Vaccinia virus recombinant expressing herpes simplex virus type 1 glycoprotein D prevents latent herpes in mice

In humans, herpes simplex virus causes a primary infection and then often a latent ganglionic infection that persists for life. Because these latent infections can recur periodically, vaccines are needed that can protect against both primary and latent herpes simplex infections. Infectious vaccinia virus recombinants that contain the herpes simplex virus type 1 (HSV-1) glycoprotein D gene under control of defined early or late vaccinia virus promoters were constructed. Tissue culture cells infected with these recombinant viruses synthesized a glycosylated protein that had the same mass (60,000 daltons) as the glycoprotein D produced by HSV-1. Immunization of mice with one of these recombinant viruses by intradermal, subcutaneous, or intraperitoneal routes resulted in the production of antibodies that neutralized HSV-1 and protected the mice against subsequent lethal challenge with HSV-1 or HSV-2. Immunization with the recombinant virus also protected the majority of the mice against the development of a latent HSV-1 infection of the trigeminal ganglia. This is the first demonstration that a genetically engineered vaccine can prevent the development of latency.     

Rapid identification of nonessential genes of herpes simplex virus type 1 by Tn5 mutagenesis

The large genome of herpes simplex virus type of (HSV-1) encodes at least 80 polypeptides, the majority of which have no recognized function. A subgroup of these gene products appears to be nonessential for virus replication in cell culture, but contributes to the complex life cycle of the virus in the host. To identify such functions, a simple insertional mutagenesis method has been used for selective inactivation of individual HSV-1 genes. The bacterial transposon Tn5 was allowed to insert randomly into cloned restriction fragments representing the entire short unique (US) region of the HSV-1 genome. Of the 12 open reading frames that were mutagenized with Tn5, mutant derivatives of US2, US4, and US5 were recombined into the virus. These three genes proved to be nonessential for HSV-1 replication in Vero (African Green monkey kidney) cells and the US4 gene appeared to be involved in viral pathogenesis in the central nervous system of mice. This rapid mutagenesis procedure should prove useful in exploring the entire HSV-1 genome as well as the genomes of other complex animal viruses.     

Protection from genital herpes simplex virus type 2 infection by vaccination with cloned type 1 glycoprotein D

Guinea pigs were vaccinated with truncated herpes simplex virus type-1 (HSV-1) glycoprotein D produced in the genetically engineered mammalian cell line gD10.2. Vaccinated animals formed antibodies that neutralized both HSV-1 and herpes simplex virus type 2 (HSV-2) in an in vitro neutralization assay. Vaccinated animals were challenged with HSV-2 by intravaginal infection. Animals that received the immunogen in Freund's complete adjuvant were completely protected from the clinical manifestations of genital HSV-2 infection. Animals that received the immunogen incorporated in alum adjuvants were partly protected from clinical disease; the infections that did develop were significantly less severe than those that occurred in control animals injected with adjuvant alone. The results demonstrate that immunization with a purified viral protein can provide significant protection against primary genital infection by HSV-2 in guinea pigs.     

Inflammation and herpes simplex virus: release of a chemotaxis-generating factor from infected cells

Infection of primary rabbit kidney cells with herpes simplex virus leads to the release of a cell factor or factors that upon incubation with serum results in the cleavage of the fifth component, C5, of complement. The product of this cleavage, C5a, is chemotactic for polymorphonuclear leukocytes and could be responsible for the accumulation of these cells at the site of herpetic lesions.     

Crystal structure of glycoprotein B from herpes simplex virus 1

Glycoprotein B (gB) is the most conserved component of the complex cell-entry machinery of herpes viruses. A crystal structure of the gB ectodomain from herpes simplex virus type 1 reveals a multidomain trimer with unexpected homology to glycoprotein G from vesicular stomatitis virus (VSV G). An alpha-helical coiled-coil core relates gB to class I viral membrane fusion glycoproteins; two extended beta hairpins with hydrophobic tips, homologous to fusion peptides in VSV G, relate gB to class II fusion proteins. Members of both classes accomplish fusion through a large-scale conformational change, triggered by a signal from a receptor-binding component. The domain connectivity within a gB monomer would permit such a rearrangement, including long-range translocations linked to viral and cellular membranes.     

Mapping of herpes simplex virus-1 neurovirulence to gamma 134.5, a gene nonessential for growth in culture

The gene designated gamma 134.5 maps in the inverted repeats flanking the long unique sequence of herpes simplex virus-1 (HSV-1) DNA, and therefore it is present in two copies per genome. This gene is not essential for viral growth in cell culture. Four recombinant viruses were genetically engineered to test the function of this gene. These were (i) a virus from which both copies of the gene were deleted, (ii) a virus containing a stop codon in both copies of the gene, (iii) a virus containing after the first codon an insert encoding a 16-amino acid epitope known to react with a specific monoclonal antibody, and (iv) a virus in which the deleted sequences were restored. The viruses from which the gene was deleted or which carried stop codons were avirulent on intracerebral inoculation of mice. The virus with the gene tagged by the sequence encoding the epitope was moderately virulent, whereas the restored virus reacquired the phenotype of the parent virus. Significant amounts of virus were recovered only from brains of animals inoculated with virulent viruses. Inasmuch as the product of the gamma 134.5 gene extended the host range of the virus by enabling it to replicate and destroy brain cells, it is a viral neurovirulence factor.     

Latent herpes simplex virus in spinal ganglia of mice

Herpes simplex virus establishes a persistent, latent infection in spinal ganglia after mice have recovered from posterior paralysis. Infectious virus is replicated when these ganglia are explanted and maintained as organ cultures in vitro.     

Three-dimensional structure of herpes simplex virus from cryo-electron tomography

Herpes simplex virus, a DNA virus of high complexity, consists of a nucleocapsid surrounded by the tegument-a protein compartment-and the envelope. The latter components, essential for infectivity, are pleiomorphic. Visualized in cryo-electron tomograms of isolated virions, the tegument was seen to form an asymmetric cap: On one side, the capsid closely approached the envelope; on the other side, they were separated by approximately 35 nanometers of tegument. The tegument substructure was particulate, with some short actin-like filaments. The envelope contained 600 to 750 glycoprotein spikes that varied in length, spacing, and in the angles at which they emerge from the membrane. Their distribution was nonrandom, suggesting functional clustering.     

Rapid diagnosis of herpes simplex virus infections with fluorescent antibody

A fluorescent microscopy technique is described which may prove useful in differentiating clinically similar lesions into lesions of herpes simplex virus etiology and nonherpetic lesions. Herpes simplex virus was isolated from the specimens which yielded positive fluorescence, and no virus was isolated from the specimens which yielded no fluorescence.     

Latency of herpes simplex virus in absence of neutralizing antibody: model for reactivation

Mice inoculated with herpes simplex virus (type 1) by the lip or corneal route and then passively immunized with rabbit antibody to herpes simplex virus developed a latent infection in the trigeminal ganglia within 96 hours. Neutralizing antibody to herpes simplex virus was cleared from the circulation and could not be detected in most of these mice after 2 months. Examination of ganglia from the antibody-negative mice revealed latent virus in over 90 percent of the animals, indicating that serum neutralizing antibody is not necessary to maintain the latent state. When the lips or corneas of these mice were traumatized, viral reactivation occurred in up to 90 percent of the mice, as demonstrated by the appearance of neutralizing antibody. This study provides a model for identifying factors that trigger viral reactivation.     

Rat transforming growth factor type 1: structure and relation to epidermal growth factor

The complete amino acid sequence of rat transforming growth factor type 1 has been determined. This growth factor, obtained from retrovirus-transformed fibroblasts, is structurally and functionally related to mouse epidermal growth factor and human urogastrone. Production of this polypeptide by various neoplastic cells might contribute to the continued expression of the transformed phenotype.     

胰岛素样生长因子-1的放射免疫测定方法的建立

胰岛素样生长因子－１（ＩＧＦ－１）分别与两种载体蛋白——牛血清白蛋白（ＢＳＡ）和卵清蛋白（ＯＡ）用戊二醛连接,经紫外分光光度仪扫描确定偶联成功。以ＩＧＦ－１－ＢＳＡ作为免疫原,皮内多点注射免疫青紫兰兔,制备ＩＧＦ－１抗体,经ＥＬＩＳＡ法测定,血清效价为１∶２０００,放免工作浓度为１∶１５０００。采用氯胺－Ｔ标记ＩＧＦ－１,用酸醇抽提法处理样品,去除ＩＧＦ－１结合蛋白。所建立的ＩＧＦ－１放免标准曲线,其最小检测量为０．０６ｎｇ／ｍＬ,批内误差为４．８％,批间误差为８．２％（ｎ＝１０）。     

Latent ganglionic infection with herpes simplex virus types 1 and 2: viral reactivation in vivo after neurectomy

Inoculation of the cornea, lip, or footpad of mice with herpes simplex virus type 1 resulted in a latent infection of the local sensory ganglia. Inoculation of the vagina and cervix with herpes simplex virus type 2, as well as type 1, also induced a latent ganglionic infection. With the use of sciatic nerve section as a stimulus, a reproducible model of viral reactivation in vivo was established.     

5-Azacytidine-induced reactivation of a herpes simplex thymidine kinase gene

Mouse cells transformed with herpes simplex virus and containing the viral thymidine kinase (TK) gene in an inactive state were treated with 5-azacytidine. The result was the reexpression of the viral TK gene. Two days of exposure to 5-azacytidine followed by 2 days of expression time was sufficient for maximal induction of the TK+ phenotype. The induction of TK expression by 5-azacytidine was concentration-dependent, with maximal induction at 10 micromoles per liter. 5-Azacytidine also inhibited the decay of TK expression in TK+ transformants removed from selective conditions. Analysis of the methylation patterns of the viral TK gene with restriction endonucleases Hpa II and Msp I showed the active gene to be unmethylated, the inactive gene methylated, and the 5-azacytidine-induced gene unmethylated.     

Purification and complementary DNA cloning of a receptor for basic fibroblast growth factor

Basic fibroblast growth factor (bFGF) participates in many processes including early developmental events, angiogenesis, wound healing, and maintenance of neuronal cell viability. A 130-kilodalton protein was isolated on the basis of its ability to specifically bind to bFGF. A complementary DNA clone was isolated with an oligonucleotide probe corresponding to determined amino acid sequences of tryptic peptide fragments of the purified protein. The putative bFGF receptor encoded by this complementary DNA is a transmembrane protein that contains three extracellular immunoglobulin-like domains, an unusual acidic region, and an intracellular tyrosine kinase domain. These domains are arranged in a pattern that is different from that of any growth factor receptor described.     

CRACM1 is a plasma membrane protein essential for store-operated Ca2+ entry

Store-operated Ca2+ entry is mediated by Ca2+ release-activated Ca2+ (CRAC) channels following Ca2+ release from intracellular stores. We performed a genome-wide RNA interference (RNAi) screen in Drosophila cells to identify proteins that inhibit store-operated Ca2+ influx. A secondary patch-clamp screen identified CRACM1 and CRACM2 (CRAC modulators 1 and 2) as modulators of Drosophila CRAC currents. We characterized the human ortholog of CRACM1, a plasma membrane-resident protein encoded by gene FLJ14466. Although overexpression of CRACM1 did not affect CRAC currents, RNAi-mediated knockdown disrupted its activation. CRACM1 could be the CRAC channel itself, a subunit of it, or a component of the CRAC signaling machinery.     

Protection of mice against fatal herpes simplex type 2 infection by liposomes containing muramyl tripeptide

Intravenous administration of liposomes containing muramyl tripeptide phosphatidylethanolamine, a lipophilic derivative of muramyl dipeptide that activates macrophages to a cytolytic state in situ, significantly protected mice against lethal challenge with herpes simplex virus type 2. These findings suggest that the systemic activation of macrophages by liposomes containing an immunomodulator can lead to prophylaxis of severe infections caused by herpesviruses.     

Vascular endothelial growth factor is a secreted angiogenic mitogen

Vascular endothelial growth factor (VEGF) was purified from media conditioned by bovine pituitary folliculostellate cells (FC). VEGF is a heparin-binding growth factor specific for vascular endothelial cells that is able to induce angiogenesis in vivo. Complementary DNA clones for bovine and human VEGF were isolated from cDNA libraries prepared from FC and HL60 leukemia cells, respectively. These cDNAs encode hydrophilic proteins with sequences related to those of the A and B chains of platelet-derived growth factor. DNA sequencing suggests the existence of several molecular species of VEGF. VEGFs are secreted proteins, in contrast to other endothelial cell mitogens such as acidic or basic fibroblast growth factors and platelet-derived endothelial cell growth factor. Human 293 cells transfected with an expression vector containing a bovine or human VEGF cDNA insert secrete an endothelial cell mitogen that behaves like native VEGF.     

The trk proto-oncogene product: a signal transducing receptor for nerve growth factor.

The trk proto-oncogene encodes a 140-kilodalton, membrane-spanning protein tyrosine kinase (p140prototrk) that is expressed only in neural tissues. Nerve growth factor (NGF) stimulates phosphorylation of p140prototrk in neural cell lines and in embryonic dorsal root ganglia. Affinity cross-linking and equilibrium binding experiments with 125I-labeled NGF indicate that p140prototrk binds NGF specifically in cultured cells with a dissociation constant of 10(-9) molar. The identification of p140prototrk as an NGF receptor indicates that this protein participates in the primary signal transduction mechanism of NGF.