Growth Of Varicella-zoster Virus Research Articles

Varicella-Zoster Virus ORF39 Transmembrane Protein Suppresses Interferon-Beta Promoter Activation by Interacting with STING.

Varicella-Zoster virus (VZV) causes varicella in primary infection of children and zoster during reactivation in adults. Type I interferon (IFN) signaling suppresses VZV growth, and stimulator of interferon genes (STING) plays an important role in anti-VZV responses by regulating type I IFN signaling. VZV-encoded proteins are shown to inhibit STING-mediated activation of the IFN-β promoter. However, the mechanisms by which VZV regulates STING-mediated signaling pathways are largely unknown. In this study, we demonstrate that the transmembrane protein encoded by VZV open reading frame (ORF) 39 suppresses STING-mediated IFN-β production by interacting with STING. In IFN-β promoter reporter assays, ORF39 protein (ORF39p) inhibited STING-mediated activation of the IFN-β promoter. ORF39p interacted with STING in co-transfection assays, and this interaction was comparable to that of STING dimerization. The cytoplasmic N-terminal 73 amino acids region of ORF39P was not necessary for ORF39 binding and suppression of STING-mediated IFN-β activation. ORF39p also formed a complex containing both STING and TBK1. A recombinant VZV expressing HA-tagged ORF39 was produced using bacmid mutagenesis and showed similar growth to its parent virus. During HA-ORF39 virus infection, the expression level of STING was markedly reduced, and HA-ORF39 interacted with STING. Moreover, HA-ORF39 also colocalized with glycoprotein K (encoded by ORF5) and STING at the Golgi during virus infection. Our results demonstrate that the transmembrane protein ORF39p of VZV plays a role in evading the type I IFN responses by suppressing STING-mediated activation of the IFN-β promoter.

Locked-nucleotide antagonists to varicella zoster virus small non-coding RNA block viral growth and have potential as an anti-viral therapy

Herpes zoster (HZ) remains a significant health burden with millions of cases in North America and Europe annually. HZ is frequently followed by long-term pain or post-herpetic neuralgia (PHN). Although effective vaccines for HZ are available, currently used nucleotide analogues often have limited effectiveness against HZ and especially PHN, so there remains a need for additional antiviral therapies for HZ. We recently identified a population of small non-coding RNA (sncRNA) encoded by Varicella Zoster Virus (VZV) and showed that single locked-nucleic acid antagonists (LNAA) to some sncRNA can modulate VZV replication in cell culture. In this work, we explored the antiviral effects of combinations of LNAA oligonucleotides targeting VZVsncRNA. Combinations of LNAA targeting three VZVsncRNA encoded in and near a critical viral regulatory gene were additive, achieving 96 % reduction in virus growth in a cell line. VZV growth was also inhibited by more than 90 % in primary human skin fibroblast cultures by individual and combinations of LNAA to VZVsncRNA. The inhibition by VZVsncRNA was specific and not a consequence of innate immune responses since LNAA to a different VZVsncRNA enhanced VZV growth. Targeted VZVsncRNA lack homologous sequences in the human transcriptome suggesting that LNAA to them would have reduced cytotoxicity if used as therapeutics. These results support further development of oligonucleotides targeting VZVsncRNA as a novel treatment for HZ.

Identification of a Hydrophobic Domain in Varicella-Zoster Virus ORF61 Necessary for ORF61 Self-Interaction, Viral Replication, and Skin Pathogenesis

The varicella-zoster virus (VZV) ORF61 protein is necessary for normal replication in vitro and virulence in human skin xenografts in the severe combined immunodeficiency mouse model in vivo. These experiments identify a hydrophobic domain that mediates ORF61 self-interaction. While not needed to inhibit host cell defenses, disruption of this domain (residues 250 to 320) severely impairs VZV growth, transactivation of the immediate early 63 and glycoprotein E genes, and the pathogenesis of VZV skin infection in vivo.

Antiviral activity of (+)-sattabacin against varicella zoster

The first report of the antiviral activity of (+)-sattabacin against varicella-zoster virus (VZV) is described. Our results show that (+)-sattabacin potently inhibits the growth of VZV at concentrations in the range of other drugs commonly prescribed for VZV infection. Experiments detailing the synthesis of (+)-sattabacin, quantification of cytotoxicity and gene expression data in human fibroblast cells are also presented. Gene expression data was obtained through microarray analysis from human fibroblast cells exposed to sattabacin in order to identify a possible mechanism by which (+)-sattabacin inhibits VZV replication.

Varicella-zoster virus ORF 58 gene is dispensable for viral replication in cell culture

BackgroundOpen reading frame 58 (ORF58) of varicella-zoster virus (VZV) lies at the 3'end of the Unique long (UL) region and its functional is unknown. In order to clarify whether ORF58 is essential for the growth of VZV, we constructed a deletion mutant of ORF58 (pOka-BACΔ58) from the Oka parental genome cloned into a bacterial artificial chromosome (pOka-BAC).ResultsThe ORF58-deleted virus (rpOkaΔ58) was reconstituted from the pOka-BACΔ58 genome in MRC-5 cells, indicating that the ORF58 gene is non-essential for virus growth. Comparison of the growth rate of rpOkaΔ58 and recombinant wild-type virus by assessing plaque sizes revealed no significant differences between them both in MRC-5 cells and malignant melanoma cells.ConclusionThis study shows that the ORF58 gene is dispensable for viral replication and does not affect the virus' ability to form plaques in vitro.

Roscovitine, a cyclin-dependent kinase inhibitor, prevents replication of varicella-zoster virus.

Understanding the interactions between varicella-zoster virus (VZV) and host cells can be addressed by using small molecule inhibitors of cellular enzymes. Roscovitine (Rosco) is a purine derivative that inhibits cyclin-dependent kinase 1 (cdk1), cdk2, cdk5, cdk7, and cdk9, which are key regulators of the cell cycle and transcription. Herpesviruses are known to interact with cell cycle proteins; thus, the antiviral effects of Rosco on VZV growth were evaluated. In a plaque reduction assay, 25 micro M Rosco prevented VZV replication, and the antiviral effect was reversible for at least up to 24 h posttreatment. Rosco also reduced expression of the major transactivator, IE62, over 48 h. Confocal microscopy studies indicated that Rosco caused the immediate-early proteins ORF4 and IE62 to abnormally localize in infected cells and prevented cell-cell spread of VZV over 48 h. Rosco was found to inhibit VZV DNA synthesis as measured by real-time PCR, and this technique was used to estimate the 50% effective concentration (EC(50)) of 14 micro M. This value was close to the EC(50) estimate of 12 micro M determined from plaque reduction assays. At 25 micro M, Rosco was not cytotoxic over 48 h in a neutral red uptake assay, and proliferation was slowed as the cells accumulated in a G(2)-like state. These results demonstrate the importance of cdk's in VZV replication and suggest that cdk inhibitors could serve as useful VZV antivirals.

Use of a rodent model to show that varicella-zoster virus ORF61 is dispensable for establishment of latency.

Varicella-zoster virus (VZV) results in a latent infection in humans after primary infection. Latency has also been established in guinea pigs and rats after inoculation with the virus. It was found that infection of cotton rats with the Oka vaccine strain of VZV results in a latent infection. To begin to identify which genes are required for latency, we infected cotton rats with VZV strain Oka that is deleted for ORF61. ORF61 protein transactivates certain VZV promoters and enhances the infectivity of viral DNA in transient transfections. Deletion of ORF61 results in abnormal syncytia and impairs the growth of VZV in vitro. Inoculation of cotton rats with ORF61-deleted Oka virus resulted in latent VZV infection in the nervous system similar to that seen for animals infected with parental virus. Thus, the cotton rat can be used to study the ability of mutants in the Oka vaccine strain of VZV to establish latent infection.

Novel human topoisomerase I inhibitors, topopyrones A, B, C and D. I. Producing strain, fermentation, isolation, physico-chemical properties and biological activity.

In the course of a screening program for specific inhibitors of human topoisomerase I using a recombinant yeast, we have discovered four new active compounds. All four compounds were isolated from the culture broth of a fungus, Phoma sp. BAUA2861, and two of them were isolated from the culture broth of a fungus, Penicillium sp. BAUA4206. We designated these compounds as topopyrones A, B, C and D. Topopyrones A, B, C and D selectively inhibited recombinant yeast growth dependent on expression of human topoisomerase I with IC50 values of 1.22, 0.15, 4.88 and 19.63 ng/ml, respectively. The activity and selectivity of topopyrone B were comparable to those of camptothecin. The relaxation of supercoiled pBR322 DNA by human DNA topoisomerase I was inhibited by these compounds, however they did not inhibit human DNA topoisomerase II. Topopyrones A, B, C and D were cytotoxic to all tumor cell lines when tested in vitro. Topopyrone B has potent inhibitory activity against herpesvirus, especially varicella zoster virus (VZV). It inhibited VZV growth with EC50 value of 0.038 microg/ml, which is 24-fold stronger than that of acyclovir (0.9 microg/ml). Topopyrones A, B, and C were inhibitory to Gram-positive bacteria.

The varicella-zoster virus ORF66 protein induces kinase activity and is dispensable for viral replication.

Varicella-zoster virus (VZV) open reading frames (ORFs) 47 and 66 encode proteins that are homologous to a family of eukaryotic serine-threonine kinases. Prior studies showed that the VZV ORF47 protein has kinase activity in vitro and is dispensable for replication in cultured cells. To examine the role of the ORF66 protein during infection, we constructed VZV recombinants that are unable to express either the ORF66 protein (ROka 66S) or both the ORF47 and ORF66 proteins (ROka 47S/66S). VZV unable to express ORF66 grew to titers similar to those of the parental VZV (ROka) in vitro; however, VZV lacking both ORF66 and ORF47 grew to titers lower than those of ROka. Nuclear extracts from ROka 66S- or ROka 47S-infected cells showed a 48-kDa phosphoprotein(s); a phosphoprotein with a similar size was not present in nuclear extracts from ROka 47S/66S-infected cells. To determine the role of the ORF66 protein in the phosphorylation of specific VZV-encoded proteins, we immunoprecipitated known VZV phosphoproteins (ORF4, ORF62, ORF63, and ORF68 proteins) from nuclear extracts of phosphate-labeled cells infected with ROka, ROka 66S, or ROka 47S/66S. Each of the VZV phosphoproteins was phosphorylated to a similar extent in the presence or absence of either the ORF66 protein or both the ORF66 and ORF47 proteins. From these studies we conclude (i) neither ORF66 alone nor ORF66 and ORF47 in combination are essential for VZV growth in cultured cells, (ii) ORF66 either is a protein kinase or induces protein kinase activity during infection, and (iii) the VZV phosphoproteins encoded by ORF4, ORF62, ORF63, and ORF68 do not require either ORF66 alone or ORF66 and ORF47 for phosphorylation in vitro.

The varicella-zoster virus (VZV) open reading frame 47 (ORF47) protein kinase is dispensable for viral replication and is not required for phosphorylation of ORF63 protein, the VZV homolog of herpes simplex virus ICP22.

To investigate the role of varicella-zoster virus (VZV) open reading frame 47 (ORF47) protein kinase during infection, a VZV mutant was generated in which two contiguous stop codons were introduced into ORF47, thus eliminating expression of the ORF47 kinase. ORF47 kinase was not essential for the growth of VZV in cultured cells, and the growth rate of the VZV mutant lacking ORF47 protein was indistinguishable from that of parental VZV. Nuclear extracts from cells infected with parental VZV contained several phosphorylated proteins which were not detected in extracts from cells infected with the ORF47 mutant. The herpes simplex virus type 1 (HSV-1) UL13 protein (the homolog of VZV ORF47 protein) is responsible for the posttranslational processing associated with phosphorylation of HSV-1 ICP22 (the homolog of VZV ORF63 protein). Immunoprecipitation of 32P-labeled proteins from cells infected with parental virus and those infected with ORF47 mutant virus yielded similar amounts of the VZV phosphoproteins encoded by ORF4, ORF62, ORF63, and ORF68 (VZV gE), and the electrophoretic migration of these proteins was not affected by the lack of ORF47 kinase. Therefore, while the VZV ORF47 protein is capable of phosphorylating several cellular or viral proteins, it is not required for phosphorylation of the ORF63 protein in virus-infected cells.

Egress of varicella-zoster virus from the melanoma cell: a tropism for the melanocyte.

The pathway of envelopment and egress of the varicella-zoster virus (VZV) and the primary site of viral production within the epidermal layer of the skin are not fully understood. There are several hypotheses to explain how the virus may receive an envelope as it travels to the surface of the monolayer. In this study, we expand earlier reports and provide a more detailed explanation of the growth of VZV in human melanoma cells. Human melanoma cells were selected because they are a malignant derivative of the melanocyte, the melanin-producing cell which originates in the neural crest. We were able to observe the cytopathic effects of syncytial formation and the pattern of egress of virions at the surfaces of infected monolayers by scanning electron microscopy and laser-scanning confocal microscopy. The egressed virions did not appear uniformly over the syncytial surface, rather they were present in elongated patterns which were designated viral highways. In order to document the pathway by which VZV travels from the host cell nucleus to the outer cell membrane, melanoma cells were infected and then processed for examination by transmission electron microscopy (TEM) at increasing intervals postinfection. At the early time points, within minutes to hours postinfection, it was not possible to localize the input virus by TEM. Thus, viral particles first observed at 24 h postinfection were considered progeny virus. On the basis of the TEM observations, the following sequence of events was considered most likely. Nucleocapsids passed through the inner nuclear membrane and acquired an envelope, after which they were seen in the endoplasmic reticulum. Enveloped virions within vacuoles derived from the endoplasmic reticulum passed into the cytoplasm. Thereafter, vacuoles containing nascent enveloped particles acquired viral glycoproteins by fusion with vesicles derived from the Golgi. The vacuoles containing virions fused with the outer plasma membrane and the particles appeared on the surface of the infected cell. Late in infection, enveloped virions were also present within the nuclei of infected cells; the most likely mechanism was retrograde flow from the perinuclear space back into the nucleus. Thus, this study suggests a role for the melanocyte in the pathogenesis of VZV infection, because all steps in viral egress can be accounted for if VZV subsumes the cellular pathways required for melanogenesis.

Deletion of the varicella-zoster virus large subunit of ribonucleotide reductase impairs growth of virus in vitro

Cells infected with varicella-zoster virus (VZV) express a viral ribonucleotide reductase which is distinct from that present in uninfected cells. VZV open reading frames 18 and 19 (ORF18 and ORF19) are homologous to the herpes simplex virus type 1 genes encoding the small and large subunits of ribonucleotide reductase, respectively. We generated recombinant VZV by transfecting cultured cells with four overlapping cosmid DNAs. To construct a virus lacking ribonucleotide reductase, we deleted 97% of VZV ORF19 from one of the cosmids. Transfection of this cosmid with the other parental cosmids yielded a VZV mutant with a 2.3-kbp deletion confirmed by Southern blot analysis. Virus-specific ribonucleotide reductase activity was not detected in cells infected with VZV lacking ORF19. Infection of melanoma cells with ORF19-deleted VZV resulted in plaques smaller than those produced by infection with the parental VZV. The mutant virus also exhibited a growth rate slightly slower than that of the parental virus. Chemical inhibition of the VZV ribonucleotide reductase has been shown to potentiate the anti-VZV activity of acyclovir. Similarly, the concentration of acyclovir required to inhibit plaque formation by 50% was threefold lower for the VZV ribonucleotide reductase deletion mutants than for parental virus. We conclude that the VZV ribonucleotide reductase large subunit is not essential for virus infection in vitro; however, deletion of the gene impairs the growth of VZV in cell culture and renders the virus more susceptible to inhibition by acyclovir.

Incomplete growth of varicella-zoster virus in human monocytes.

Infection of human peripheral blood monocytes by varicella-zoster virus (VZV) was investigated. When freshly isolated monocytes of young adult volunteers were infected with cell-free VZV and examined by indirect immunofluorescence, specific antigens appeared at 8 hr and the number of antigen-positive cells reached the maximum between 24 and 48 hr postinfection. The proportion of antigen-positive cells to total cells was similar to that of the permissive control (HeLa cells), while very few infectious centers (IC) of monocytes were formed in comparison with the infected control cells. Monocytes isolated from infants and old persons formed a larger number of IC than those of young adults. Electron microscopic study of VZV-infected monocytes from three young adult volunteers demonstrated that the proportion of VZV particle-positive cells to total cells was similar to that of antigen-positive cells, and most of the particles seen in the nuclei were low in density and lacked a central core. These results suggest that the growth of VZV in human adult monocytes is incomplete. This restriction of VZV growth by monocytes may play an important role in defense against VZV infection.

Varicella-zoster virus infection of human brain cells and ganglion cells in tissue culture

The growth of varicella-zoster virus (VZV) in cultures of human brain (HB) and human ganglion (HG) cells was compared to VZV growth in human fibroblasts. Infected cultures were monitored by histologic, electron microscopic (EM), and virologic techniques. Two to three days after VZV infection of all cell cultures at a multiplicity of infection (MOI) of 0.1, a multifocal cytopathic effect (CPE) developed. CPE was characterized by multinucleated cells and virus-specific intranuclear inclusions as determined by immunofluorescence and EM. In VZV- infected HB and HG cells only, large vacuoles were also seen in the cytoplasm of dying cells. Some vacuoles were almost devoid of structures. Within and at the limiting membranes of other vacuoles, aggregates of VZV particles (measuring 210--230 nm) were seen enveloped in osmiophilic material. VZV infection of HB and HG cultures was strongly cell-associated. Clarified tissue culture medium removed at maximum CPE failed to infect homologous HB or HG cells. When an inoculum of VZV-infected HB or HG cells was transferred to homologous uninfected cultures for 10--15 passages, the incubation period for CPE remained constant, and the titer of VZV in cells sampled randomly corresponded to the amount of virus that was used for original infection.