African green monkey kidney Vero cells require de novo protein synthesis for efficient herpes simplex virus 1-dependent apoptosis

Department of Microbiology, Mount Sinai School of Medicine, One Gustave L. Levy Place, New York, NY 10029, USA.
Virology (Impact Factor: 3.28). 07/2005; 336(2):274-90. DOI: 10.1016/j.virol.2005.03.026
Source: PubMed

ABSTRACT During HSV-1 infection, IE gene expression triggers apoptosis, but subsequent synthesis of infected cell proteins blocks apoptotic death from ensuing. This "HSV-1-dependent" apoptosis was identified in HEp-2/HeLa cells infected with wild-type HSV-1 in the presence of an inhibitor of protein synthesis or a virus lacking ICP27 {HSV-1(vBSDelta27)}. Unlike HEp-2/HeLa cells, vBSDelta27-infected Vero cells fail to exhibit dramatic apoptotic morphologies at times prior to 24 hpi. Here, we examined the basis of these different apoptotic responses to HSV-1. We found that infected Vero cells take substantially longer than HEp-2/HeLa cells to display membrane blebbing, chromatin condensation, DNA laddering, and PARP cleavage. Vero, but not HEp-2/HeLa, cells required de novo protein synthesis to exhibit efficient HSV-1-dependent apoptosis, which included changes in mitochondrial membrane potential, and these factors were produced prior to 3 hpi. Vero cells infected with recombinant viruses devoid of the ICP27 and ICP4 proteins alone or both the ICP27 and ICP22 proteins were apoptotic. These results indicate a requirement for cellular or other viral protein synthesis in Vero cells and provide insight into cell type differences in HSV-1-dependent apoptosis.

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    ABSTRACT: The herpes simplex virus type 1 (HSV-1) ICP27 protein is an immediate early protein, conserved through the Herpesviridae family, which is essential for HSV lytic replication. ICP27 is a key regulator of viral and cellular gene expression, with known roles in the nucleus, where it seems to exert its effects by controlling and coordinating a complex coupled network of gene expression machines, which includes transcription, mRNA 3’ end formation, splicing and mRNA export. These properties make of ICP27 a target for the development of antiviral drugs and vaccines against the Herpesviridae family. In addition, ICP27 constitutes a good example of a multifunctional protein, with roles in the nucleus and the cytoplasm (identified here), thus it can be used as a model to study gene regulation in eukaryotes. This work was initially aimed at identifying ICP27’s cellular partners that may be involved in the modulation of the length of the poly(A) tail, a phenomenon observed with some ICP27 viral mutants. Consequently, poly(A) binding protein 1 (PABP1), a cytoplasmic multifunctional protein, was identified as a new ICP27 interacting protein. Since it is known that other viral families regulate PABP1 activity to hijack cellular translation, my work was subsequently aimed to determine whether ICP27 exerts effects on the translational machinery and to study how PABP1 activity could possibly be regulated during HSV-1 infection. I present here data that show that ICP27 protein interacted directly with PABP1 in vitro and an interaction occurred occurred in vivo at 5-7 hours post-inoculation (hpi), but the interaction in vivo was partially RNA-mediated. The regions of interaction were mapped in vitro and in vivo, using a panel of HSV-1 ICP27 recombinant viruses that contain deletion and missense mutations. Interestingly, the ICP27 C-terminus interacting region seemed to be involved in an essential function for viral lytic replication, as all the mutants that failed to interact with PABP1 were defective for growth and for the induction of a subclass of late viral proteins. I also show that early in HSV-1 infection (5 hpi) both wild type (WT) ICP27 and PABP1 were associated with polyribosomes, whereas ICP27 protein from a missense HSV-1 mutant that failed to interact with PABP1 was excluded from polyribosomes and ribosomal fractions. These results strongly suggest that ICP27 protein might have cytoplasmic activities, in addition to its known nuclear roles. This hypothesis was confirmed using the tethered function assay, where it was shown that ICP27 can directly stimulate the translation of mRNAs to which it is bound, in the absence of any other viral factors. ICP27 C-terminus was also required for ICP27’s ability to stimulate translation; and two C-terminus missense mutants that failed to interact with PABP1, also failed to stimulate translation. The correlation of protein-protein interaction techniques, polysome experiments and tethered function assay, led us to formulate the hypothesis that ICP27 might increase translation efficiency of viral mRNAs by recruiting PABP1. Nevertheless, further experiments refuted this hypothesis. First, it was found that a fraction of PABP1 was redistributed from the cytoplasm to the nucleus at 10.5 hpi. Second, PABP1 expression was significantly reduced late in HSV-1 infection (after 12 hpi). Third, PABP1 was phosphorylated early in infection (7 hpi), an event that was dependent on UL13 kinase expression, a viral kinase that is packaged in the tegument. Finally, infection of HeLa cells, depleted of PABP1 by RNA interference, demonstrated that PABP1 is not required for efficient HSV-1 replication. I found no statistical difference in the viral titres of WT virus and two competent ICP27 N-terminus deletion mutants, which have a reduced interaction with PABP1 compared to WT, when they were inoculated in mock-transfected HeLa cells or in HeLa cells knockdown for PABP1. In addition, PABP1 was not required for optimal expression of VP16 protein, a viral tegument protein that is regulated at the translational level by ICP27 protein. In conclusion, PABP1 activity seems to be regulated during HSV-1 infection by phosphorylation that might change its affinity of interaction for translation factors or perhaps it might contribute to its unusual nuclear localisation or degradation late in infection. These events seem to contribute to cellular translation shut off because HSV-1 WT replicates very efficiently in HeLa cells depleted of PABP1. It is very likely that ICP27 protein acts in concert with other viral proteins to enhance the translation of viral mRNAs by an unidentified mechanism that does not require PABP1.
    10/2006, Degree: PhD in Molecular Virology, Supervisor: Prof. J. Barklie Clements
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    ABSTRACT: Reaching the right destination is of vital importance for molecules, proteins, organelles, and cargoes. Thus, intracellular traffic is continuously controlled and regulated by several proteins taking part in the process. Viruses exploit this machinery, and viral proteins regulating intracellular transport have been identified as they represent valuable tools to understand and possibly direct molecules targeting and delivery. Deciphering the molecular features of viral proteins contributing to (or determining) this dynamic phenotype can eventually lead to a virus-independent approach to control cellular transport and delivery. From this virus-independent perspective we looked at US9, a virion component of Herpes Simplex Virus involved in anterograde transport of the virus inside neurons of the infected host. As the natural cargo of US9-related vesicles is the virus (or its parts), defining its autonomous, virus-independent role in vesicles transport represents a prerequisite to make US9 a valuable molecular tool to study and possibly direct cellular transport. To assess the extent of this autonomous role in vesicles transport, we analyzed US9 behavior in the absence of viral infection. Based on our studies, Us9 behavior appears similar in different cell types; however, as expected, the data we obtained in neurons best represent the virus-independent properties of US9. In these primary cells, transfected US9 mostly recapitulates the behavior of US9 expressed from the viral genome. Additionally, ablation of two major phosphorylation sites (i.e. Y32Y33 and S34ES36) have no effect on protein incorporation on vesicles and on its localization on both proximal and distal regions of the cells. These results support the idea that, while US9 post-translational modification may be important to regulate cargo loading and, consequently, virion export and delivery, no additional viral functions are required for US9 role in intracellular transport.
    PLoS ONE 08/2014; 9(8):e104634. DOI:10.1371/journal.pone.0104634 · 3.53 Impact Factor
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    ABSTRACT: While the mechanism of viral reactivation from latency remains unclear, the process may involve transfer of a neuronal signal to the viral repli-cation machinery. For this reason, we have focused our work on viral proteins which play significant roles in regulating HSV replication as these likely represent novel targets for antiviral therapies. In this Review, we initially describe our biochemical and genetic studies which have elucidated the biological functions of the HSV major tegument structural protein. We also discuss our discoveries of novel posttransla-tionally modified forms of major HSV regulatory proteins. We summarize what we have discovered about the molecular mechanisms of apoptosis modulation by HSV. Finally, we review the discovery of Oncoapoptosis and discuss its potential as a new class of novel viroceutical biotherapies.


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May 30, 2014