The equine herpesvirus 2 E1 open reading frame encodes a functional chemokine receptor.
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ABSTRACT:
SUBMITTER: Camarda G
PROVIDER: S-EPMC113033 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:The open reading frame K8 of Kaposi's sarcoma-associated herpesvirus (KSHV) encodes a basic leucine zipper (bZip) protein that binds to the origin of viral DNA replication and is an integral component of viral lytic DNA replication complex. Moreover, K8 physically interacts with replication and transcription activator (RTA) and represses its transactivation activity on several viral promoters. To investigate the role of this protein in viral life cycle, we constructed two K8-null recombinant mutant viruses (BAC-ΔK8 and BAC-stopK8) by using a bacterial artificial chromosome (BAC) system. Latent viral infection can be reconstituted in 293T and BJAB cells with wild-type and the K8-null recombinant viruses by introducing the cloned viral genomes into the cells. When the cells carrying these viruses were induced with 12-O-tetradecanoylphorbol-13-acetate (TPA) and sodium butyrate, no significant difference was seen in overall viral gene expression between wild-type and K8-null viruses, with lytic DNA replication still active in the latter. However, 293T cells harboring K8-null mutant viruses, either BAC-ΔK8 or BAC-stopK8, displayed lower copy numbers of latent KSHV genome in comparison with wild-type viruses. Furthermore, although K8 deficiency appeared to not affect infectivity when K8-null viruses were used to infect 293T, primary human microvascular dermal endothelial and human foreskin fibroblast cells, they exhibited much lower viral genome copy numbers in all types of cell compared to wild-type viruses. Taken together, these data suggest a possible role of K8 in abortive lytic DNA replication occurring in early stages of de novo infection or in the maintenance of latent viral genomes.
| S-EPMC3067771 | biostudies-literature
Project description:Viruses have long been studied not only for their pathology and associated disease but also as model systems for understanding cellular and immunological processes. Rodent herpesvirus Peru (RHVP) is a recently characterized rhadinovirus related to murine gammaherpesvirus 68 (MHV68) and Kaposi's sarcoma-associated herpesvirus (KSHV) that establishes acute and latent infection in laboratory mice. RHVP encodes numerous unique proteins that we hypothesize might facilitate host immune evasion during infection. We report here that open reading frame (ORF) R17 encodes a high-affinity chemokine binding protein that broadly recognizes human and murine CC and C chemokines. The interaction of R17 with chemokines is generally characterized by rapid association kinetics, and in the case of CCL3, CCL4, CCL5, CCL24, and XCL1, extremely stable complexes are formed. Functionally, R17 potently inhibited CCL2-driven chemotaxis of the human monocytic cell line THP-1, CCL3-driven chemotaxis of peripheral blood mononuclear cells, and CCL2-mediated calcium flux. Our studies also reveal that R17 binds to glycosaminoglycans (GAGs) in a process dependent upon two BBXB motifs and that chemokine and GAG binding can occur simultaneously at distinct sites. Collectively, these studies suggest that R17 may play a role in RHVP immune evasion through the targeted sabotage of chemokine-mediated immune surveillance.
| S-EPMC3911705 | biostudies-literature
Project description:Herpesviruses alternate between the latent and the lytic life cycle. Switching into the lytic life cycle is important for herpesviral replication and disease pathogenesis. Activation of a transcription factor replication and transcription activator (RTA) has been demonstrated to govern this switch in Kaposi's sarcoma-associated herpesvirus (KSHV). The protein encoded by open reading frame 49 from KSHV (ORF49KSHV) has been shown to upregulate lytic replication in KSHV by enhancing the activities of the RTA. We have solved the crystal structure of the ORF49KSHV protein to a resolution of 2.4 Å. The ORF49KSHV protein has a novel fold consisting of 12 alpha-helices bundled into two pseudodomains. Most notably are distinct charged patches on the protein surface, which are possible protein-protein interaction sites. Homologs of the ORF49KSHV protein in the gammaherpesvirus subfamily have low sequence similarities. Conserved residues are mainly located in the hydrophobic regions, suggesting that they are more likely to play important structural roles than functional ones. Based on the identification and position of three sulfates binding to the positive areas, we performed some initial protein-DNA binding studies by analyzing the thermal stabilization of the protein in the presence of DNA. The ORF49KSHV protein is stabilized in a dose-responsive manner by double-stranded oligonucleotides, suggesting actual DNA interaction and binding. Biolayer interferometry studies also demonstrated that the ORF49KSHV protein binds these oligonucleotides. Kaposi's sarcoma-associated herpesvirus (KSHV) is a tumorigenic gammaherpesvirus that causes multiple cancers and lymphoproliferative diseases. The virus exists mainly in the quiescent latent life cycle, but when it is reactivated into the lytic life cycle, new viruses are produced and disease symptoms usually manifest. Several KSHV proteins play important roles in this reactivation, but their exact roles are still largely unknown. In this study, we report the crystal structure of the open reading frame 49 protein encoded by KSHV (ORF49KSHV). Possible regions for protein interaction that could harbor functional importance were found on the surface of the ORF49KSHV protein. This led to the discovery of novel DNA binding properties of the ORF49KSHV protein. Evolutionary conserved structural elements with the functional homologs of ORF49KSHV were also established with the structure.
| S-EPMC5215346 | biostudies-literature
Project description:We have previously examined the transcription and splicing of open reading frames (ORFS) 71 (K13), 72, and 73 of Kaposi's sarcoma-associated herpesvirus (KSHV) in the primary effusion lymphoma cell line BCP-1 (latently infected with KSHV) (45). The three genes encoded by these ORFs (for vFLIP, vCyclin, and latency-associated nuclear antigen [LANA]) are transcribed from a common transcription start site in BCP-1 cells during both latency and the lytic cycles. The resulting transcript is spliced to yield a 5.32-kb message encoding LANA, vCyclin, and vFLIP and a 1.7-kb bicistronic message encoding vCyclin and vFLIP. To investigate whether the vFLIP protein could be expressed from this vCyclin/vFLIP message, we utilized a bicistronic luciferase reporter system. The genes for Renilla and firefly luciferases (which utilize different substrates) were cloned in tandem downstream from a T7 RNA polymerase promoter. Fragments of DNA immediately upstream from the initiating codon of vFLIP were cloned between the two luciferase genes. The relative expression of the two luciferases, one directed by the putative internal ribosome entry site (IRES) sequences and the other by cap-dependent ribosome scanning, was used to compare the activities of the different DNA fragments. A minimum fragment of 233 bp within the coding region of vCyclin was found to direct efficient expression of the downstream cistron (firefly luciferase). The activity of this IRES was orientation dependent and unaffected by methods used to inhibit cap-dependent translation. This is the first demonstration of an IRES element encoded by a DNA virus and may represent a novel mechanism through which KSHV controls protein expression.
| S-EPMC114096 | biostudies-literature
Project description:Equine herpesvirus type 1 (EHV-1) outbreaks continue to occur despite widely used vaccination. Therefore, development of EHV-1 vaccines providing improved immunity and protection is ongoing. Here, an open reading frame 2 deletion mutant of the neuropathogenic EHV-1 strain Ab4 (Ab4ΔORF2) was tested as a vaccine candidate. Three groups of horses (n = 8 each) were infected intranasally with Ab4ΔORF2 or the parent Ab4 virus or were kept as noninfected controls. Horses infected with Ab4ΔORF2 had reduced fever and nasal virus shedding compared to those infected with Ab4 but mounted similar adaptive immunity dominated by antibody responses. Nine months after the initial infection, all horses were challenged intranasally with Ab4. Previously noninfected horses (control/Ab4) displayed clinical signs, shed large amounts of virus, and developed cell-associated viremia. In contrast, 5/8 or 3/8 horses previously infected with Ab4ΔORF2 or Ab4, respectively, were fully protected from challenge infection as indicated by the absence of fever, clinical disease, nasal virus shedding, and viremia. All of these outcomes were significantly reduced in the remaining, partially protected 3/8 (Ab4ΔORF2/Ab4) and 5/8 (Ab4/Ab4) horses. Protected horses had EHV-1-specific IgG4/7 antibodies prior to challenge infection, and intranasal antibodies increased rapidly postchallenge. Intranasal inflammatory markers were not detectable in protected horses but quickly increased in control/Ab4 horses during the first week after infection. Overall, our data suggest that preexisting nasal IgG4/7 antibodies neutralize EHV-1, prevent viral entry, and thereby protect from disease, viral shedding, and cell-associated viremia. In conclusion, improved protection from challenge infection emphasizes further evaluation of Ab4ΔORF2 as a vaccine candidate.IMPORTANCE Nasal equine herpesvirus type 1 (EHV-1) shedding is essential for virus transmission during outbreaks. Cell-associated viremia is a prerequisite for the most severe disease outcomes, abortion and equine herpesvirus myeloencephalopathy (EHM). Thus, protection from viremia is considered essential for preventing EHM. Ab4ΔORF2 vaccination prevented EHV-1 challenge virus replication in the upper respiratory tract in fully protected horses. Consequently, these neither shed virus nor developed cell-associated viremia. Protection from virus shedding and viremia during challenge infection in combination with reduced virulence at the time of vaccination emphasizes ORF2 deletion as a promising modification for generating an improved EHV-1 vaccine. During this challenge infection, full protection was linked to preexisting local and systemic EHV-1-specific antibodies combined with rapidly increasing intranasal IgG4/7 antibodies and lack of nasal type I interferon and chemokine induction. These host immune parameters may constitute markers of protection against EHV-1 and be utilized as indicators for improved vaccine development and informed vaccination strategies.
| S-EPMC6819910 | biostudies-literature
Project description:BackgroundHuman pathogenic coronavirus NL63 (hCoV-NL63) is a group 1 (alpha) coronavirus commonly associated with respiratory tract infections. In addition to known non-structural and structural proteins all coronaviruses have one or more accessory proteins whose functions are mostly unknown. Our study focuses on hCoV-NL63 open reading frame 3 (ORF 3) which is a highly conserved accessory protein among coronaviruses.ResultsIn-silico analysis of the 225 amino acid sequence of hCoV-NL63 ORF 3 predicted a triple membrane-spanning protein. Expression in infected CaCo-2 and LLC-MK2 cells was confirmed by immunofluorescence and Western blot analysis. The protein was detected within the endoplasmatic reticulum/Golgi intermediate compartment (ERGIC) where coronavirus assembly and budding takes place. Subcellular localization studies using recombinant ORF 3 protein transfected in Huh-7 cells revealed occurrence in ERGIC, Golgi- and lysosomal compartments. By fluorescence microscopy of differently tagged envelope (E), membrane (M) and nucleocapsid (N) proteins it was shown that ORF 3 protein colocalizes extensively with E and M within the ERGIC. Using N-terminally FLAG-tagged ORF 3 protein and an antiserum specific to the C-terminus we verified the proposed topology of an extracellular N-terminus and a cytosolic C-terminus. By in-vitro translation analysis and subsequent endoglycosidase H digestion we showed that ORF 3 protein is N-glycosylated at the N-terminus. Analysis of purified viral particles revealed that ORF 3 protein is incorporated into virions and is therefore an additional structural protein.ConclusionsThis study is the first extensive expression analysis of a group 1 hCoV-ORF 3 protein. We give evidence that ORF 3 protein is a structural N-glycosylated and virion-incorporated protein.
| S-EPMC2819038 | biostudies-literature
Project description:Open reading frame 11 (ORF11) is a conserved gammaherpesvirus gene that remains undefined. We identified the product of murine gammaherpesvirus 68 (MHV-68) ORF11, p43, as a virion component with a predominantly perinuclear distribution in infected cells. MHV-68 lacking p43 grew normally in vitro but showed reduced lytic replication in vivo and a delay in seeding to the spleen. Subsequent latency amplification was normal. Thus, MHV-68 ORF11 encoded a virion component that was important for in vivo lytic replication but dispensable for the establishment of latency.
| S-EPMC548481 | biostudies-other
Project description:Human herpesvirus 8 (HHV-8) can be classified into distinct subtypes on the basis of sequence polymorphisms in several open reading frames (ORFs). We analyzed the subtypes of HHV-8 in 59 human immunodeficiency virus-infected Japanese patients by using polymorphisms in ORF26 and found that over two-thirds of the HHV-8 isolates fell into major subtype A. We also found that single nucleotide polymorphisms (SNPs) at nucleotide positions 1032 (C-to-A substitution) and 1055 (G-to-T substitution) in HHV-8 ORF26 were correlated with increased susceptibility to Kaposi's sarcoma, compared to the results obtained with HHV-8 with wild-type nucleotides at these positions (P = 0.0106). This observation suggests that molecular heterogeneity of the HHV-8 genome affects the biological properties of HHV-8, resulting in different clinical phenotypes of HHV-8 infection. Since sensitive PCR of ORF26 allowed us to analyze the SNPs by using peripheral blood from HHV-8-infected patients, the ORF26 SNPs will be a potent tool for investigating the pathogenesis of HHV-8 infection.
| S-EPMC156527 | biostudies-literature
Project description:Human herpesvirus 8 (HHV-8) is likely to be involved in the pathogenesis of Kaposi's sarcoma (KS) and body cavity-based lymphomas (BCBLs). The HHV-8 genome is primarily in a latent state in BCBL-derived cell lines like BCBL-1, but lytic replication can be induced by phorbol esters (R. Renne, W. Zhang, B. Herndier, M. McGrath, N. Abbey, D. Kedes, and D. E. Ganem, Nat. Med. 2:342-346, 1996). A 35- to 37-kDa glycoprotein (gp35-37) is the polypeptide most frequently and intensively recognized by KS patient sera on Western blots with induced BCBL-1 cells. Its apparent molecular mass is reduced to 30 kDa by digestion with peptide-N-glycosidase F. By searching the known HHV-8 genomic sequence for open reading frames (ORF) with the potential to encode such a glycoprotein, an additional, HHV-8-specific reading frame was identified adjacently to ORF K8. This ORF, termed K8.1, was found to be transcribed primarily into a spliced mRNA encoding a glycoprotein of 228 amino acids. Recombinant K8.1 was regularly recognized by KS patient sera in Western blots, and immunoaffinity-purified antibodies to recombinant K8.1 reacted with gp35-37. This shows that the immunogenic gp35-37 is encoded by HHV-8 reading frame K8.1, which will be a useful tool for studies of HHV-8 epidemiology and pathogenesis.
| S-EPMC109880 | biostudies-literature
Project description:Tegument is a unique structure of herpesvirus, which surrounds the capsid and interacts with the envelope. Morphogenesis of gammaherpesvirus is poorly understood due to lack of efficient lytic replication for Epstein-Barr virus and Kaposi's sarcoma-associated herpesvirus/human herpesvirus 8, which are etiologically associated with several types of human malignancies. Murine gammaherpesvirus 68 (MHV-68) is genetically related to the human gammaherpesviruses and presents an excellent model for studying de novo lytic replication of gammaherpesviruses. MHV-68 open reading frame 33 (ORF33) is conserved among Alpha-, Beta-, and Gammaherpesvirinae subfamilies. However, the specific role of ORF33 in gammaherpesvirus replication has not yet been characterized. We describe here that ORF33 is a true late gene and encodes a tegument protein. By constructing an ORF33-null MHV-68 mutant, we demonstrated that ORF33 is not required for viral DNA replication, early and late gene expression, viral DNA packaging or capsid assembly but is required for virion morphogenesis and egress. Although the ORF33-null virus was deficient in release of infectious virions, partially tegumented capsids produced by the ORF33-null mutant accumulated in the cytoplasm, containing conserved capsid proteins, ORF52 tegument protein, but virtually no ORF45 tegument protein and the 65-kDa glycoprotein B. Finally, we found that the defect of ORF33-null MHV-68 could be rescued by providing ORF33 in trans or in an ORF33-null revertant virus. Taken together, our results indicate that ORF33 is a tegument protein required for viral lytic replication and functions in virion morphogenesis and egress.
| S-EPMC2753129 | biostudies-literature