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At least 127 records · Page 7Linked to original sources
PubMed2004

Spliced spleen necrosis virus vector RNA is not encapsidated: implications for retroviral replication and vector design.

RNA splicing is a complex event in the retroviral life cycle and can involve multiple steps, as well as cis-acting sequences, to maintain a proper balance of spliced and unspliced viral RNA for translation and encapsidation. The retroviral RNA can be processed by cellular machinery and enables the removal of intronic sequences. We aimed to utilize the removal of a synthetic intron for targeted gene expression. To analyze intron removal and gene expression, we have constructed a novel self-inactivating gene-activating (SIGA) vector for potential universal gene therapy. New vectors for gene therapy are necessary for safe and effective gene delivery in humans. The SIGA vector is derived from spleen necrosis virus (SNV), which is an avian reticuloendotheliosis virus. The vector was designed so that expression of a therapeutic gene is blocked in helper cell lines due to an intervening sequence containing various blocks in transcription and translation. However, after one round of retroviral replication, the intervening sequence should be removed by the cellular machinery and the therapeutic gene will be selectively expressed in target cells. Our studies show that the intervening sequence in SIGA vector RNA is partially spliced. However, spliced vector RNA was not transduced to target cells. Previous studies showed that an infectious SNV vector enabled transduction of spliced RNA. However, yet-undefined differences in infectious and replication-deficient retroviral replication may have an effect on the transduction of spliced RNA. The results of this study present key information on spliced RNA and its encapsidation, as well as data for the construction of a new generation of SNV-derived retroviral vectors.

Animals↗
PubMed2000

Vasoactive intestinal peptide (VIP) prevents neurotoxicity in neuronal cultures: relevance to neuroprotection in Parkinson's disease.

Vasoactive intestinal peptide (VIP) provides neuroprotection against beta-amyloid toxicity in models of Alzheimer's disease. A superactive analogue, stearyl-Nle17-VIP (SNV) is a 100-fold more potent than VIP. In primary neuronal cultures, VIP protective activity may be mediated by femtomolar-acting glial proteins such as activity-dependent neurotrophic factor (ADNF), activity-dependent neuroprotective protein (ADNP), peptide derivatives ADNF-9 (9aa) and NAP (8aa), respectively. It has been hypothesized that beta-amyloid induces oxidative stress leading to neuronal cell death. Similarly, dopamine and its oxidation products were suggested to trigger dopaminergic nigral cell death in Parkinson's disease. We now examined the possible protective effects of VIP against toxicity of dopamine, 6-hydroxydopamine (6-OHDA) and 1-methyl-4-phenylpyridinium ion (MPP+) in neuronal cultures [rat pheochromocytoma (PC12), human neuroblastoma (SH-SY5Y) and rat cerebellar granular cells]. Remarkably low concentrations of VIP (10(-16)-10(-8) M), ADNF-9 and NAP (10(-18)-10(-10) M) protected against dopamine and 6-OHDA toxicity in PC12 and neuroblastoma cells. VIP (10(-11)-10(-9) M) and SNV (10(-13)-10(-11) M), protected cerebellar granule neurons against 6-OHDA. In contrast, VIP did not rescue neurons from death associated with MPP+. Since dopamine toxicity is linked to the red/ ox state of the cellular glutathione, we investigated neuroprotection in cells depleted of reduced glutathione (GSH). Buthionine sulfoximine (BSO), a selective inhibitor of glutathione synthesis, caused a marked reduction in GSH in neuroblastoma cells and their viability decreased by 70-90%. VIP, SNV or NAP (over a wide concentration range) provided significant neuroprotection against BSO toxicity. These results show that the mechanism of neuroprotection by VIP/SNV/NAP may be mediated through raising cellular resistance against oxidative stress. Our data suggest these compounds as potential lead compounds for protective therapies against Parkinson's disease.

1-Methyl-4-phenylpyridinium↗
PubMed2000

VIP and the potent analog, stearyl-Nle(17)-VIP, induce proliferation of keratinocytes.

Vasoactive intestinal polypeptide (VIP) exhibits effects on cell proliferation. Here, VIP, as well as the related peptide, pituitary adenylate cyclase activating peptide (PACAP), promoted human keratinocyte division. Stearyl-Nle(17)-VIP (SNV) was identified as a superior mitogen for the keratinocytic cell line, HaCaT, both in potency (fM-nM concentrations) and efficacy. Reverse transcription-polymerase chain reaction detected in keratinocytes only PACAP mRNA and the relevant type 1 (VPAC(1)R) and type 2 (VPAC(2)R) receptors, while VIP and the third receptor (PAC(1)) transcripts were absent. Upon serum deprivation of HaCaT, the VPAC(1)R mRNA was apparently increased, while the VPAC(2)R transcript remained constant. Incubation of HaCaT with VIP or SNV increased nitric oxide and cGMP formation. In contrast to VIP, SNV did not augment cAMP. Thus, the paracrine VIP, and autocrine PACAP, related pathways leading to keratinocyte proliferation may involve VPAC(1)R/VPAC(2)R and nitric oxide/cGMP production.

Cell Division↗
PubMed2001

Vasoactive intestinal peptide and related molecules induce nitrite accumulation in the extracellular milieu of rat cerebral cortical cultures.

Nanomolar concentrations of vasoactive intestinal peptide (VIP), picomolar concentrations of stearyl-norleucine17-VIP (SNV) and femtomolar concentrations of NAPVSIPQ (NAP), an 8-amino-acid peptide derived from the VIP-responsive activity-dependent neuroprotective protein, provide broad neuroprotection. In rat cerebral cortical cultures, 10(-16)-10(-7) M NAP increased intracellular cyclic guanosine monophosphate (cGMP) (2.5-4-fold) and 10(-10) M NAP increased extracellular nitric oxide (NO) by 60%. In the same culture system, VIP and SNV (at micromolar concentrations) increased extracellular NO by 45-55%. The NAP dose required for cGMP increases correlated with the dose providing neuroprotection. However, the concentrations of NAP, SNV and VIP affecting NO production did not match the neuro-protective doses. Thus, NO may mediate part of the cell-cell interaction and natural maintenance activity of VIP/SNV/NAP, while cGMP may mediate neuroprotection.

Animals↗
PubMed1982

Encapsidation sequences for spleen necrosis virus, an avian retrovirus, are between the 5' long terminal repeat and the start of the gag gene.

The minimal cis-acting sequences outside the long terminal repeat (LTR) required for formation of an infectious retrovirus cloning vector were determined with recombinants of spleen necrosis virus (SNV) DNA and herpes simplex virus type 1 thymidine kinase gene. The 3' end of SNV DNA was removed to within 40 base pairs (bp) from the 3' LTR with only a 2-fold effect on the recovery of infectious recombinant virus. However, when the 5' end of SNV DNA was removed to within 100 bp from the 5' LTR, infectious recombinant virus was not recovered. Deletion mutants constructed around this latter region showed that nucleotides between 100 and 285 bp from the 5' LTR are necessary for encapsidation of genomic viral RNA. We call this region required for encapsidation E.

Animals↗
PubMed1986

Nondefective spleen necrosis virus-derived vectors define the upper size limit for packaging reticuloendotheliosis viruses.

We constructed a nondefective retrovirus vector based on spleen necrosis virus (SNV), a replication-competent reticuloendotheliosis virus. We introduced different DNA sequences into this vector and studied the ability of the resulting viruses to replicate in chicken embryo fibroblasts. The replication efficiency of SNV-derived viruses decreased with increasing virus size. Viruses larger than 9.4 kilobases (kb) were rapidly overgrown by replication-competent deletion mutants. The size restriction for the efficient replication of nondefective SNV-derived viruses prevented the production of viruses larger than 10.0 kb. Analysis of the kinetics of virus particle release indicated that the size restriction occurred during virus encapsidation.

Capsid↗
PubMed1999

High levels of viremia in patients with the Hantavirus pulmonary syndrome.

Hantavirus pulmonary syndrome (HPS) is a rare but acute fulminant disease caused by Sin Nombre virus (SNV). To understand the role of the viral load in the pathogenesis of HPS, the load of virus in the blood of patients with HPS was measured. A quantitative reverse transcription-polymerase chain reaction assay was developed for SNV, because SNV is difficult to grow in cell culture. Thirty-eight samples from 26 patients with HPS were analyzed. Twenty of the 26 initial samples were positive for viral RNA (7 of 9 samples were obtained from patients with fatal cases, and 13 of 17 were obtained from survivors). Mean viral RNA copy numbers were 106.1+/-1.4/mL in positive cases (106.7+/-1.4/mL in fatal cases, 105.8+/-1.3/mL in survivors) and were correlated with peak hematocrit (P<.05) and with the lowest platelet count (P=.05). In 8 survivors who had serial samples obtained, viral RNA copy numbers decreased promptly after resolution of fever.

Blotting, Southern↗
PubMed1998

Tissue distribution of hantavirus antigen in naturally infected humans and deer mice.

The Sin Nombre virus (SNV) is the etiologic agent of hantavirus pulmonary syndrome in humans but does not cause disease in chronically infected deer mice (Peromyscus maniculatus), the natural host. In this study, murine antiserum raised against recombinant SNV nucleocapsid protein was utilized to localize viral antigen immunohistochemically in tissues from both humans (n = 20; 11 positive, 9 negative) and deer mice (n = 6; 4 positive, 2 negative). Viral infection status was confirmed by Western blot or reverse transcriptase-polymerase chain reaction. SNV antigen was detected in pulmonary and cardiac endothelium in both species, but positive cells in deer mice were rare. Other deer mouse tissues, including kidney, were negative; in contrast, vascular elements of several tissues from infected humans were positive, with strong staining of renal endothelium. The paucity of positive cells in chronically infected mice suggests a low virus burden compared with that of acutely infected humans.

Animals↗
PubMed1985

Dietary and hormonal interrelationships among vegetarian Seventh-Day Adventists and nonvegetarian men.

The relationship between dietary nutrients and plasma testosterone, 5 alpha-dihydrotestosterone, estradiol-17 beta, luteinizing hormone, and prolactin levels was investigated in 12 Seventh-Day Adventist (SDA) vegetarian (SV), 10 SDA nonvegetarian (SNV), and 8 non-SDA nonvegetarian (NV) men. Fasting blood samples and 3-day dietary intake information were obtained from each subject. The SV subjects consumed significantly more crude and dietary fiber than the SNV and NV subjects, respectively. Plasma levels of testosterone and estradiol-17 beta were significantly lower in the SV than in the omnivores. Additionally, the plasma levels of testosterone and estradiol-17 beta of the combined groups (SV, SNV, and NV) revealed a significant negative relationship with their crude and dietary fiber intakes. These subjects hormonal milieu was related to specific dietary constituents, possibly leading to a decreased plasma concentration of androgen and estrogen in vegetarians. Implications include the possible modification of prostate cancer risk through dietary intervention.

Body Height↗
PubMed1996

Evidence against infection with hantaviruses among forest and park workers in the southwestern United States.

To determine if individuals with extensive exposure to rodent habitats were infected with Sin Nombre hantavirus (SNV), we evaluated forest and park service personnel from the region of endemicity in the southwestern United States. Information about work and recreational activities, including exposure to rodents and a history of recent illnesses, was obtained via a standardized questionnaire. Serum specimens were also collected. Of 140 participating workers, 84 (60%) were primarily engaged in outdoor work activities, 14 (10%) were office-based supervisors, and 42 (30%) were office workers. Of the 140 employees, 89 (64%) reported repeated exposures to rodents, rodent nests, and/or rodent droppings; 22 (16%) reported trapping or handling wild rodents. Hantavirus antibodies were not detected in any park employee. These data suggest that transmission of SNV is a rare event even among persons in the southwestern United States who have a high level of exposure to this virus. Although park employees and visitors in this region are at low risk of infection with SNV, these persons should continue to use recommended measures for risk reduction.

Animals↗
PubMed1996

Epidemiologic linkage of rodent and human hantavirus genomic sequences in case investigations of hantavirus pulmonary syndrome.

Sin Nombre virus (SNV) causes the zoonotic disease hantavirus pulmonary syndrome (HPS). Its mechanisms of transmission from rodent to human are poorly understood. It is possible that specific genetic signature sequences could be used to determine the probable site of each case-patient's exposure. Environmental assessments suggested 12 possible sites of rodent exposure for 6 HPS patients. Rodents were captured at 11 of the 12 sites and screened for SNV infection within 2 weeks of the patient's diagnosis. Viral sequences amplified from tissues of rodents at each site were compared with those from case-patients' tissues. Rodents bearing viruses with genetic sequence identity to case-patients' viruses across 2 genomic segments were identified in 4 investigations but never at >1 site. Indoor exposures to rodents were especially common at implicated sites. By distinguishing among multiple possible sites of exposure, viral genotyping studies can enhance understanding of the conditions associated with infection by SNV.

Animals↗
PubMed1997

Assessment of occupational risk for hantavirus infection in Arizona and New Mexico.

Differentiating occupational exposure from other potential domestic or recreational exposure(s) for Sin Nombre virus (SNV) infection is an epidemiologic challenge. Interviews on work-related activities were conducted, and serum specimens were obtained from 494 workers in Arizona and New Mexico. These workers may have been exposed to rodents and rodent excreta at work, but their primary occupation did not require rodent contact (National Park Service [n = 193]; Navajo Agricultural Product Industry [n = 65], utility companies [n = 169] and plumbing and heating contractors [n = 67]. Within each occupational group (farm workers [n = 57], laborers [n = 20], professionals [n = 70], repairers [n = 211], service industry workers [n = 83], and technicians [n = 53], the majority of workers reported working in areas that had rodent droppings (range, 75 to 95%); 70% of laborers and 64% of service industry workers reported handling rodents. More than 60% of workers in each group, except technicians, reported reopening and cleaning or working in closed spaces. Approximately 90% of laborers, repairers, and farm workers reported hand-plowing. Although the risk for occupationally related SNV infection appears to be low, workers frequently performed risk activities associated with hantavirus pulmonary syndrome (HPS). All workers were seronegative for SNV by enzyme-linked immunoassay or Western blot testing. These findings, the known occupational exposure of some HPS cases, and the high HPS case-fatality rate (52%) support the need for recommendations to reduce human contact with rodents in the workplace. Increased understanding of hantavirus transmission to humans will help focus future recommendations to minimize human exposures effectively.

Adolescent↗
PubMed2002

Characterization of HLA-A2.1-restricted epitopes, conserved in both Hantaan and Sin Nombre viruses, in Hantaan virus-infected patients.

Nine different CTL epitopes, conserved in both Hantaan virus (HTNV) and Sin Nombre virus (SNV), were selected for study. The binding affinity of each peptide with HLA-A2.1 molecules in vitro was determined and antigen-specific responses from seven donors who had a previous field infection with HTNV were examined. Although the strength or frequency of CTL activity showed different patterns in the seven patients, five of seven patients showed significant activity against at least one or more epitope peptides. In particular, the peptide ILQDMRNTI (HTNV, aa 334-342; SNV, aa 333-341), which elicited CTL activity in five patients, was shown to be specifically HLA-A2.1-restricted in partially cloned CD8+ T cells and also induced activated and effector CD8+ T cell-producing T cytotoxic (Tc) type 1 cytokines, such as IL-2 and IFN-gamma. The results suggest that this epitope would serve as a useful component for the intervention of both HTNV and SNV infection.

Epitopes, T-Lymphocyte↗
PubMed2002

Identification of R-peptides in envelope proteins of C-type retroviruses.

Activation of the murine leukaemia virus (MLV) envelope protein (Env) requires proteolytic cleavage of the R-peptide, a 16 amino acid C-terminal part of the cytoplasmic tail (C-tail) of Env. This paper demonstrates the presence of R-peptides in Env proteins of C-type retroviruses of simian, avian and porcine origin. Sequence alignment with the MLV C-tail led to the identification of a conserved hydrophobic protease cleavage motif located in the centre of retroviral Env protein C-tails. Expression of Env proteins, truncated at the predicted cleavage sites, of spleen necrosis virus (SNV), gibbon ape leukaemia virus and porcine endogenous retroviruses resulted in cell-cell fusion as monitored by microscopy and reporter gene fusion assays. Western blot analysis of MLV particles pseudotyped with the SNV Env protein demonstrated proteolytic cleavage of the SNV R-peptide by the MLV protease. Our data suggest that activation of membrane fusion by R-peptide cleavage is a common mode in C-type retroviruses.

Amino Acid Sequence↗
PubMed1979

Pheasant virus DNA polymerase is related to avian leukosis virus DNA polymerase at the active site.

The DNA polymerase from Amherst pheasant virus (APV), a member of the pheasant virus species of retroviruses, was compared to the DNA polymerases of avian leukosis viruses (ALV) and a reticuloendotheliosis virus (spleen necrosis virus (SNV)). Immunoglobulin inhibition tests and competition immunoassays showed that APV and ALV DNA polymerases are closely related at their active sites. The determinants common to their active sites are not shared by SNV DNA polymerase. Bu using a species-specific radioimmunoassay, it was shown that both APV and SNV DNA polymerases are grossly different from ALV DNA polymerase. The specificity of the relationship of the active sites of APV and ALV DNA polymerases was confirmed by a heterologous radioimmunoassay. Our data indicate that pheasant viruses are evolutionarily linked to ALV.

Avian Leukosis Virus↗
PubMed1994

Lower mutation rate of bovine leukemia virus relative to that of spleen necrosis virus.

Genetic variation of the more complex retroviruses in the human T-cell leukemia virus/bovine leukemia virus (HTLV/BLV) group is less than in some other retroviral genera. To test whether reverse transcription of HTLV/BLV group members is less error prone than that of members of other groups, we developed an assay for detecting forward mutations in BLV, similar to that developed for the simpler spleen necrosis virus (SNV). We used this system to study the rates and types of mutations that occur during a single replication cycle. We found that BLV reverse transcription is approximately two and one-half times less error prone than SNV reverse transcription (4.8 x 10(-6) versus 1.2 x 10(-5) mutation per bp per cycle, respectively). The relative numbers of all types of observed mutations (that is, base pair substitutions, frameshifts, deletions, and deletions with insertions) were similar for BLV and SNV.

Animals↗
PubMed1994

Improved self-inactivating retroviral vectors derived from spleen necrosis virus.

Self-inactivating (SIN) retroviral vectors contain a deletion spanning most of the right long terminal repeat's (LTR's) U3 region. Reverse transcription copies this deletion to both LTRs. As a result, there is no transcription from the 5' LTR, preventing further replication. Many previously developed SIN vectors, however, had reduced titers or were genetically unstable. Earlier, we reported that certain SIN vectors derived from spleen necrosis virus (SNV) experienced reconstitution of the U3-deleted LTR at high frequencies. This reconstitution occurred on the DNA level and appeared to be dependent on defined vector sequences. To study this phenomenon in more detail, we developed an almost completely U3-free retroviral vector. The promoter and enhancer of the left LTR were replaced with those of the cytomegalovirus immediate-early genes. This promoter swap did not impair the level of transcription or alter its start site. Our data indicate that SNV contains a strong initiator which resembles that of human immunodeficiency virus. We show that the vectors replicate with efficiencies similar to those of vectors possessing two wild-type LTRs. U3-deleted vectors carrying the hygromycin B phosphotransferase gene did not observably undergo LTR reconstitution, even when replicated in helper cells containing SNV-LTR sequences. However, vectors carrying the neomycin resistance gene did undergo LTR reconstitution with the use of homologous helper cell LTR sequences as template. This supports our earlier finding that sequences within the neomycin resistance gene can trigger recombination.

Base Sequence↗
PubMed1998

Deoxyribonucleoside triphosphate pool imbalances in vivo are associated with an increased retroviral mutation rate.

Deoxyribonucleoside triphosphate (dNTP) pool imbalances are associated with an increase in the rate of misincorporation and hypermutation during in vitro reverse transcription reactions. However, the effects of in vivo dNTP pool imbalances on the accuracy of reverse transcription are unknown. We sought to determine the effects of in vivo dNTP pool imbalances on retroviral mutation rates and to test our hypothesis that 3'-azido-3'-deoxythymidine (AZT) increases the retroviral mutation rates through induction of dNTP pool imbalances. D17 cells were treated with thymidine, hydroxyurea (HU), or AZT, and the effects on in vivo dNTP pools were measured. Thymidine and HU treatments induced significant dNTP pool imbalances. In contrast, AZT treatment had very little effect on the dNTP pools. The effects of in vivo dNTP pool imbalances induced by thymidine and HU treatments on the retroviral mutation rates were also determined. Spleen necrosis virus (SNV)-based and murine leukemia virus (MLV)-based retroviral vectors that expressed the lacZ mutant reporter gene were used. The frequencies of inactivating mutations introduced in the lacZ gene in a single replication cycle provided a measure of the retroviral mutation rates. Treatment of D17 target cells with 500 microM thymidine increased the SNV and MLV mutant frequencies 4.7- and 4-fold, respectively. Treatment of D17 target cells with 2 mM HU increased the SNV and MLV mutant frequencies 2.1- and 2.7-fold, respectively. These results demonstrate that dNTP pool imbalances are associated with an increase in the in vivo retroviral mutation rates, but AZT treatment results in an increase in the retroviral mutation rates by a mechanism not involving alterations in dNTP pools.

Cell Line↗