The Kaposi's Sarcoma-Associated Herpesvirus ORF34 Protein Interacts and Stabilizes HIF-2α via Binding to the HIF-2α bHLH and PAS Domains.

Hypoxia and hypoxia inducible factors (HIFs) play important roles in the Kaposi's sarcoma-associated herpesvirus (KSHV) life cycle. KSHV is the causative agent of Kaposi's sarcoma (KS) and other AIDS-related malignancies. Kaposi's sarcoma is a highly vascular tumor, which preferentially develops in the lower extremities of the body where blood vessels are often poorly oxygenated. The main cellular responses to hypoxia are mediated mainly by two isoforms of HIF, HIF-1α and HIF-2α. HIF-1α and HIF-2α have common as well as distinct functions, although they are similar in structure and function. Previously, we showed that the KSHV ORF34 protein binds HIF-1α and facilitates its degradation through the ubiquitin-proteasome pathway causing negative regulation of HIF-1α-dependent genes (Haque and Kousoulas, J Virol 87:2164-2173, 2013, https://www.doi.org/10.1128/JVI.02460-12). Herein, we show that the ORF34 gene is involved in the regulation of KSHV lytic gene expression, since deletion of ORF34 resulted in reduced immediate early and early lytic gene expression and blocked late gene expression. Coimmunoprecipitation experiments revealed that the ORF34 protein physically interacted with HIF-2α in transfected as well as in KSHV-infected cells. Utilization of ORF34 truncations revealed that three distinct domains bind HIF-2α and that both bHLH and PAS domains of HIF-2α interacted with ORF34. Unlike HIF-1α, dose-dependent coexpression of ORF34 stabilized the HIF-2α protein, ensuring HIF-2α-dependent transcriptional activity. The ORF34 protein enhanced HIF-2α ubiquitination at the bHLH and PAS domains. The results show that the KSHV ORF34 protein is involved in the KSHV life cycle by regulating the expression of HIF-1α and HIF-2α proteins.IMPORTANCE Hypoxia inducible factor 1α (HIF-1α) and HIF-2α are transcription factors which play important roles in the Kaposi's sarcoma-associated herpesvirus (KSHV) latent and lytic gene replication. Herein, we show that the ORF34 gene is involved in the regulation of KSHV lytic gene expression, since deletion of ORF34 resulted in reduced immediate early and early lytic gene expression and blocked late gene expression. In addition, we demonstrate that the KSHV ORF34 protein binds and stabilizes HIF-2α, in contrast to its role in binding HIF-1α and causing its degradation via the proteasome pathway. Thus, the KSHV ORF34 protein plays a regulatory role in the KSHV life cycle by regulating HIF-1α and HIF-2α expression.

The Amino Terminus of Herpes Simplex Virus 1 Glycoprotein K (gK) Is Required for gB Binding to Akt, Release of Intracellular Calcium, and Fusion of the Viral Envelope with Plasma Membranes.

Previously, we have shown that the amino terminus of glycoprotein K (gK) binds to the amino terminus of gB and that deletion of the amino-terminal 38 amino acids of gK prevents herpes simplex virus 1 (HSV-1) infection of mouse trigeminal ganglia after ocular infection and virus entry into neuronal axons. Recently, it has been shown that gB binds to Akt during virus entry and induces Akt phosphorylation and intracellular calcium release. Proximity ligation and two-way immunoprecipitation assays using monoclonal antibodies against gB and Akt-1 phosphorylated at S473 [Akt-1(S473)] confirmed that HSV-1(McKrae) gB interacted with Akt-1(S473) during virus entry into human neuroblastoma (SK-N-SH) cells and induced the release of intracellular calcium. In contrast, the gB specified by HSV-1(McKrae) gKΔ31-68, lacking the amino-terminal 38 amino acids of gK, failed to interact with Akt-1(S473) and induce intracellular calcium release. The Akt inhibitor miltefosine inhibited the entry of McKrae but not the gKΔ31-68 mutant into SK-N-SH cells. Importantly, the entry of the gKΔ31-68 mutant but not McKrae into SK-N-SH cells treated with the endocytosis inhibitors pitstop-2 and dynasore hydrate was significantly inhibited, indicating that McKrae gKΔ31-68 entered via endocytosis. These results suggest that the amino terminus of gK functions to regulate the fusion of the viral envelope with cellular plasma membranes.IMPORTANCE HSV-1 glycoprotein B (gB) functions in the fusion of the viral envelope with cellular membranes during virus entry. Herein, we show that a deletion in the amino terminus of glycoprotein K (gK) inhibits gB binding to Akt-1(S473), the release of intracellular calcium, and virus entry via fusion of the viral envelope with cellular plasma membranes.

Kaposi's sarcoma-associated herpesvirus glycoproteins B and K8.1 regulate virion egress and synthesis of vascular endothelial growth factor and viral interleukin-6 in BCBL-1 cells.

Kaposi's sarcoma-associated herpesvirus (KSHV) viral glycoproteins play important roles in the infectious life cycle and have been implicated in KSHV-associated endothelial cell transformation, angiogenesis, and KS-induced malignancies. KSHV-associated primary effusion lymphomas (PELs) secrete high levels of vascular endothelial growth factor (VEGF) and viral interleukin-6 (vIL-6) in vitro and VEGF, vIL-6, and basic-fibroblast growth factor (b-FGF) in mouse xenografts. KSHV-encoded glycoproteins B (gB) and K8.1 stimulate VEGF secretion, most likely mediated by direct or indirect binding to cell surface receptors, including the gB-specific alphaVbeta3 and alpha3beta1 integrins. In this study, the short interfering RNA (siRNA)-mediated inhibition of either gB or K8.1 transcription by anti-gB or -K8.1 siRNAs caused a substantial reduction in virion egress and a decrease in both vIL-6 and VEGF production. Similarly, the treatment of BCBL-1 cells with anti-gB or anti-K8.1 antibodies caused a substantial reduction in vIL-6 and VEGF production. Codon-optimized versions of either wild-type gB, mutant gB having the RGD amino acid motif changed to RAA, or K8.1 efficiently rescued virion egress and VEGF and vIL-6 production. These results suggest that the binding of gB via its RGD motif to integrin receptors was not responsible for the observed gB-associated regulation of VEGF and vIL-6 transcription. Conditioned medium collected from BCBL-1 cells transfected with anti-gB and anti-K8.1 siRNAs or treated with anti-gB and anti-K8.1 antibodies exhibited a significantly reduced ability to induce the formation of the capillary network of endothelial cells compared to the ability of medium from mock-infected BCBl-1 cells. Furthermore, medium obtained from BCBL-1 cells expressing smaller amounts of gB and K8.1 produced a substantial reduction in endothelial cell migration in a vertical migration assay compared to that of control medium containing wild-type levels of gB and K8.1. These results suggest a functional linkage between gB/K8.1 synthesis and VEGF/vIL-6 transcriptional regulation via paracrine and/or autocrine signaling pathways.

The herpes simplex virus type 1 glycoprotein D (gD) cytoplasmic terminus and full-length gE are not essential and do not function in a redundant manner for cytoplasmic virion envelopment and egress.

Herpes simplex virus type 1 (HSV-1) acquires its final envelope by budding into cytoplasmic vesicles thought to be derived from trans-Golgi network membranes. This process is facilitated by interactions among the carboxyl termini of viral glycoproteins and tegument proteins. To directly investigate the relative importance of the carboxyl terminus of glycoprotein D (gD) in the presence or absence of gE, a recombinant virus (gDDeltact) was constructed to specify a truncated gD lacking the carboxy-terminal 29 amino acids. Furthermore, two additional recombinant viruses were constructed by mutating from ATG to CTG the initiation codons of gE (gEctg) or both gE and gM (gEctg+gMctg), causing lack of expression of gE or both gE and gM, respectively. A fourth mutant virus was constructed to specify the gEctg+gDDeltact mutations. The replication properties of these viruses were compared to those of a newly constructed recombinant virus unable to express UL20 due to alteration of the two initiation codons of UL20 (UL20ctgctg). All recombinant viruses were constructed by using the double-Red, site-directed mutagenesis system implemented on the HSV-1(F) genome cloned into a bacterial artificial chromosome. The gEctg, gEctg+gMctg, gDDeltact, and gEctg+gDDeltact viruses produced viral plaques on African monkey kidney cells (Vero), as well as other cells, that were on average approximately 30 to 50% smaller than those produced by the wild-type virus HSV-1(F). In contrast, the UL20ctgctg virus produced very small plaques containing three to five cells, as reported previously for the DeltaUL20 virus lacking the entire UL20 gene. Viral replication kinetics of intracellular and extracellular viruses revealed that all recombinant viruses produced viral titers similar to those produced by the wild-type HSV-1(F) virus intracellularly and extracellularly at late times postinfection, with the exception of the UL20ctgctg and DeltaUL20 viruses, which replicated more than two-and-a-half logs less efficiently than HSV-1(F). Electron microscopy confirmed that all viruses, regardless of their different gene mutations, efficiently produced enveloped virions within infected cells, with the exception of the UL20ctgctg and DeltaUL20 viruses, which accumulated high levels of unenveloped virions in the cytoplasm. These results show that the carboxyl terminus of gD and the full-length gE, either alone or in a redundant manner, are not essential in cytoplasmic virion envelopment and egress from infected cells. Similarly, gM and gE do not function alone or in a redundant manner in cytoplasmic envelopment and virion egress, confirming previous findings.

Functional and physical interactions of the herpes simplex virus type 1 UL20 membrane protein with glycoprotein K.

Herpes simplex virus type 1 glycoprotein K (gK) and the UL20 protein (UL20p) are coordinately transported to the trans-Golgi network (TGN) and cell surfaces and are required for cytoplasmic virion envelopment at the TGN. In addition, cell surface expression of gK and UL20p is required for virus-induced cell fusion. Previously, confocal microscopy colocalization and intracellular transport experiments strongly suggested direct protein-protein interactions between gK and UL20p. Direct protein-protein interactions between gK and UL20p were demonstrated through reciprocal coimmunoprecipitation experiments, as well as with glutathione S-transferase (GST) pull-down experiments. A fusion protein consisting of the amino-terminal 66 amino acids of UL20p fused in-frame with GST was expressed in Escherichia coli and purified via glutathione column chromatography. Precipitation of GST-UL20p from mixtures of GST-UL20p fusion protein with cellular extracts containing gK specifically coprecipitated gK but not other viral glycoproteins. The purified UL20p-GST fusion protein reacted with all gK-associated protein species. It was concluded that the amino terminus of UL20p, most likely, interacted with gK domain III, which is predicted to lie intracellularly. UL20p-gK domain-specific interactions must serve important functions in the coordinate transport of UL20p and gK to the TGN, because retention of UL20p in the endoplasmic reticulum (ER) via the addition of an ER retention signal at the carboxyl terminus of UL20p forced the ER retention of gK and drastically inhibited intracellular virion envelopment and virus-induced cell fusion.

The cytoplasmic terminus of Kaposi's sarcoma-associated herpesvirus glycoprotein B is not essential for virion egress and infectivity.

Kaposi's sarcoma-associated herpesvirus (KSHV)-encoded glycoprotein B (gB) is an important determinant of viral infectivity and virion egress. A small interfering RNA (siRNA)-based strategy was devised to inhibit KSHV gB gene expression. Transient cotransfection of plasmids constitutively expressing gB and anti-gB siRNAs in 293 cells substantially inhibited gB mRNA levels and protein production. Similarly, transient expression of siRNAs into the primary effusion lymphoma cell line BCBL-1 caused a substantial reduction of gB transcripts and protein synthesis. TaqMan real-time PCR assays against the lytic KSHV gene ORF59 and infectivity assays on 293 cells were employed to assess the effect of inhibiting gB synthesis on virion egress from BCBL-1 cells and infectivity on 293 cells, respectively. These experiments showed that gB was essential for virion egress and infectivity. Transfection of a codon-optimized gB gene with the first 540 nucleotides altered, and therefore not recognized by anti-gB siRNAs that target the native but not the codon-optimized sequence, efficiently rescued virion egress and infectivity in BCBL-1 cells in the presence of siRNAs inhibiting wild-type gB expression. To assess the role of the cytoplasmic domain of gB in virion egress, mutant gB genes were generated specifying carboxyl terminal truncations of 25 and 58 amino acids disrupting two prominent predicted alpha-helical domains associated with virus-induced cell fusion. A third truncation removed the entire predicted cytoplasmic terminus of 84 amino acids, while a fourth truncation removed 110 amino acids, including the terminal most hydrophobic, intramembrane anchoring sequence. Virion egress experiments revealed that all truncated gBs facilitated virion egress from BCBL-1 cells, with the exception of the largest 110-amino-acid truncation, which removed the gB anchoring sequence. Importantly, the gB truncation that removed the entire predicted cytoplasmic domain increased virion egress, suggesting the presence of a egress regulation domain located proximal to the intramembrane sequence within the cytoplasmic domain of gB. All supernatant virions were infectious on 293 cells, indicating that the carboxyl terminus of gB is not essential for either virion egress or virus infectivity.

Efficient long-PCR site-specific mutagenesis of a high GC template.

A long PCR method was developed for the efficient site-specific mutagenesis of herpes simplex virus (HSV-1) DNA fragments with high GC content. In this protocol, a PCR product was partially extended first using a cloned DNA fragment. The final mutagenized fragment was produced after a second extension using another PCR product and final amplification using external primers. The sequential use of two extension reactions increased the predicted frequency of the engineered mutation in the final product to 100%. This method was used to generate a mutated glycoprotein K (gK) gene specified by HSV-1. A recombinant virus that carried the mutated gK gene caused extensive cell fusion of infected cells.

Efficient production of single-stranded DNA as long as 2 kb for sequencing of PCR-amplified DNA.

A modification of the asymmetric PCR method is described, which reliably facilitates sequencing of PCR-amplified DNA. This procedure produces single-stranded DNA fragments as long as two kilobases that are suitable for dideoxy DNA sequencing. First, a PCR for double-stranded DNA is preformed under optimal conditions (double-stranded PCR). Then, a 5-10-microliters fraction of the double-stranded PCR and a single primer are used to generate single-stranded DNA in a separate PCR (single-stranded PCR). The concentration of the single primer are used to generate single-stranded DNA in a separate PCR (single-stranded PCR). The concentration of the single primer is approximately 0.4 microM. Usually 15 to 25 cycles of single-stranded PCR are optimal to produce single-stranded DNA for four to eight sequencing reactions. The single-stranded DNA is purified by centrifugal ultrafiltration and used directly in dideoxy sequencing. This method was employed to produce high-quality single-stranded DNA templates from a variety of organisms for efficient DNA sequencing of PCR-amplified DNA.

An amphipathic alpha-helical synthetic peptide analogue of melittin inhibits herpes simplex virus-1 (HSV-1)-induced cell fusion and virus spread.

The effects of hecate, a 23-amino acid synthetic peptide analogue of melittin, on HSV-1-induced cell fusion and virus multiplication was investigated. Hecate completely inhibited cell fusion induced by HSV-1 syncytial (syn) mutants HSV-1 HFEM (tsB5) and HSV-1 mp (MP) at a concentration of 5.0 microM. Metabolic labeling experiments indicated that hecate did not adversely affect cellular growth and protein synthesis. The synthesis of virus-specified glycoproteins B, C, D, and H was reduced in the presence of hecate; however, the transport of these glycoproteins to the surface of infected cells was not affected. Production of infectious virions for wild-type and syn mutants tsB5 and MP was reduced in the presence of hecate. The effect of hecate on virus titer was dependent on the multiplicity of infection. Virus titers were reduced 2-28-fold at an M.O.I. of 0.1, 3-6-fold at an M.O.I. of 0.5, and 0-2.5-fold at an M.O.I. of 2.5. Direct treatment of semipurified virions with hecate reduced titers by approximately 4-fold for KOS, 2-fold for tsB5, and over 30-fold for MP.

Temperature-sensitive acetylesterase activity of haemagglutinin-esterase specified by respiratory bovine coronaviruses.

Numerous respiratory bovine coronaviruses (RBCV) were isolated recently from nasal swab samples and lung tissues of feedlot cattle with acute respiratory tract disease. These newly emerging RBCV isolates exhibited distinct phenotypic features that differentiated them from enteropathogenic bovine coronaviruses (EBCV). The RBCV strains had a receptor-destroying enzyme function mediated by acetylesterase (AE) activity of the haemagglutinin-esterase (HE) glycoprotein. The HE genes of wild-type EBCV strain LY138 and RBCV strains OK-0514 (OK) and LSU-94LSS-051 (LSU) were cloned, sequenced and transiently expressed in COS-7 cells. The enzymic properties of HE proteins in COS-7 cellular extracts and in purified virus preparations were assayed at room temperature, 37 degrees C and 39 degrees C by two different assays. One assay used p-nitrophenyl acetate (PNPA) as substrate and detected serine-esterase activity; the second assay monitored AE function with bovine submaxillary mucin (BSM) as substrate. The PNPA tests confirmed that HE proteins of EBCV and RBCV were functionally expressed in transfected COS-7 cells. Time-dependent determination of the AE activity of purified RBCV OK and LSU particles showed lower AE activity at 39 degrees C than at 37 degrees C, whereas the purified EBCV LY particles retained full AE activity at both 37 degrees C and 39 degrees C. Transiently expressed RBCV HE exhibited a marked reduction of AE activity after 40 min of assay time at 37 degrees C. In contrast, the AE activity of the transiently expressed EBCV HE remained stable beyond 40 min. The deduced amino-acid sequences of the HE proteins specified by the RBCV strains OK and LSU contained specific amino-acid changes in comparison with the EBCV LY strain, which may be responsible for the observed enzymic differences. These results are consistent with the hypothesis that RBCV strains have evolved to selectivelyreplicate in respiratory tissues and that HE may play a role in this tissue tropism.

Expression of the enhanced green fluorescent protein by herpes simplex virus type 1 (HSV-1) as an in vitro or in vivo marker for virus entry and replication.

Glycoprotein K (gK) is involved in membrane fusion phenomena during infectious virus production and egress and is an important determinant for neurovirulence. To assess better the in vitro and in vivo roles of gK in virus replication, a recombinant virus was constructed expressing an engineered enhanced green fluorescent protein (EGFP) under the control of the human cytomegalovirus immediate early gene promoter (HCMV-IEP) inserted in place of the gK gene. The EGFP gene insertion was confirmed by diagnostic polymerase chain reaction (PCR), and the presence of the EGFP protein was detected by western immunoblot analysis using anti-GFP monoclonal antibody. Fluorescence microscopy revealed that virus infected cells emitted bright fluorescence when examined using filters for fluorescein. Fluorescence emission was detected as early as 4 h post-infection. Fluorescence intensity increased over time and was stable at late times after infection at which point viral plaques continued to emit bright green fluorescence. The amount of fluorescence emitted by virus infected Vero cells was monitored by fluorescence cytometry using a FACS cytometer. At an MOI of 3, all infected cells emitted strong green fluorescence as quantified by cytometry at 48 h post-infection. The deltagK-EGFP expressing recombinant virus will enable the determination of the role of gK in virus entry and egress as well as the role of gK in the molecular pathogenesis of herpes simplex virus type 1 (HSV-1).

Cross-species PCR cloning of gerbil (Meriones unguiculatus) interleukin-2 cDNA and its expression in COS-7 cells.

A cDNA clone of the gerbil interleukin-2 (IL-2) gene was isolated by cross-species PCR cloning, and demonstrated to produce a functional gerbil IL-2 protein when inserted in the eucaryotic expression vector pSV-SPORT1 and transfected into COS-7 monkey cells. The open reading frame codes for a polypeptide of 155 amino acid residues with a molecular weight (MW) of 17,601 which includes a putative signal peptide. The mature gerbil IL-2 is deduced to contain 135 amino acid residues and has a calculated MW of 15,496. Culture supernatant of COS-7 cells transfected with pSV-SPORT1-GIL-2, but not pSV-SPORT1 stimulates the proliferation of the IL-2 dependent murine CTLL-2 cells. Molecular characteristics of gerbil IL-2 have been compared with IL-2 of mouse, rat, human, bovine, ovine and porcine origin. The mature form of gerbil IL-2 is similar in molecular weight to all species except the mouse. A N-glycosylation site present in bovine, ovine and porcine IL-2 respectively, is absent in gerbil. Three Cys residues are conserved in all compared mature IL-2 molecules. In these comparisons, gerbil IL-2 has highest identity with rat IL-2 for both nucleotide and amino acid sequence.

Production of a rabbit anti-cockatiel immunoglobulin G and characterization of its cross-reactivities with immunoglobulin G of other psittacine species.

The purpose of this work was to produce rabbit anti-cockatiel immunoglobulin G (IgG) and compare its cross-reactivity with sera from eight other psittacine birds: Quaker parakeet, budgerigar, green-wing macaw, blue-fronted Amazon parrot, eclectus parrot, African grey parrot, Patagonian conure, Moluccan cockatoo. Cockatiel IgG did not bind to protein A or G; therefore, these proteins could not be used in column chromatography to isolate the IgG. A combination of serum IgG precipitation by ammonium sulfate and yolk IgG extraction from egg was loaded in sodium dodecyl sulfate-polyacrylamide gel upon which the IgG was resolved by electrophoresis. The resolved IgG in sodium dodecyl sulfate-polyacrylamide gel was stained with Coomassie blue, cut, crushed in phosphate-buffered saline, and injected into rabbits. The rabbit anti-cockatiel IgG produced in this way reacted with a single protein in gel immunodiffusion assay with all nine psittacine bird sera but not with those of chicken and ostrich. Immunoelectrophoresis confirmed the cross-reactivity of different psittacine sera with the anti-cockatiel IgG serum but not with ostrich and chicken sera. This antiserum detected antibody responses in sera from cockatiels vaccinated against chlamydial major outer membrane protein in an immunoblot assay.

Active immunization of Japanese quail hens with a recombinant chicken inhibin fusion protein enhances production performance.

The effects of active immunization against inhibin on production performance in female Japanese quail (Coturnix coturnix japonica) were assessed in two separate trials using an MBP-cINA521 fusion protein as an immunogen. The fusion protein, MBP-cINA521, consisted of the bacterial maltose binding protein (MBP) and a truncated form of the mature alpha-subunit of chicken inhibin (cINA521). MBP-cINA1521 was constructed by: 1) excising a 521-bp PstI fragment from a chicken inhibin alpha-subunit cDNA (cINA6; gift of P. A. Johnson), 2) cloning this fragment, which encodes all but the first 11 amino acid residues of the mature alpha-subunit, into the pMal-c2 vector of the MBP fusion expression system, and 3) expressing the fusion protein (MBP-cINA521) from the Escherichia coli and purifying it using affinity chromatography. In each trial, quail were randomly and equally assigned to one of two injection treatments as follows: 1) MBP-cINA521 in Freund's adjuvant, or 2) Freund's adjuvant (vehicular controls; CON). All immunizations were given subcutaneously and Freund's complete and incomplete adjuvant were used for primary and booster injections, respectively. In Trial 1, birds were given a primary challenge of 0.2 mg MBP-cINA521 per bird at 25 d of age, followed by booster immunizations (0.1 mg MBP-cINA521 per bird) at 33, 40, 47, 54 and 61 d of age and every 35 d thereafter. The CON birds received vehicular immunizations at the same time intervals. In Trial 2, birds treated with MBP-cINA521 received a primary challenge of 0.2 mg MBP-cINA521 per bird at 26 d of age, followed by booster immunizations (0.1 mg MBP-cINA521 per bird) using the same schedule as that used in Trial 1, with the exception that no boosters were given after 61 d of age. The CON birds received vehicular immunizations at the same time intervals. Collection of production performance data was initiated coincident with the laying of the first egg in each trial (i.e., beginning at 41 and 44 d of age for Trials 1 and 2, respectively) and continued for 30 1-wk periods of lay. Combined data from Trials 1 and 2 indicated that the mean +/- SE age at first egg lay was markedly decreased (P < 0.005) in MBP-cINA521-treated quail (53.4 +/- 0.9 d of age) when compared to the CON (57.6 +/- 1.3 d of age). Likewise, the mean +/- SE age at 50% egg production was reduced (P < 0.03) in quail immunized against inhibin (65.4 +/- 2.1 d of age) when compared to the CON (77.6 +/- 4.7 d of age). Total hen-day egg production was also higher (P < 0.05, Trial 1; P < 0.01, Trial 2) in MBP-cINA521-treated quail (88.7 +/- 1.4%, Trial 1; 90.1 +/- 1.2%, Trial 2) than in the CON birds (81.9 +/- 2.9%, Trial 1; 73.6 +/- 6.5%, Trial 2). Collectively, these findings provide evidence that inhibin immunoneutralization accelerated puberty and enhanced hen-day egg production during a 30-wk period of egg lay in Japanese quail.

A replication competent HSV-1(McKrae) with a mutation in the amino-terminus of glycoprotein K (gK) is unable to infect mouse trigeminal ganglia after cornea infection.

PURPOSE: To determine the role of the amino terminus of herpes simplex virus-1 (HSV-1) glycoprotein K (gK) in corneal infection, neuroinvasion, and establishment of virus latency in trigeminal ganglia of mice. METHODS: The recombinant virus HSV-1 (McKΔgK31-68) was constructed by engineering gK genes encoding gK lacking 38 amino acids immediately after the gK signal sequence. A rescued virus was also produced. Mouse eyes were scarified and infected with 10(5) plaque forming units (PFU) in each eye. Clinical signs of ocular disease were monitored daily. Thirty days postinfection trigeminal ganglia were collected and processed for quantitative PCR (qPCR) analysis of viral DNA and recovery of infectious virions by cell culture of ganglionic tissues. RESULTS: Deletion of the amino terminus of gK encoded by the McKΔgK31-68 mutant virus did not substantially affect its replication kinetics on African green monkey kidney cells (Vero), while it reduced cell-to-cell spread. McK viral infection of scarified mouse corneas with 10(5) PFU produced severe ocular disease. In contrast, McKΔgK31-68 viral infection with 10(5) PFU produced no significant ocular disease symptoms. All ganglia from mice infected with the McK virus produced high numbers of infectious virions upon explant culture in Vero cells, in agreement with qPCR results detecting high number of HSV-1 viral DNA in ganglionic tissues. In contrast, qPCR failed to detect any viral genomes in McKΔgK31-68 ganglia, while two of the ten ganglia revealed the presence of low numbers of infectious virions upon explant culture in Vero cells. CONCLUSIONS: The results show that the amino terminus of gK is essential for neuroinvasiveness and acute herpes keratitis in the mouse eye model. It is likely that gK is involved in efficient infection of axonal termini, since mouse eye scarification provided a direct access to the high density of neuronal axons innervating mouse corneas.

The herpes simplex virus type 1 (HSV-1) glycoprotein K(gK) is essential for viral corneal spread and neuroinvasiveness.

PURPOSE: To determine the role of herpes simplex virus-1 (HSV-1) glycoprotein K(gK) in corneal infection, neuroinvasion, and virus latency in trigeminal ganglia of mice. METHODS: The recombinant virus HSV-1 (McKrae) Delta gK (MKDelta gK) carrying a deletion of the gK gene was constructed by insertional/deletion mutagenesis and replaced by a gene cassette constitutively expressing the enhanced green fluorescence protein. The gK deletion of the MKDelta gK virus was rescued to produce the wild-type-like virus MKgK. Balb/c mice were infected ocularly with either virus, and the infection pattern in the eye, clinical disease progression, and establishment of viral latency was monitored. RESULTS: Mice infected with the MKDelta gK strain produced in a gK complementing cell line did not exhibit clinical signs when compared with mice infected with the MKgK virus. Direct visualization of infected eyes revealed that the MKDelta gK virus was unable to spread in mouse corneas, while the MKgK rescued virus spread efficiently. Nineteen of 20 scarified and 5/12 unscarified mice infected with the MKgK virus produced infectious virus after coculture with permissive cells, while 0/20 scarified and 0/12 unscarified mice infected with the MKDelta gK virus produced infectious virus. HSV DNA was detected in trigeminal ganglia by PCR in 19/20 scarified and 9/12 unscarified mice inoculated with MKgK, while HSV DNA was detected in the trigeminal ganglia of 3/20 scarified and 0/12 unscarified mice inoculated with MKDelta gK. CONCLUSIONS: The results show that HSV-1 gK is essential for efficient replication and spread in the corneal epithelium and trigeminal ganglia neuroinvasion in MKDelta gK inoculated mice.

Palmitoylation of the cysteine-rich endodomain of the SARS-coronavirus spike glycoprotein is important for spike-mediated cell fusion.

The SARS-coronavirus (SARS-CoV) is the etiological agent of the severe acute respiratory syndrome (SARS). The SARS-CoV spike (S) glycoprotein mediates membrane fusion events during virus entry and virus-induced cell-to-cell fusion. The cytoplasmic portion of the S glycoprotein contains four cysteine-rich amino acid clusters. Individual cysteine clusters were altered via cysteine-to-alanine amino acid replacement and the modified S glycoproteins were tested for their transport to cell-surfaces and ability to cause cell fusion in transient transfection assays. Mutagenesis of the cysteine cluster I, located immediately proximal to the predicted transmembrane, domain did not appreciably reduce cell-surface expression, although S-mediated cell fusion was reduced by more than 50% in comparison to the wild-type S. Similarly, mutagenesis of the cysteine cluster II located adjacent to cluster I reduced S-mediated cell fusion by more than 60% compared to the wild-type S, while cell-surface expression was reduced by less than 20%. Mutagenesis of cysteine clusters III and IV did not appreciably affect S cell-surface expression or S-mediated cell fusion. The wild-type S was palmitoylated as evidenced by the efficient incorporation of (3)H-palmitic acid in wild-type S molecules. S glycoprotein palmitoylation was significantly reduced for mutant glycoproteins having cluster I and II cysteine changes, but was largely unaffected for cysteine cluster III and IV mutants. These results show that the S cytoplasmic domain is palmitoylated and that palmitoylation of the membrane proximal cysteine clusters I and II may be important for S-mediated cell fusion.