Influence of cell surface glycoprotein gC produced by pseudorabies virus on cytopathic effect

The wild-type pseudorabies virus (WT-PRV) produced a round-type cytopathic effect (CPE) in PK-15 cell line of porcine kidney origin, while PRVgCs lacking in gC-transmembrane-anchor region and PRVgC-defecting in gC gene produced a syncytium-type CPE. The mouse embryo cell line (BALB/3T3 clone A31) were transfected with recombinant plasmid of pcDNA3 which incorporated with gC gene. The transfected A31/gC cells were stably expressing gC. Only a round-type CPE was observed in these cells infected with WT-PRV, while a syncytium-type CPE was observed in the cells infected with each of the PRVgCs and PRVgC-. Any viruses described above induced a syncytium-type CPE in A31/pcDNA cells transfected with a plasmid without gC gene. By WT-PRV infection, PK-15 cells generated about 2- or 8-fold more gC than the A31/gC and A31/pcDNA cells when gC was measured by hemagglutination test. Flowcytometric analysis revealed that amount of gC on the cell surface of A31/gC and PK-15 cells increased after infection with WT-PRV. Round-type CPE was observed with the increase of gC. These results suggest that the type of CPE formation induced by PRV is dominated by the amount of gC on the infected cell surface.     

Enhanced cytopathic effect of human cytomegalovirus on a retinal pigment epithelium cell line, K-1034, by serum-free medium

Although human cytomegalovirus (HCMV) predominantly infects epithelial cells in vivo, the majority of studies of HCMV gene expression and replication have been conducted using non-epithelial cell lines in part because of the absence of a good experimental system using epithelial cells. To address the nature of epithelial cell infection, we investigated the susceptibility of an epithelial cell line (K-1034) established from the retinal pigment epithelium to HCMV infection. This cell line exhibited high susceptibility to HCMV, as evidenced by detection of one of the immediate early antigens, IE2, in the nuclei of more than 80% of K-1034 cells at 24 h following inoculation at a multiplicity of infection of 3 plaque forming units per cell. However, the yield after one-step growth of HCMV in K-1034 cells was about twenty-fold less than that in human embryonic lung fibroblast cells. Cytopathic effect (CPE) on K-1034 cells was not prominent in medium supplemented with 10% fetal bovine serum and viral late antigens were detected in less than 5% of K-1034 cells. Interestingly, infected cells expressing late antigens and exhibiting CPE were markedly increased in serum-free medium, even though the yield of infectious HCMV and viral genome copy numbers were almost the same in the different serum concentrations, due to viral instability in the absence of serum. Thus, the progression of late antigens expression and the induction of CPE in infected epithelial cells is influenced by physiological conditions, and are negatively regulated by some serum factor.     

Expression of measles virus V protein is associated with pathogenicity and control of viral RNA synthesis

Nonstructural proteins encoded by measles virus (MV) include the V protein which is translated from an edited P mRNA. V protein is not associated with intracellular or released viral particles and has recently been found to be dispensable for MV propagation in cell culture (H. Schneider, K. Kaelin, and M. A. Billeter, Virology 227:314-322, 1997). Using recombinant MVs (strain Edmonston [ED]) genetically engineered to overexpress V protein (ED-V+) or to be deficient for V protein (ED-V-), we found that in the absence of V both MV-specific proteins and RNAs accumulated to levels higher than those in the parental MV molecular clone (ED-tag), whereas MV-specific gene expression was strongly attenuated in human U-87 glioblastomas cells after infection with ED-V+. The titers of virus released from these cells 48 h after infection with either V mutant virus were lower than those from cells infected with ED-tag. Similarly, significantly reduced titers of infectious virus were reisolated from lung tissue of cotton rats (Sigmodon hispidus) after intranasal infection with both editing mutants compared to titers isolated from ED-tag-infected animals. In cell culture, expression of V protein led to a redistribution of MV N protein in doubly transfected Cos-7 cells, indicating that these proteins form heterologous complexes. This interaction was further confirmed by using a two-hybrid approach with both proteins expressed as Gal4 or VP16 fusion products. Moreover, V protein efficiently competed complexes formed between MV N and P proteins. These findings indicate that V protein acts to balance accumulation of viral gene products in cell culture, and this may be dependent on its interaction with MV N protein. Furthermore, expression of V protein may contribute to viral pathogenicity in vivo.     

Antisense-mediated repression of DNA topoisomerase II expression leads to an impairment of HIV-1 replicative cycle

Previously, we have observed a strong restriction of the Moloney murine leukemia virus (MoMLV) replicative cycle in a cell line displaying resistance to topoisomerase II (topo II)-interactive drugs. Resistance towards these antitumoral inhibitors was associated with decreased expression and activity of topo II, suggesting that such a decrease may be responsible for MoMLV restriction. To more specifically assess the role of topo II during the retroviral cycle, we have used the antisense strategy to obtain a selective decrease of cellular topo II expression. The RNA antisense was isolated from a retroviral library expressing random fragments of human topo II (alpha form). This system allowed us to investigate the HIV-1 replicative cycle in two related human CEM cell lines expressing different levels of topo II. Expression of the enzyme is decreased four- to sixfold following formation of a sense-antisense RNA hybrid. Repression of the topo II enzyme results in an impairment of the HIV-1 replicative cycle. Using the polymerase chain reaction, we showed that the number of integration events was decreased in cells repressing the enzyme, although viral DNA synthesis and circularization were equivalent to those in the parent cells.     

Rabbit cells expressing human CD4 and human CCR5 are highly permissive for human immunodeficiency virus type 1 infection

To evaluate the feasibility of using transgenic rabbits expressing CCR5 and CD4 as a small-animal model of human immunodeficiency virus type 1 (HIV) disease, we examined whether the expression of the human chemokine receptor (CCR5) and human CD4 would render a rabbit cell line (SIRC) permissive to HIV replication. Histologically, SIRC cells expressing CD4 and CCR5 formed multinucleated cells (syncytia) upon exposure to BaL, a macrophagetropic strain of HIV that uses CCR5 for cell entry. Intracellular viral capsid p24 staining showed abundant viral gene expression in BaL-infected SIRC cells expressing CD4 and CCR5. In contrast, neither SIRC cells expressing CD4 alone nor murine 3T3 cells expressing CCR5 and CD4 exhibited significant expression of p24. These stably transfected rabbit cells were also highly permissive for the production of virions upon infection by two other CCR5-dependent strains (JR-CSF and YU-2) but not by a CXCR4-dependent strain (NL4-3). The functional integrity of these virions was demonstrated by the successful infection of human peripheral blood mononuclear cells (PBMC) with viral stocks prepared from these transfected rabbit cells. Furthermore, primary rabbit PBMC were found to be permissive for production of infectious virions after circumventing the cellular entry step. These results suggest that a transgenic rabbit model for the study of HIV disease may be feasible.     

In vitro infection of primary and retrovirus-infected human leukocytes by human foamy virus

The infectivity of human foamy virus (HFV) was examined in primary and cultured human leukocytes. Cell-free infectious viral stocks of HFV were prepared from the human kidney cell line 293 transfected with an infectious molecular clone of HFV. HFV productively infects a variety of human myeloid and lymphoid cell lines. In addition, primary cell cultures enriched for human CD4+, monocytes and brain-derived microglial cells, were readily infected by HFV. Interestingly, while infected primary CD4+ lymphocytes and microglial cells showed marked cytopathology characteristic of foamy virus, HFV-infected monocyte-derived macrophages failed to show any cytopathology. In addition, marked cytotoxicity due to HFV infection was seen in both human T-cell leukemia virus type 1- and human immunodeficiency virus type 1-infected T-cell lines and in human immunodeficiency virus type 1-infected monocytoid cell lines. Thus, HFV infection produces differential cytopathology in a wide host range of primary human leukocytes and hematopoietic cell lines.     

Enhanced human T-cell lymphotropic virus type I expression following induction of the cellular stress response

Human T-cell lymphotropic virus type I (HTLV-I) infection is typically associated with long incubation periods between virus exposure and disease manifestation. Although viral protein expression is considered to play an important role in the pathogenesis of HTLV-I-associated diseases, limited information is known regarding host cell mechanisms that control viral gene expression. This study was designed to evaluate modulation of HTLV-I gene expression following induction of the cellular stress response in HTLV-I-infected lymphocytes. The cellular stress response was elicited by treatment with either Na arsenite or thermal stress and was monitored by demonstrating increased expression of the 72-kDa heat shock protein. Induction of the cellular stress response in HTLV-I-infected lymphocytes resulted in significantly increased HTLV-I-mediated syncytia formation due to enhanced HTLV-I envelope (gp46) expression. Intracellular viral proteins and released p24 capsid protein were increased in stressed infected lymphocytes as compared to nonstressed infected lymphocytes. Furthermore, HTLV-I-LTR reporter gene constructs had increased activity (three- to sixfold) in a transiently transfected, uninfected lymphocyte cell line following induction of the cellular stress response. Quantitation of HTLV-I RNA expression by slot blot analysis of infected lymphocytes suggested variable increases in RNA accumulation. Northern blot analysis demonstrated no qualitative changes in expression of RNA species. These data suggest a relationship between modulation of viral replication and a basic cellular response to stress and have important implications for understanding host cell control mechanisms of HTLV-I expression.     

Plants transformed with a mutant alfalfa mosaic virus coat protein gene are resistant to the mutant but not to wild-type virus

Transgenic tobacco plants expressing the wild-type (wt) coat protein (CP) gene of alfalfa mosaic virus (AIMV) have been shown to be resistant to infection with viral particles and RNAs or to infection with viral particles only. The difference in resistance of these plants to RNA inocula was found to correlate with a difference in the expression level of the transgene. Plants expressing a mutant AIMV CP with the N-terminal serine residue changed to glycine have been shown to be susceptible to infection with wt viral particles or RNAs. By site-directed mutagenesis of AIMV cDNA a viable mutant virus encoding CP with the same N-terminal mutation was obtained. Plants expressing wt or mutant CP were resistant to the mutant virus, demonstrating that a single amino acid substitution in CP did not permit the virus to overcome CP-mediated resistance. Although the mutant CP did not confer resistance to wt virus when expressed in transgenic plants, it was still effective in classical cross-protection: plants infected with the mutant virus were resistant to severe strain of AIMV. A model to explain the data is discussed.     

Measles virus infects human dendritic cells and blocks their allostimulatory properties for CD4+ T cells

Measles causes a profound immune suppression which is responsible for the high morbidity and mortality induced by secondary infections. Dendritic cells (DC) are professional antigen-presenting cells required for initiation of primary immune responses. To determine whether infection of DC by measles virus (MV) may play a role in virus-induced suppression of cell-mediated immunity, we examined the ability of CD1a+ DC derived from cord blood CD34+ progenitors and Langerhans cells isolated from human epidermis to support MV replication. Here we show that both cultured CD1a+ DC and epidermal Langerhans cells can be infected in vitro by both vaccine and wild type strains of MV. DC infection with MV resulted within 24-48 h in cell-cell fusion, cell surface expression of hemagglutinin, and virus budding associated with production of infectious virus. MV infection of DC completely abrogated the ability of the cells to stimulate the proliferation of naive allogeneic CD4+ T cell as early as day 2 of mixed leukocyte reaction (MLR) (i.e., on day 4 of DC infection). Mannose receptor-mediated endocytosis and viability studies indicated that the loss of DC stimulatory function could not be attributed to the death or apoptosis of DC. This total loss of DC stimulatory function required viral replication in the DC since ultraviolet (UV)-inactivated MV or UV-treated supernatant from MV-infected DC did not alter the allostimulatory capacity of DC. As few as 10 MV- infected DC could block the stimulatory function of 10(4) uninfected DC. More importantly, MV-infected DC, in which production of infectious virus was blocked by UV treatment or paraformaldehyde fixation, actively suppressed allogeneic MLR upon transfer to uninfected DC-T-cultures. Thus, the mechanisms which contribute to the loss of the allostimulatory function of DC include both virus release and active suppression mediated by MV-infected DC, independent of virus production. These data suggest that carriage of MV by DC may facilitate virus spreading to secondary lymphoid organs and that MV replication in DC may play a central role in the general immune suppression observed during measles.     

Persistent echovirus infection of mouse cells expressing the viral receptor VLA-2

Mouse cells are not susceptible to infection with echovirus 1 (EV-1) because they lack the viral receptor, human VLA-2. Two mouse fibroblast cell lines, L cells and 3T3 cells, were made susceptible to EV-1 infection after transformation with cDNAs of human VLA-2. After EV-1 infection, L cell transformants of human VLA-2 (alpha2beta1 L cells) develop cytopathic effect (CPE) as expected, while 3T3 cell transformants of human VLA-2 (alpha2beta1 3T3 cells) or the alpha2 subunit of human VLA-2 (alpha2 3T3 cells) become persistently infected. The distinct outcome is not a result of differential virus growth on these transformants because one-step growth curve analysis reveals little difference in EV-1 replication in both cell lines. In addition, 3T3 cell transformants expressing the poliovirus receptor (Pvr 3T3 cells) are lysed during poliovirus infection, suggesting that 3T3 cells are not intrinsically resistant to CPE caused by enterovirus infection. The results of limit dilution assays indicate that all EV-1-infected alpha2 3T3 cells produce infectious virus. All EV-1-infected alpha2 3T3 cells remain viable after EV-1 infection, and the kinetics of cell growth were not altered. FACS analysis reveals that receptor down-regulation is not involved in the establishment of persistent infection. Furthermore, inhibition of host protein synthesis was not observed in EV-1-infected alpha2 3T3 or alpha2beta1 L cells. Since alpha2beta1 L cells are lysed by EV-1 infection, these findings suggest that virus-induced translation inhibition is not a determinant of cell killing.     

Virus RNA persists within the retina in coronavirus-induced retinopathy

The murine coronavirus, mouse hepatitis virus (MHV), JHM strain, induces a biphasic retinal disease in adult BALB/c mice. In the early phase, Day 1 to Day 7, a retinal vasculitis is noted which is associated with the presence of viral proteins and infectious virus. In the late phase, Day 10 to Day 140, a retinal degeneration is associated with the absence of viral proteins, infectious virus, and inflammatory cells. The purpose of this study was to determine if viral RNA persists within the retina during the retinal degenerative phase of the disease. BALB/c mice were inoculated by the intravitreal route with 10(4.0) TCID50/5 microliters of virus. The presence of viral RNA was detected by in situ hybridization with a viral cDNA probe and viral proteins were identified by immunocytochemical staining. During the acute phase of the infection, viral RNA was found in the retina, RPE, ciliary body epithelium, and the iris epithelium. During the late phase of the infection, viral RNA was almost exclusively found within the retina and RPE and not in the anterior segment of the eye. Within the retina, viral RNA was detected in the ganglion cell layer, the inner retina, the outer retina, and the RPE cell. Immunocytochemical staining identified viral protein within the retina only from Day 1 to Day 8. This ocular disease was also associated with a persistent systemic infection. Both viral RNA and viral proteins were identified within the liver during the first 8 days. However, only viral RNA was detected in the liver from Day 8 to Day 60. These studies demonstrate that MHV established an acute infection (Day 1-8) where infectious virus and viral proteins were identified. This was followed by a persistent infection within the retina and liver where only viral RNA were detected by in situ hybridization.     

Measles virus infection of cerebral endothelial cells and effect on their adhesive properties

Measles virus (MV) RNA is present in endothelial cells (Ec) in brain tissue from cases of subacute sclerosing panencephalitis (SSPE) and relatively high titres of infectious virus are produced in human cerebral Ec in vitro. Infection of Ec at the blood-brain barrier could therefore provide the opportunity for entry of virus to the CNS. Adhesion of syngeneic splenocytes to MV infected murine (Balb/c) cerebral Ec is found to be upregulated. Increased expression of endothelial adhesion molecules, following virus infection at the blood-brain-barrier, may be an important mechanism in inducing inflammatory infiltration of the CNS in SSPE.     

Susceptibility of the Sf9 insect cell line to infection with adventitious viruses

Sf9, the insect cell line commonly used for gene expression by recombinant baculovirus (BV), can be infected by St. Louis encephalitis (SLE) virus, a flavivirus, resulting in a persistent, productive, and cytopathic infection, while retaining the ability to be infected with a recombinant baculovirus (rBV). We now demonstrate using double immunofluorescence that single cells are dually infected with SLE virus and rBV. Fourteen additional viruses including additional flaviviruses, other arbovirus classes, vesicular stomatitis virus (VSV), and herpes simplex virus, type 1 (HSV-1) failed to produce a cytopathic effect (CPE) in Sf9 cells. Plaque assays indicated infectious virus was present for several weeks post-inoculation for Yellow fever (YF), Dengue types 1 and 2 (DEN-1 and DEN-2), Gumbo limbo (GL), Eastern equine encephalomyelitis virus (EEE), Western equine encephalomyelitis virus (WEE), HSV-1, and VSV viruses. For HSV-1, GL, EEE, WEE and VSV, but not for YF, DEN-1 or DEN-2 viruses, this could be attributed solely to survival in the Sf9 cell culture media. Of the 14 viruses tested, only HSV-1 could be detected after 2 weeks in serum-free media. The data indicate that several viruses which are pathogenic for humans are stable for long periods of time at 27 degrees C in the serum-containing media used for cultivation of Sf9 cells. YF, DEN-1 and DEN-2 viruses may replicate in Sf9 cells at extremely low levels. This suggests that adventitious agents which do not produce obvious CPE or interfere with rBV infection or recombinant protein expression could contaminate Sf9 cell cultures or media.     

Are laryngeal papilloma virus-infected cells viable in the plume derived from a continuous mode carbon dioxide laser, and are they infectious? A preliminary report on one laser mode

The purpose of this study was to determine the potential risk of transmitting viable viral infected cells as well as viral infectivity of laryngeal papilloma in the plume derived from a continuous mode carbon dioxide laser. Each of 10 juvenile recurrent laryngeal papilloma specimens was divided into two equal parts, and one part was irradiated with a carbon dioxide laser employing a continuous mode at the power setting of 10 watts with 0.5 mm spot size and a power density of 1667 W/cm2. The resultant laser plume was trapped and was cultured simultaneously with the other part of the specimen which served as the control. All irradiated specimens tested yielded negative culture results while all the control counterparts revealed viable cell growth. To detect the viral infectivity, laser plume was cultured with two separate cell systems, one was the porcine PS clone D cell line and the other normal mucosal cells obtained from the same patient, and to control these systems both cell lines were also designed to be infected with polio virus. Both cell lines in the viral infectivity testing systems revealed no sign of viral infection. The results suggest that papilloma virus-infected cells cannot survive the continuous mode of carbon dioxide laser irradiation. We primarily conclude that, to avoid airborne transmission of plume containing laryngeal papilloma viral-infected cells and infectious viral particles, the carbon dioxide laser parameters should be in a continuous mode with the power density equal to, or more than, 1667 W/cm2.     

Theiler's murine encephalomyelitis virus kills restrictive but not permissive cells by apoptosis

Theiler's murine encephalomyelitis viruses (TMEV), genus Cardiovirus, family Picorniviridae, are natural enteric pathogens of mice which cause central nervous system demyelination similar to that seen in multiple sclerosis. TMEV can be classified into two groups based on neurovirulence: a highly virulent group, e.g., GDVII virus, and a less virulent group, e.g., BeAn virus. Both viruses, depending on the multiplicity of infection, produced cytopathology in BSC-1 cells similar to that in BHK-21 cells. Since apoptosis has been reported as a mechanism of cell death after infection with many viruses, we examined infected BHK-21 and BSC-1 cells for morphological and biochemical changes consistent with apoptosis. Only the restrictive BSC-1 cells showed evidence of nuclear morphology and internucleosomal DNA degradation indicative of apoptosis. Interestingly, the more virulent GDVII virus was at least 50-fold more efficient in inducing apoptosis than the less virulent BeAn virus. This difference was not due to greater GDVII viral RNA replication or production of infectious virus, since the two viruses were similarly restricted in BSC-1 cells. Apoptosis in BSC-1 cells appears to be triggered by a cytoplasmic event, since inactivation of GDVII viral RNA by UV light abolished the ability of the virus to induce apoptosis. The possible role of apoptosis in the pathogenesis of TMEV infection in mice, especially virus persistence in central nervous system macrophages, is discussed.     

A phenotypic host range alteration determines RD114 virus restriction in feline embryonic cells

We have characterized the restriction mechanism for RD114 virus replication in embryonic feline cells (FeF). By comparing growth properties of the virus in FeF cells with its behavior in a fetal feline glial cell line (G355) permissive for RD114, we showed that both cell lines were readily infectible by virus grown in permissive cells and that no significant differences in viral integration or viral RNA expression could be detected. However, analysis of viral protein expression revealed differences in viral env gene processing in the two cell types. Envelope precursor pR85 was produced, but the expected processed gp70 product was detectable only in permissive (G355) cells. An envelope product of 85 kDa was packaged into virions produced by FeF cells, while virions produced by G355 cells contained the expected RD114 gp70. While the gp85 env-containing virions were infectious for permissive G355 cells, they were unable to infect FeF cells. The block to infection by the gp85-containing particles in FeF cells could be abrogated by treatment with the glycosylation inhibitor tunicamycin. Our results indicate that restriction of RD114 virus involves a novel mechanism dependent on two factors: altered glycosylation of the envelope to a gp85 form and an altered RD114 receptor in FeF cells.