neutralize the RNase activity of VHS. The results suggest that early in infection the VHS RNase degrades RNAs that activate PKR. Coupled with published data, the results suggest that inhibition of activation of PKR or its effect on viral replication is staged early in infection by VHS, postsynthesis of VP16 and VP22 by the γ134.5 protein, and very late in infection by the US11 protein.