Permissive Cell Types Research Articles

Alpha/beta interferon protects adult mice from fatal Sindbis virus infection and is an important determinant of cell and tissue tropism.

Infection of adult 129 Sv/Ev mice with consensus Sindbis virus strain TR339 is subclinical due to an inherent restriction in early virus replication and viremic dissemination. By comparing the pathogenesis of TR339 in 129 Sv/Ev mice and alpha/beta interferon receptor null (IFN-alpha/betaR(-/-)) mice, we have assessed the contribution of IFN-alpha/beta in restricting virus replication and spread and in determining cell and tissue tropism. In adult 129 Sv/Ev mice, subcutaneous inoculation with 100 PFU of TR339 led to extremely low-level virus replication and viremia, with clearance under way by 96 h postinoculation (p.i.). In striking contrast, adult IFN-alpha/betaR(-/-) mice inoculated subcutaneously with 100 PFU of TR339 succumbed to the infection within 84 h. By 24 h p.i. a high-titer serum viremia had seeded infectious virus systemically, coincident with the systemic induction of the proinflammatory cytokines interleukin-12 (IL-12) p40, IFN-gamma, tumor necrosis factor alpha, and IL-6. Replicating virus was located in macrophage-dendritic cell (DC)-like cells at 24 h p.i. in the draining lymph node and in the splenic marginal zone. By 72 h p.i. virus replication was widespread in macrophage-DC-like cells in the spleen, liver, lung, thymus, and kidney and in fibroblast-connective tissue and periosteum, with sporadic neuroinvasion. IFN-alpha/beta-mediated restriction of TR339 infection was mimicked in vitro in peritoneal exudate cells from 129 Sv/Ev versus IFN-alpha/betaR(-/-) mice. Thus, IFN-alpha/beta protects the normal adult host from viral infection by rapidly conferring an antiviral state on otherwise permissive cell types, both locally and systemically. Ablation of the IFN-alpha/beta system alters the apparent cell and tissue tropism of the virus and renders macrophage-DC-lineage cells permissive to infection.

Replication of murine cytomegalovirus in differentiated macrophages as a determinant of viral pathogenesis.

Blood monocytes or tissue macrophages play a pivotal role in the pathogenesis of murine cytomegalovirus (MCMV) infection, providing functions beneficial to both the virus and the host. In vitro and in vivo studies have indicated that differentiated macrophages support MCMV replication, are target cells for MCMV infection within tissues, and harbor latent MCMV DNA. However, this cell type presumably initiates early, antiviral immune responses as well. In addressing this paradoxical role of macrophages, we provide evidence that the proficiency of MCMV replication in macrophages positively correlates with virulence in vivo. An MCMV mutant from which the open reading frames M139, M140, and M141 had been deleted (RV10) was defective in its ability to replicate in macrophages in vitro and was highly attenuated for growth in vivo. However, depletion of splenic macrophages significantly enhanced, rather than deterred, replication of both wild-type (WT) virus and RV10 in the spleen. The ability of RV10 to replicate in intact or macrophage-depleted spleens was independent of cytokine production, as this mutant virus was a poor inducer of cytokines compared to WT virus in both intact organs and macrophage-depleted organs. Macrophages were, however, a major contributor to the production of tumor necrosis factor alpha and gamma interferon in response to WT virus infection. Thus, the data indicate that tissue macrophages serve a net protective role and may function as "filters" in protecting other highly permissive cell types from MCMV infection. The magnitude of virus replication in tissue macrophages may dictate the amount of virus accessible to the other cells. Concomitantly, infection of this cell type initiates the production of antiviral immune responses to guarantee efficient clearance of acute MCMV infection.

G alpha 13 stimulates gene expression and increases cell size in cultured neonatal rat ventricular myocytes.

Constitutively-active G alpha 13 causes permissive cell types to proliferate or undergo phenotypic transformation implying a role for G13 in the control of cell growth. Cardiac myocytes are terminally-differentiated cells which respond to growth stimuli by increasing in size rather than by cell division. The objective of this study was to determine whether constitutively-active G alpha 13 is able to induce a hypertrophic phenotype in cardiac myocytes. Cultured neonatal rat ventricular myocytes were transiently transfected with an expression vector (pRC/RSV) encoding wild-type G alpha 13 or constitutively-active G alpha 13Q226L. Effects on transcription were monitored by co-transfected luciferase (LUX) reporter genes under the control of promoters responsive to hypertrophic stimuli. Cell size was determined by planimetry. Transfection of neonatal myocytes with G alpha 13Q226L, but not wild-type G alpha 13, stimulated ANF638LUX and ANF3003LUX expression to 3.0 +/- 0.3- and 4.3 +/- 0.6-fold of the control, respectively. Likewise, G alpha 13Q226L stimulated vMLC250LUX and vMLC2700LUX expression to 3.9 +/- 1.0- and to 7.7 +/- 1.7-fold of controls, respectively, but there was relatively little effect of G alpha 13Q226L on c-fos-SRE- and beta-MHC promoter activity. The effects of G alpha 13Q226L on ANF3003LUX were inhibited by expression of C3 exoenzyme. Wild-type G alpha 13 and G alpha 13Q226L increased myocyte area from 869 +/- 43 micron 2 in control tranfections to 1287 +/- 64 micron 2 and 1278 +/- 59 microns, respectively. We conclude that G alpha 13Q226L is able to induce gene expression and morphological changes associated with a hypertrophic response in cardiac myocytes and that the transcriptional effects may be mediated through a Rho-dependent mechanism.

A simple procedure to determine the biological titer of recombinant retroviral vectors.

Retroviral vectors are widely used to deliver genetic material to live cells both in experimental and clinical settings. The ability of these vectors to transduce target cells is an important aspect of their clinical applicability and one of the factors determining their transduction efficiency is vector functional titer. Current methods for titrating retroviral vectors involve measuring the number of target cells in culture transduced by a given volume of vector solution. In this report, we describe a new procedure which allows one to estimate the actual number of infectious particles capable of transducing a permissive cell type. Vector biological titer is calculated from the fractional decline in transduction efficiency observed when a given volume of vector solution is sequentially added to multiple dishes containing permissive cells. Values determined this way are greater than those obtained from a single transduction experiment, with the difference being inversely proportional to the degree of cell permissiveness for vector entry. The present procedure is simple, reliable and expeditious. It will be useful to standardize vector biological titers determined in different laboratories, and help implement strategies for efficient gene delivery protocols.

Isolation and characterization of a low-abundance splice variant from the human cytomegalovirus major immediate-early gene region.

The major immediate-early (IE) gene region of human cytomegalovirus (HCMV) encodes several proteins as a result of differential RNA splicing events. By expression vector cloning of HCMV IE mRNA, we isolated and characterized a cDNA for a novel splice variant from the major IE gene region. The RNA product is a derivative of the IE55 mRNA and contains an additional splice from nucleotides 170,635 to 170,307 in the IE2 gene region (UL122), resulting in a 1.4-kb mRNA. The predicted open reading frame codes for a 164-amino-acid protein with a calculated molecular mass of 18 kDa (IE18). Mung bean nuclease analysis and PCR were used to characterize expression of IE18 mRNA in HCMV-infected cells. While the 1.4-kb mRNA was detected in infected human fibroblasts in the presence of a protein synthesis inhibitor, it was not detectable during a normal infection. However, the 1.4-kb mRNA was readily detected in infected human monocyte-derived macrophages at IE times. These results suggest that the novel IE18 mRNA exhibits cell type-specific expression indicating differential regulation of the major IE gene region in different permissive cell types.

Priming Antiviral Cytotoxic T Lymphocytes: Requirement for CD4 + Cells Is Dependent on the Antigen Presenting Cell in Vivo

We have analyzed cytotoxic thymus-derived lymphocyte (CTL) responses to vesicular stomatitis virus (VSV) to determine whether VSV precursor CTL (pCTL) can be primed in vivo in the absence of CD4 + cells. Our studies demonstrated that secondary anti-VSV CTL responses in vitro were markedly reduced by CD4-depletion prior to priming in vivo with VSV. Limiting dilution analysis indicated that the vast majority (>90%) of VSV pCTL failed to become primed when exposed to VSV in the absence of CD4 + cells. A second minor population (5-10%) of pCTL was identified that was reproducibly primed in CD4-deficient mice. In contrast to CD4-depleted mice infected with free, infectious virus, CD4-deficient mice primed with VSV-infected, activated B cells mounted normal secondary anti-VSV CTL responses in vitro . Precursor estimates indicated that virtually all VSV pCTL became primed using this cellular immunogen. CD4-independent priming could not be achieved using VSV-infected, activated T cells, another permissive cell type for VSV replication. Thus, most VSV pCTL require inductive signals from classical CD4 + helper T cells in order to become primed in vivo and this requirement may be regulated in vivo by the antigen presenting cell.

Expression of Rous sarcoma virus-derived retroviral vectors in the avian blastoderm: potential as stable genetic markers.

Retroviruses are valuable tools in studies of embryonic development, both as gene expression vectors and as cell lineage markers. In this study early chicken blastoderm cells are shown to be permissive for infection by Rous sarcoma virus and derivative replication-defective vectors, and, in contrast to previously published data, these cells will readily express viral genes. In cultured blastoderm cells, Rous sarcoma virus stably integrates and is transcribed efficiently, producing infectious virus particles. Using replication-defective vectors encoding the bacterial lacZ gene, we further show that blastoderms can be infected in culture and in ovo. In ovo, lacZ expression is seen within 24 hr of virus inoculation, and by 96 hr stably expressing clones of cells are observed in diverse tissues throughout the embryo, including epidermis, somites, and heart, as well as in extraembryonic membranes. Given the rapid onset of vector expression and the broad range of permissive cell types, it should be feasible to use Rous sarcoma virus-derived retroviruses as early lineage markers and expression vectors beginning at the blastoderm stage of avian embryogenesis.

Regulation of herpes simplex virus immediate-early gene promoters in mouse neuroblastoma cells

The non-permissivity of C1300 mouse neuroblastoma cells for herpes simplex virus (HSV) infection is due to a failure of such cells to transcribe the immediate-early (IE) genes following viral infection. We have transfected both C1300 cells and permissive cells with constructs in which each of the 5 IE promoters drives expression of the readily assayable chloramphenicol acetyl transferase (CAT) gene. These experiments show that the lack of IE gene transcription in C1300 cells is due to the weak activity of the five IE promoters in these cells compared to that observed in a range of permissive cell types. This effect is mediated both by up-stream elements and by sequences present in the minimal promoter. The different effects of DNA concentration on the activities of the minimal and complete promoters suggests that the up-stream sequences act by binding a repressor factor present in C1300 cells whilst the weak activity of the minimal promoter results from the absence of a positive factor in such cells.

Mononuclear Phagocytes and HSV-1 Infection: Increased Permissivity in Differentiated U937 Cells Is Mediated by Post-Transcriptional Regulation of Viral Immediate-Early Gene Expression

Undifferentiated U937 cells are non-permissive for herpes simplex virus (HSV) infection but can be rendered permissive by treatment with phorbol myristate acetate (PMA), which causes them to differentiate to a macrophage-like phenotype. Following infection with HSV, both PMA--treated and untreated cells correctly transcribe the viral immediate-early genes at levels comparable to those observed in fully permissive cell types, but immediate-early RNA and protein are detected only in the PMA-treated cells. Hence PMA acts by relieving an early block to HSV infection caused by the rapid turnover of immediate-early RNA. This block is not caused by the production of soluble inhibitors and can also be relieved by treatment with other agents that cause macrophage differentiation such as 1, 25 dihydroxycholecalciferol. These findings therefore indicate that the non-permissivity of undifferentiated U937 cells for HSV is mediated by post-transcriptional regulation of immediate-early gene expression.

Construction and Characterization of Herpes Simplex Virus Type 1 Mutants with Defined Lesions in Immediate Early Gene 1

Transcription from the early and late classes of the herpes simplex virus type 1 (HSV-1) promoters requires prior immediate early (IE) gene expression. Although the product of IE gene 1, Vmw110, is not absolutely essential for virus growth in tissue culture, transfection experiments have demonstrated that Vmw110 can activate gene expression both by itself and in a synergistic manner with the product of IE gene 3, Vmw175. This paper describes the construction of 10 mutant HSV-1 viruses with deletion and insertion mutations in Vmw110. The mutant viruses were then studied in single-step growth curve experiments, by assaying for plaques in a variety of cell types and by analysis of viral polypeptide synthesis during productive infection at high and low multiplicities. The results show that mutations in Vmw110 reduce the efficiency of plaque formation by HSV-1; the extent of this reduction depends on cell type and the position of the mutation in the polypeptide. In particular, a potential zinc finger domain is crucial for Vmw110 function. The patterns and amounts of viral polypeptide synthesis during high multiplicity infections with mutant and wild-type viruses were similar in all cell types. At low multiplicity, mutations in Vmw110 reduced viral gene expression in the least permissive cell type. The data suggest that the role of Vmw110 during virus infection in tissue culture is at a very early stage of low multiplicity infections; its inactivity leads to the failure to express viral genes so that the virus does not enter the lytic cycle.

ROLE OF EPITHELIUM IN EBV PERSISTENCE AND PATHOGENESIS OF B-CELL TUMOURS

EBV has long been thought to be primarily a B-lymphotropic virus. A manifestation of this tropism is the association of the virus with a variety of B-cell lymphoproliferative disorders and tumours. However, there is now considerable evidence to suggest that the permissive cell type for EBV replication is epithelial and that infection of B cells may be a secondary, and, from the point of view of the virus, an unimportant consequence. A re-evaluation of the role that epithelium plays in viral persistence and of the importance of the immune response in the development of persistent infection indicates that T-cell-mediated immunity to the epithelial infection is critical in maintaining the normal delicate balance between virus and host. It also suggests that uncontrolled EBV replication in pharyngeal epithelium may be central to the evolution of some (or all) EBV-associated lymphomas.

Protection of C58 mice from lactate dehydrogenase-elevating virus-induced motor neuron disease by non-neutralizing antiviral antibodies without interference with virus replication

The paralytic poliomyelitis induced in old, immunosuppressed C58 mice by a primary infection with the lactate dehydrogenase-elevating virus (LDV) is prevented by the presence of anti-LDV antibodies in the virus inoculum or by passive transfer of LDV-free plasma from chronically LDV-infected mice one day before infection. Non-neutralizing antibodies were protective and specifically directed to the lowest molecular weight form of the envelope glycoprotein of LDV (VP-3), which seems to exist in virions in at least ten molecular forms ranging from 24 to 44 kDa. The antibodies did not prevent the productive infection of the subpopulation of macrophages that represents the primary permissive cell type in the mouse as evidenced by normal plasma LDV levels nor the spread of LDV to the central nervous system. Many non-neuronal cells containing LDV RNA were detected by in situ hybridization in the spinal cords of mice that had been infected with LDV in the presence of protective antibodies. However, no LDV RNA-positive neurons were detected, which are normally found coincidental with the development of paralytic symptoms in LDV-infected C58 mice. We propose that an early even after infection is critical for the infection of neurons and is inhibited by the presence of non-neutralizing antibodies to the LDV glycoprotein.

Host cell receptors for two strains of Sindbis virus.

Experiments were performed to determine whether neuronal cells have different numbers of receptors for a neurovirulent and an avirulent strain of the same virus and whether neurotropic strains of different viruses share the same cellular receptors. Attempts were also made to characterize the receptors for the two strains of Sindbis virus on viable cells by studying their enzyme sensitivity. The number of cellular receptors available for Sindbis virus attachment to several cell lines was determined by saturation studies using two virus strains differing in their pathogenicity for adult mice. Cultured neuronal cells had 1.3 x 10(6) receptors for a neurovirulent strain (SaAr86) and only 5 x 10(4) for the avirulent prototype strain (EgAr339) of Sindbis virus. A refractory Lepidopteran cell type possessed 1 x 10(5) surface receptors for the neurovirulent variant while the average number of receptors on five permissive cell types was 1.5 x 10(6). Cellular receptors for the two strains of Sindbis virus on rat glioma cells were found to be distinct. The cellular receptors for EgAr339 on viable mammalian cells were sensitive to proteolytic cleavage, while those on living mosquito cells were insensitive to proteases, phospholipases and neuraminidase. The receptors for the neurovirulent variant on three mammalian cell types were less sensitive to enzymatic inactivation than those for the avirulent counterpart. After cleavage, the receptors for EgAr339 reappeared rapidly at 37 degrees and 4 degrees C, apparently in the absence of cellular synthesis.

Isolation and characterization of two new herpes-like viruses from capuchin monkeys.

Two herpes-like viruses were isolated from capuchin monkey (Cebus apella) brain and (Cebus albifrons) spleen cell cultures, respectively. Both isolates induced similar cytopathic effects consisting of rounded and ballooned cells in the original monkey cell cultures and in a wide range of permissive cell types. Neutralizing antibody to each virus was present in serum from the capuchin monkey from which it was isolated, but the two viruses did not cross-react by neutralization. Fluorescein isothiocyanate conjugates of hyperimmune rabbit serum to one of the isolates showed an antigenic cross relationship between the two isolates. By electron microscopy, herpes-like virus particles were observed in the nucleus and cytoplasm of infected human diploid fibroblast cell cultures. Virus-infected cell cultures stained with acridine orange revealed small deoxyribonucleic acid-containing intranuclear inclusion bodies. Both viruses were inhibited by 5-fluorodeoxyuridine and inactivated by chloroform or exposure to 56 degrees C for 30 min. Antisera prepared against 16 prototype herpesviruses and cytomegaloviruses did not neutralize approximately 100 50% tissue culture infective doses of either capuchin isolate. Neutralizing antibody to the capuchin isolates was detected in sera from 8 of 17 capuchin monkeys but not in sera from 16 humans, 15 chimpanzees, and 10 spider, 6 rhesus, and 5 squirrel monkeys.

Enhancement of adenovirus infection in WI-38 and AGMK cells by pretreatment of cells with 5-lododeoxyuridine

Adenovirus replicates inefficiently in WI-38 and AGMK cells. Treatment of these cells with 5-iododeoxyuridine (IdU) prior to infection enhances the yield of adenovirus by 10- to 1000-fold. IdU-pretreated cultures demonstrate an increased percentage of adenovirus T-antigen-positive cells in comparison to untreated controls. IdU treatment has no effect on virus adsorption, and in AGMK cells IdU enhancement is additive to SV40 enhancement of adenovirus yield. Thus, IdU treatment relieves a restriction to virus growth that occurs prior to T-antigen synthesis. IdU pretreatment has no effect on virus growth in HEK, a permissive cell type. Incorporation of IdU into cellular DNA is required for enhancement.