Action of Interferon in Enucleated Cells

Interferon induces protection of enucleated BSC-1 cells against infectious vesicular stomatitis virus production if cells are treated before, but not after, enucleation.     

Requirement of cell nucleus for Sindbis virus replication in cultured Aedes albopictus cells.

The ability of Sindbis virus to grow in enucleated BHK-21 (vertebrate) and Aedes albopictus (invertebrate) cells was tested to determine the dependence of this virus upon nuclear function in these two phylogenetically unrelated hosts. Although both cell types could be demonstrated to produce viable cytoplasts (enucleated cells) which produced virus-specific antigen subsequent to infection. BHK cytoplasts produced a significant number of progeny virions, whereas mosquito cytoplasts did not. The production of vesicular stomatitis virus in mosquito cells was not significantly reduced by enucleation. That such a host function was not essential for vesicular stomatitis virus growth in insect cells is supported by the observation that the production of this virus by mosquito cells is not actinomycin D sensitive. This result agrees with a previously published report in which it was shown that Sindbis virus maturation in invertebrate cells is inhibited by actinomycin D, indicating a possible requirement for host cell nuclear function (Scheefers-Borchel et al., Virology, 110:292-301, 1981).     

Nucleotide sequence of a cDNA clone carrying the glycoprotein gene of infectious hematopoietic necrosis virus, a fish rhabdovirus.

The nucleotide sequence of the mRNA encoding the glycoprotein of infectious hematopoietic necrosis virus was determined from a cDNA clone containing the entire coding region. The G-protein cDNA is 1,609 nucleotides long (excluding the polyadenylic acid) and encodes a protein of 508 amino acids. The predicted amino acid sequence was compared with that of the glycoprotein of the Indiana and New Jersey serotypes of vesicular stomatitis virus and with the glycoprotein of rabies virus, using a computer program which determined optimal alignment. An amino acid identity of approximately 20% was found between infectious hematopoietic necrosis virus and the two vesicular stomatitis virus serotypes and between infectious hematopoietic necrosis virus and rabies virus. The positions and sizes of the signal sequence and transmembrane domain and the possible glycosylation sites were determined.     

Isolation and characterization of human T cell lines and clones reactive to rabies virus: antigen specificity and production of interferon-gamma

By using a preparation of inactivated rabies virus, the blood mononuclear cells from five rabies vaccine recipients were stimulated in vitro in the presence of interleukin 2. T cell lines that displayed significant proliferative responses to whole rabies virus and to preparations of rabies glycoprotein and nucleocapsid were obtained from all the individuals. Other antigens, such as diphtheria and tetanus toxoids, influenza A virus, hepatitis B surface antigen, and serum albumin, failed to induce the proliferation of the T cell lines. One of these rabies-specific T cell lines was found to proliferate in response to rabies antigens only when the antigen-presenting cells expressed homologous HLA-DR antigens. The use of mouse monoclonal antibodies specific for human T cell surface markers revealed that most of the cells of these rabies-reactive lines were of the helper/inducer class of T lymphocytes. Stimulation of the T cell lines with the rabies antigens induced the production of interferon-gamma, a lymphokine with potent antiviral activity. Several T cell clones were isolated from two of these cell lines, and most of them appeared to be specific for the antigenic components of the viral nucleocapsid. Two T cell clones specific for the rabies glycoprotein were also isolated from one of these lymphocyte interleukin 2-dependent lines. Further in vitro studies with rabies-specific T cells could help us to understand in more depth the role of regulatory T cells in the human immune response to rabies virus.     

Production of neutralizing antibody to rabies and vesicular stomatitis viruses by hybrid cell lines

Hybrid cell lines were obtained following fusion of P 3 × 63 Ag-8 myeloma cells with spleen cells derived from BALB/c mice immunized either with rabies virus or with vesicular stomatitis virus. Hybrid cell lines were selected which continued to secrete rabies virus or vesicular stomatitis virus neutralizing antibody specifically directed against coat glycoprotein of respective viruses.     

Inhibition of group B arbovirus antigen production and replication in cells enucleated with cytochalasin B.

A comparative study of the growth of Sindbis (SIN) virus, a group A arbovirus (togavirus), and Japanese encephalitis (JE) virus, a representative group B arbovirus (togavirus), was conducted in enucleate and nucleate cells. Immunofluorescent tests and yield measurements demonstrated that chicken embryo cells which had been enucleated and subsequently infected with SIN virus produced virus-specific antigens and infectious virus. By contrast, JE failed to replicate or produce virus-specific antigen in cells which had been enucleated before or even 2 h post infection. Studies of the effect of enulceation at various times after infection demonstrated that a nucleus must be present at least 2 and possibly as long as 4 h after infection to produce either JE-specific antigen or infectious JE virus. These studies demonstrate that the replication of SIN, a group A arbovirus (togavirus), which has no nuclear requirement, contrasts sharply with that of a group B arbovirus (togavirus), JE, which may have an initial dependence on a nucleus-associated process.     

Characterization of a porcine kidney cell line resistant to influenza virus infection.

A mutant cell line of porcine kidney cells that resists the cytopathic effect of influenza virus has been obtained and characterized. These cells, designated ESK-R, were originally obtained by prolonged cultivation of cells surviving influenza B/Kanagawa/73 virus infection. No infectious virus was recovered from ESK-R cells, and no evidence for the presence of virus antigens in the cells was demonstrated by immunofluorescent staining. ESK-R cells also showed a distinct resistance to various other strains of both types A and B influenza viruses. The growth of mumps, Sendai, or Newcastle disease virus was considerably restricted, but the cell line normally supported the replication of vesicular stomatitis virus. ESK-R cells were found to lack specific receptors for influenza virus as determined by fluorescence-activated cell sorter analyses. The membrane barrier of ESK-R cells was successfully overcome by nonspecific endocytosis of calcium-coprecipitated virus particles followed by production of an appreciable amount of progeny virus.     

The association of the rabies glycoprotein with liposome (immunosome) induces an in vitro specific release of interleukin 2

BALB/c mice were primed by receiving a unique intraperitoneal injection of rabies virus antigens presented as complete inactivated virus (P.V. strain) or as purified glycoproteins either in the aggregated form or in physical combination with liposomes (i.e., in the form of "immunosomes"). The splenocytes of these mice were restimulated, 6-15 days after priming, in culture with rabies virus antigens, and antigen-specific IL-2 production was measured. It was found that rabies antigens presented as immunosomes were as active as the inactivated virus, whereas equivalent amounts of purified glycoproteins were inactive. The optimal amounts of rabies immunosomes used for priming was found to be 0.5 to 0.05 micrograms per mouse.     

Characterization of saturable binding sites for rabies virus.

A specific, saturable receptor for rabies virus was analyzed on cultured cells of neural or non-neural origin. Viral attachment kinetics were enhanced by DEAE-dextran, an effect which in turn enhanced the apparent infectivity of the virus inoculum. Under optimized conditions, the attachment of metabolically labeled ERA strain rabies virus obeyed the laws of mass action, whereby the amount of virus bound to cells varied proportionally with the concentration of cells or virus. Attachment was sensitive to changes of temperature and pH, did not require divalent cations such as Mg2+ or Ca2+, and occurred despite prior treatment of cells with proteolytic or sialic acid-specific enzymes. Saturation of the cell surface with rabies virus could be accomplished with 3 X 10(3) to 15 X 10(3) attached virions per cell. Competition for the rabies receptor occurred with rabies nonpathogenic variant virus, RV194 -2, and vesicular stomatitis virus. Reovirus type 3, another neurotropic virus, failed to inhibit rabies virus binding, and West Nile virus only slightly inhibited rabies virus binding, suggesting independent cellular receptors were recognized by these viruses. Isolated rabies virus glycoprotein failed to compete in an equivalent manner. However, solubilization of BHK-21 cells with octylglucoside yielded a chloroform-methanol-soluble extract which blocked rabies virus attachment. The binding inhibition activity of this extract was resistant to proteases but could be destroyed by phospholipases and neuraminidase, suggesting a phospholipid or glycolipid component at the receptor site. These data provide evidence for a rhabdovirus-common mechanism for cellular attachment to cells in culture.     

Comparison of vesicular stomatitis virus defective interfering particle synthesis in chick embryo and L cells.

A comparison of the ability of vesicular stomatitis virus (VSV) to generate and replicate defective interfering (DI) particles in primary chick embryo (CE) and mouse L cells was investigated as a means of analyzing host control over DI-particle synthesis and interfering capacity. Serial undiluted passage of VSV in CE and L cells indicate that VSV-DI particles are generated and (or) replicate with greater efficiency in CE than in L cells. When DI particles accumulate in L cells, they are able to interfere with infectious particle replication. The DI particles from CE cells interfered to the same extent with infectious particle replication in both CE and L cells. L cells, therefore, are not considered 'low-interference' hosts in which DI particles are produced and do not interfere with infectious virus replication, but rather hosts which restrict the production of DI particles.     

Virus-lymphocyte interactions: inductive signals necessary to render B lymphocytes susceptible to vesicular stomatitis virus infection.

We examined the inductive signals necessary to render B lymphocytes capable of supporting a productive vesicular stomatitis virus infection. Small murine splenic B cells in the G0 phase of the cell cycle were cultured with stimulators which allow progression through various stages in the activation and/or differentiation pathway leading to antibody secretion. We found that vesicular stomatitis virus expression is dependent on the state of B-cell activation and that three distinct phases can be defined. A nonsupportive state, which is defined by the failure to produce infection centers, viral proteins, or PFUs, is characteristic of freshly isolated small B cells, B cells cultured 48 h without further stimulation, or B cells in the G1 phase of the cell cycle induced by culture with T-cell-derived lymphokines. This refractory state was not due to a failure of virus uptake. Activation of G0 B cells with anti-immunoglobulin at doses which allow entry into the S phase rendered them capable of synthesizing viral proteins and increased the number of B cells producing infection centers, without enhancing PFU production on a per cell basis. In contrast, B cells stimulated with multiple inductive signals provided by anti-immunoglobulin and lymphokines showed increased infectious particle production (7 PFU per infection center). Lipopolysaccharide stimulation, acting through another induction pathway, caused the maximum increase in the number of infected B cells and production of infectious particles (25 PFU per infection center).     

Nucleotide sequence and host La protein interactions of rabies virus leader RNA.

Rabies virus leader RNA was detected in infected BHK-21 cell extracts by hybridization to end-labeled genomic RNA. Similar to the leader RNA of vesicular stomatitis virus, the leader RNA of rabies virus was also found to be associated with the La protein by specific immunoprecipitation with antisera from lupus patients. The 3' end of the genomic RNA of rabies virus was sequenced, and the size and termination site of leader RNA were determined. In addition, extension of the sequence into the nucleocapsid gene of rabies virus showed an open reading frame for at least 37 amino acid residues. Sequence relationships between rabies virus and vesicular stomatitis virus leader genes and the possible involvement of the La protein in rhabdovirus biology are discussed.     

Inhibition of N-linked oligosaccharide trimming does not interfere with surface expression of certain integral membrane proteins

The effects of 1-deoxynojirimycin (dNM) and 1-deoxymannojirimycin (dMM), inhibitors of oligosaccharide trimming glucosidase I and mannosidase I, respectively, on the biosynthesis of vesicular stomatitis virus G protein, influenza virus hemagglutinin, and human class I histocompatibility antigens were investigated. Although the oligosaccharides of these membrane glycoproteins were greatly altered, neither dNM nor dMM interferred with their surface expression, as determined by a variety of assays, including accessibility to proteases and antibodies; neither did these drugs inhibit production of infectious virus particles.     

Vaccinia Virus Replication in Enucleate BSC-1 Cells: Particle Production and Synthesis of Viral DNA and Proteins

The growth of vaccinia virus in monolayers of BSC-1 cells enucleated by centrifugation in the presence of cytochalasin B has been studied. No evidence for the production of infectious virus in these cells was obtained, and the production of virus particles was reduced to 8.3% compared with the yield from cytochalasin-treated, uncentrifuged cells. Virus DNA and early and late polypeptides were synthesized with normal timing in enucleate cells, but in reduced amounts; cleavage of structural polypeptide precursors P4a and Px also occurred in enucleate cells. Factories containing immature virus particles were demonstrated in enucleate cells by electron microscopy; these factories were reduced in number and size compared with those found in cytochalasin-treated, uncentrifuged cells.     

Effects of rabies infection on the metabolism of the host-cell: does inhibition of cellular RNA synthesis take place?

Acute infection of cloned BHK21 cells with rabies virus (CVS strain) resulted in a reduction in the amount of cellular RNA, not so rapid and pronounced as with another rhabdovirus, vesicular stomatitis virus (VSV). This decrease in the amount of cellular RNA was shown to be caused by a differential membrane permeability of infected and uninfected BHK21 cells to [3H]-uridine and by a real inhibition of cellular RNA synthesis.     

Virus Replication in Enucleate Cells: Vesicular Stomatitis Virus and Influenza Virus

The requirement of the presence of a nucleus for the replication of vesicular stomatitis virus and influenza virus has been examined by following the growth and development of these viruses in enucleate BS-C-1 cells. Vesicular stomatitis virus replicates normally in enucleate cells with the rate of production of infectious virus, the amount of virus-specific protein synthesis, and the type of proteins produced being essentially the same in nucleate and enucleate cells. Influenza virus does not replicate in enucleate cells, no virus gene products can be detected, and there is no inhibition of cellular protein synthesis.     

Rabies virus replication in primary murine bone marrow macrophages and in human and murine macrophage-like cell lines: implications for viral persistence.

To determine whether rabies viruses replicate in macrophage or macrophage-like cells, several human and murine macrophage-like cell lines, as well as primary cultures of murine bone marrow macrophages, were incubated with the Evelyn-Rokitnicki-Abelseth (ERA) virus and several different street rabies viruses (SRV). ERA rabies virus replicated well in human monocytic U937 and THP-1 cells and murine macrophage IC-21 cells, as well as primary cultures of murine macrophages. Minimal replication was detected in murine monocytic WEHI-3BD- and PU5-1R cells, and ERA virus did not replicate in murine monocytic P388D1 or J774A.1 cells. A tissue culture-adapted SRV of bat origin also replicated in IC-21 and U937 cells. Non-tissue culture-adapted SRV isolated from different animal species, particularly bats, replicated minimally in U937, THP-1, IC-21 cells and primary murine bone marrow macrophages. To determine whether rabies virus replication is dependent upon the state of differentiation of the macrophage-like cell, human promyelocytic HL-60 cells were differentiated with 12-O-tetradecanoylphorbol-13-acetate (TPA). ERA rabies virus replicated in the differentiated HL-60 cells but not in undifferentiated HL-60 cells. Persistent infections were established in macrophage-like U937 cells with ERA rabies virus and SRV, and infectious SRV was isolated from adherent bone marrow cells of mice that had been infected 96 days previously. Virus harvested from persistently infected U937 cells and the adherent bone marrow cells had specifically adapted to each cell. This specificity was shown by the inability of the viruses to infect macrophages other than U937 cells and primary bone marrow macrophages, respectively. Virus titers of the persistently infected U937 cells fluctuated with extended cell passage. After 30 passages, virus released from the cells had lost virulence as shown by its inability to kill intracranially inoculated mice. However, the avirulent virus released from the persistently infected cells was more efficient in infecting and replicating in naive U937 cells than the virus which was used to establish the persistent infection. These results suggest that macrophages may serve as reservoirs of infection in vivo, sequestering virus which may subsequently be activated from its persistent state, resulting in clinical infection and death.     

The Production of Antibody by Invading B Cells Is Required for the Clearance of Rabies Virus from the Central Nervous System

Background

The pathogenesis of rabies is associated with the inability to deliver immune effectors across the blood-brain barrier and to clear virulent rabies virus from CNS tissues. However, the mechanisms that facilitate immune effector entry into CNS tissues are induced by infection with attenuated rabies virus.

Methodology/Principal Findings

Infection of normal mice with attenuated rabies virus but not immunization with killed virus can promote the clearance of pathogenic rabies virus from the CNS. T cell activity in B cell–deficient mice can control the replication of attenuated virus in the CNS, but viral mRNA persists. Low levels of passively administered rabies virus–neutralizing antibody reach infected cells in the cerebellum of B cell–deficient mice but are not sufficient to mediate virus clearance. Production of rabies virus-specific antibody by B cells invading CNS tissues is required for this process, and a substantial proportion of the B cells that accumulate in the CNS of mice infected with attenuated rabies virus produce virus-specific antibodies.

Conclusions/Significance

The mechanisms required for immune effectors to enter rabies virus-infected tissues are induced by infection with attenuated rabies virus but not by infection with pathogenic rabies viruses or immunization with killed virus. T cell activities can inhibit rabies virus replication, but the production of rabies virus–specific antibodies by infiltrating B cells, as opposed to the leakage of circulating antibody across the BBB, is critical to elimination of the virus. These findings suggest that a pathogenic rabies virus infection may be treatable after the virus has reached the CNS tissues, providing that the appropriate immune effectors can be targeted to the infected tissues.