Production Of Cell-free Virus Research Articles

A Novel Peptide Derived from the Fusion Protein Heptad Repeat Inhibits Replication of Subacute Sclerosing Panencephalitis Virus In Vitro and In Vivo.

Subacute sclerosing panencephalitis (SSPE) is a persistent, progressive, and fatal degenerative disease resulting from persistent measles virus (MV) infection of the central nervous system. Most drugs used to treat SSPE have been reported to have limited effects. Therefore, novel therapeutic strategies are urgently required. The SSPE virus, a variant MV strain, differs virologically from wild-type MV strain. One characteristic of the SSPE virus is its defective production of cell-free virus, which leaves cell-to-cell infection as the major mechanism of viral dissemination. The fusion protein plays an essential role in this cell-to-cell spread. It contains two critical heptad repeat regions that form a six-helix bundle in the trimer similar to most viral fusion proteins. In the case of human immunodeficiency virus type-1 (HIV-1), a synthetic peptide derived from the heptad repeat region of the fusion protein enfuvirtide inhibits viral replication and is clinically approved as an anti-HIV-1 agent. The heptad repeat regions of HIV-1 are structurally and functionally similar to those of the MV fusion protein. We therefore designed novel peptides derived from the fusion protein heptad repeat region of the MV and examined their effects on the measles and SSPE virus replication in vitro and in vivo. Some of these synthetic novel peptides demonstrated high antiviral activity against both the measles (Edmonston strain) and SSPE (Yamagata-1 strain) viruses at nanomolar concentrations with no cytotoxicity in vitro. In particular, intracranial administration of one of the synthetic peptides increased the survival rate from 0% to 67% in an SSPE virus-infected nude mouse model.

Trehalose, an mTOR-Independent Inducer of Autophagy, Inhibits Human Cytomegalovirus Infection in Multiple Cell Types.

Human cytomegalovirus (HCMV) is the major viral cause of birth defects and a serious problem in immunocompromised individuals and has been associated with atherosclerosis. Previous studies have shown that the induction of autophagy can inhibit the replication of several different types of DNA and RNA viruses. The goal of the work presented here was to determine whether constitutive activation of autophagy would also block replication of HCMV. Most prior studies have used agents that induce autophagy via inhibition of the mTOR pathway. However, since HCMV infection alters the sensitivity of mTOR kinase-containing complexes to inhibitors, we sought an alternative method of inducing autophagy. We chose to use trehalose, a nontoxic naturally occurring disaccharide that is found in plants, insects, microorganisms, and invertebrates but not in mammals and that induces autophagy by an mTOR-independent mechanism. Given the many different cell targets of HCMV, we proceeded to determine whether trehalose would inhibit HCMV infection in human fibroblasts, aortic artery endothelial cells, and neural cells derived from human embryonic stem cells. We found that in all of these cell types, trehalose induces autophagy and inhibits HCMV gene expression and production of cell-free virus. Treatment of HCMV-infected neural cells with trehalose also inhibited production of cell-associated virus and partially blocked the reduction in neurite growth and cytomegaly. These results suggest that activation of autophagy by the natural sugar trehalose or other safe mTOR-independent agents might provide a novel therapeutic approach for treating HCMV disease. HCMV infects multiple cell types in vivo, establishes lifelong persistence in the host, and can cause serious health problems for fetuses and immunocompromised individuals. HCMV, like all other persistent pathogens, has to finely tune its interplay with the host cellular machinery to replicate efficiently and evade detection by the immune system. In this study, we investigated whether modulation of autophagy, a host pathway necessary for the recycling of nutrients and removal of protein aggregates, misfolded proteins, and pathogens, could be used to target HCMV. We found that autophagy could be significantly increased by treatment with the nontoxic, natural disaccharide trehalose. Importantly, trehalose had a profound inhibitory effect on viral gene expression and strongly impaired viral spread. These data constitute a proof-of-concept for the use of natural products targeting host pathways rather than the virus itself, thus reducing the risk of the development of resistance to treatment.

HIV-1 Cell-Free and Cell-to-Cell Infections Are Differentially Regulated by Distinct Determinants in the Env gp41 Cytoplasmic Tail.

The HIV-1 envelope (Env) glycoprotein mediates viral entry during both cell-free and cell-to-cell infection of CD4(+) T cells. The highly conserved long cytoplasmic tail (CT) of Env is required in a cell type-dependent manner for optimal infectivity of cell-free virus. To probe the role of the CT in cell-to-cell infection, we tested a panel of mutations in the CT region that maintain or attenuate cell-free infection to investigate whether the functions of the CT are conserved during cell-free and cell-to-cell infection. The mutations tested included truncations of structural motifs in the gp41 CT and two point mutations in lentiviral lytic peptide 3 (LLP-3) previously described as disrupting the infectivity of cell-free virus. We found that small truncations of 28 to 43 amino acids (aa) or two LLP-3 point mutations, YW_SL and LL_RQ, severely impaired single-round cell-free infectivity 10-fold or more relative to wild-type full-length CT. These mutants showed a modest 2-fold reduction in cell-to-cell infection assays. Conversely, large truncations of 93 to 124 aa severely impaired cell-to-cell infectivity 20-fold or more while resulting in a 50% increase in infectivity of cell-free viral particles when produced in 293T cells. Intermediate truncations of 46 to 90 aa showed profound impairment of both modes of infection. Our results show that the abilities of Env to support cell-free and cell-to-cell infection are genetically distinct. These differences are cell type dependent for large-CT-truncation mutants. Additionally, point mutants in LLP-3 can maintain multiround propagation from cell-to-cell in primary CD4(+) T cells. The functions of HIV Env gp41 CT remain poorly understood despite being widely studied in the context of cell-free infection. We have identified domains of the gp41 CT responsible for striking selective deficiencies in either cell-free or cell-to-cell infectivity. These differences may reflect a different intrinsic regulatory influence of the CT on cell-associated versus particle-associated Env or differential interaction with host or viral proteins. Our findings provide novel insight into the key regulatory potential of the gp41 CT in cell-free and cell-to-cell HIV-1 infection, particularly for short-truncation mutants of ≤43 amino acids or mutants with point mutations in the LLP-3 helical domain of the CT, which are able to propagate via cell-to-cell infection in the absence of infectious cell-free virus production. These mutants may also serve as tools to further define the contributions of cell-free and cell-to-cell infection in vitro and in vivo.

The ULb′ Region of the Human Cytomegalovirus Genome Confers an Increased Requirement for the Viral Protein Kinase UL97

We report a requirement for the viral protein kinase UL97 in human cytomegalovirus (HCMV) replication that maps to the ULb' region of the viral genome. A UL97-null (Δ97) mutant of strain TB40/E, which encodes a full-length ULb' region, exhibited replication defects, particularly in production of cell-free virus, that were more severe than those seen with a Δ97 mutant of laboratory strain AD169, which harbors extensive deletions in its ULb' region. These differences were recapitulated with additional HCMV strains by treatment with a UL97 kinase inhibitor, 1-(β-L-ribofuranosyl)-2-isopropylamino-5,6-dichlorobenzimidazole (maribavir). We observed lower levels of viral DNA synthesis and an increased requirement for UL97 in viral late gene expression in strains with full-length ULb' regions. Analysis of UL97-deficient TB40/E infections by electron microscopy revealed fewer C-capsids in nuclei, unusual viral particles in the cytoplasmic assembly compartment, and defective viral nuclear egress. Partial inhibition of viral DNA synthesis caused defects in production of cell-free virus that were up to ≈ 100-fold greater than those seen with cell-associated virus in strains TB40/E and TR, suggesting that UL97-dependent defects in cell-free virus production in strains with full-length ULb' regions were secondary to DNA synthesis defects. Accordingly, a chimeric virus in which the ULb' region of TB40/E was replaced with that of AD169 showed reduced effects of UL97 inhibition on viral DNA synthesis, late gene expression, and production of cell-free virus compared to parental TB40/E. Together, these results argue that the ULb' region encodes a factor(s) which invokes an increased requirement for UL97 during viral DNA synthesis.

Single-point mutations of the M protein of a measles virus variant obtained from a patient with subacute sclerosing panencephalitis critically affect solubility and subcellular localization of the M protein and cell-free virus production

Subacute sclerosing panencephalitis (SSPE) is caused by variants of wild-type measles virus (MV). Such MV variants lack almost completely the ability to produce cell-free progeny virus. We recently isolated an MV variant that has only three amino acid mutations (L165P,L250P and Y282H) in the M protein compared with MV field isolates of the same genotype. In the present study, we analyzed the significance of these mutations with regard to the characteristics of the M protein and progeny virus production. We found that each of the three mutations rendered the M protein insoluble in 0.5% Triton X-100 and altered its subcellular localization, either when ectopically expressed alone using a plasmid-based expression system or when expressed in the context of viral replication. Moreover, each of the three mutations markedly, but not completely, impaired the ability of MV to produce cell-free progeny virus, with the degree of impairment being the same as for all three mutations together. These results suggest the possibility that the changes in the solubility and subcellular localization of the M protein determine the ability to produce cell-free progeny virus, at least to some extent, and play a role in the pathogenicity of variants causing SSPE.

Infection of different cell lines of neural origin with subacute sclerosing panencephalitis (SSPE) virus.

Measles virus is the causative agent of subacute sclerosing panencephalitis (SSPE). The viruses isolated from brain cells of patients with SSPE (called SSPE viruses) are defective in cell-free virus production in vitro. To investigate the cell tropism of three strains of SSPE virus (Osaka-1, Osaka-2, Osaka-3), SSPE virus-infected cell cultures were treated with cytochalasin D to prepare virus-like particles (CD-VLPs). All CD-VLPs formed syncytia after infection in CHO cells expressing CD150 but not in those expressing CD46. In addition, an antibody to CD46 did not block the infection of Vero cells by SSPE CDVLPs. The results were consistent with our previous suggestion that one or more unidentified receptors might be involved in the entry process. Infection with the CD-VLPs from three SSPE strains was further examined in different human cell lines, including those of neural origin, and was found to induce syncytia in epithelial cells (HeLa and 293T) as well as neuroblastoma cells (IMR-32 and SK-N-SH) with varying efficiency. SSPE CD-VLPs also infected glioblastoma cells (A172) and astrocytoma cells (U-251) but syncytial formation was rarely induced. These epithelial and neural cell lines were not permissive for the replication of wild-type MV. Together with our previous observations, these results suggest that the cell entry receptor is the major factor determining the cell tropism of SSPE viruses. Further studies are necessary to identify other viral and/or cellular factors that might be involved in the replication of SSPE virus in specific neural cells and in the brain.

Plasma viraemia as a marker of viral replication in HIV-infected individuals

Two hundred and twenty-eight blood samples from 154 HIV-seropositive subjects were investigated for the presence of HIV in peripheral blood mononuclear cells (PBMC) and plasma (without prior ultracentrifugation or filtration), using normal PBMC as the target for replication. HIV was recovered from PBMC and plasma in 80.5 and 19.5% of the patients, respectively. Plasma viraemia was significantly associated with clinical manifestations of HIV infection, indicating that HIV replication increased as disease progressed. This was confirmed by the statistically significant correlations between plasma viraemia and low CD4 cell counts (P less than 0.01) and low anti-p24 antibody titers (P less than 0.01). On patient follow-up, detection of HIV in plasma was transient. p24 antigenaemia was only detected in 13.6% of cases and was also associated with advanced clinical stages of the disease. When HIV RNA detection by polymerase chain reaction (PCR) was compared with plasma viraemia, HIV RNA was detected in plasma in all symptomatic cases and in 53.8% (seven out of 13) of asymptomatic patients [Centers for Disease Control (CDC) stages II and III], confirming that PCR was far more sensitive than plasma culture. These results indicate that cell-free virus production is associated with the clinical stage of HIV infection and may serve as a marker for disease progression. Detection of HIV RNA by PCR appears to be the most sensitive method to detect viraemia.

Variability in radioimmunoprecipitation assays of HIV

Variation of the rate of production of cell-free virus from human immunodeficiency virus (HIV)-infected transformed T-lymphocyte cultures alters the relative proportions of viral antigens obtained in cell-lysate preparations commonly used in radioimmunoprecipitation assays.

Diversity of Matrix Protein in Subacute Sclerosing Panencephalitis and Measles Virus‐Infected Cells

Expression of the viral matrix (M) proteins in Vero cells infected with 18 strains of subacute sclerosing panencephalitis (SSPE) virus and measles virus was examined by immunocytochemistry and Western blot analysis using an anti-M monospecific serum and two sera against the M protein specific synthetic peptides. By immunocytochemistry using the anti-M monospecific serum, M protein was detected in all of the virus-infected cells regardless of cell-free virus production. M proteins of the seven non-productive strains were found to vary significantly in their epitope, in their reactivity to different assay systems, and in their molecular weight, whereas M proteins of the other 11 productive strains were detected consistently. These results suggest diversification of M protein of the non-productive strains.

Detection of Cell-Free Human Immunodeficiency Virus in Cerebrospinal Fluid by Using Immune Scanning Electron Microscopy

The cerebrospinal fluid of 52 patients with human immunodeficiency virus type 1 (HIV-1) infection was incubated with polystyrene microspheres coated with monoclonal antibodies specific for HIV-1 core or envelope antigens. The beads were then collected on a filter surface and inspected by scanning electron microscopy (SEM). In 37 of 51 samples from subjects with chronic HIV-1 infection and from all three patients with primary HIV-1 infection, with or without neurologic symptoms, particles of 50-75 nm and 100-125 nm were visualized on beads coated with antibodies to HIV-1 core and envelope antigens, respectively. No such binding of particles was detected in the controls. Findings using immune SEM, which was found to be as sensitive as virus isolation, indicate that HIV-1 can replicate in the central nervous system (CNS) of patients with primary or chronic HIV-1 infection without causing neurologic symptoms. Production of cell-free virus seems to occur in the CNS of the majority of HIV-1-infected patients.

Varicella-zoster virus infection of diploid and chemically transformed guinea-pig embryo cells: factors influencing virus replication.

Factors influencing the replication of varicella-zoster virus (VZV) in guinea-pig embryo cells were evaluated using both diploid cells (GPEC) and a chemically transformed cell line (GPT). Wild-type and vaccine strains of VZV were successfully isolated and serially propagated in GPEC prepared from early gestation whole embryos (less than 2 cm in length). Low passage GPEC (less than or equal to 5 subcultivations) were more susceptible to VZV infection than high passage GPEC (greater than 5 subcultivations), and guinea-pig cells were consistently less permissive than human diploid cells. Cell-free virus was produced from VZV-infected GPEC cultures by sonication and peak yields of 10(3) p.f.u./ml were obtained. In addition, we report the isolation and propagation of VZV, as well as production of cell-free virus, in GPT. Both GPEC and GPT cells were less susceptible to VZV infection than human cells. However, viral replication was enhanced by incubation of VZV-infected GPT cultures at 32 degree C rather than 36 degree C.

Cell-Free Varicella-Zoster Virus in Cultured Human Melanoma Cells

Varicella-zoster virus (VZV) has been isolated and serially propagated in a continuous cell line derived from a human malignant melanoma tumour. Human melanoma cells (HMC) have been further evaluated as a substrate for the production of cell-free virus and compare favourably with human embryo cells. Within 60 h after inoculation with VZV-infected cells, HMC monolayers incubated at 32 degrees C exhibited advanced syncytial cytopathic effect, and the overlying culture medium contained greater than 10(2) p.f.u./ml. The cell pellet from a mechanically dispersed 150 cm2 monolayer yielded 10(5) p.f.u. after sonic disruption, while the medium ('scraping medium') in which the cells had been harvested contained up to one log more infectious virus than was found in the cells from the same monolayer. When infected cells were subjected to Dounce homogenization, most of the infectivity was found in the nuclear fraction. The concentration and purification of cell-free virus were also investigated. Concentration was carried out by three methods: ultracentrifugation, dialysis against hydrophilic compounds and liquid polymer phase separation. The first two procedures caused considerable loss of biological activity, whereas precipitation with 8% polyethylene glycol resulted in a 50-fold increase in titre. Purification of cell-free virus with retention of infectivity was achieved by rate zonal centrifugation in linear potassium tartrate gradients. Infectious virus was also recovered after sedimentation in combination equilibrium-viscosity gradients of potassium tartrate and glycerol, but not after centrifugation to equilibrium in caesium chloride gradients.

Effect of Passively Transferred Immunoglobulins on Marek's Disease

Early events in the pathogenesis of Marek's disease (MD) were studied in chicks with no passive MD antibodies, with congenitally derived parental antibodies, and with injected antibodies (immunoglobulins). MD responses in chicks inoculated with immunoglobulins at hatching were virtually identical to responses in chicks with parental antibodies. In vivo neutralization indices of 1.0 log1l lesionproducing-50% dose (LPD50) were obtained for cell-associated MD virus inocula in chicks with passive antibodies directed against either MD virus or an antigenically related herpesvirus of turkeys (HVT). Parallel studies with cell-free MD virus gave neutralization indices of 2.1 log1o LPD50. Latent periods (50% response times) for both lympholiferative lesions and for degenerative skin and bursal lesions were approximately 50-70% greater in antibody-free chicks. Also noted in MD-infected chicks with passive antibody were absence of bursal atrophy, delayed latent periods for precipitin antigen and cell-free virus production in the skin, and lower frequency of virus isolation from buffy coat cells. These data suggest possible mechanisms for the inhibitory role of passive antibody on the in vivo dissemination and primary oncogenic functions of MD virus.