John L. Portis

Characterization of mouse monoclonal antibodies specific for friend murine leukemia virus-induced erythroleukemia cells: Friend-specific and FMR-specific antigens

Abstract Monoclonal antibodies were derived from hybrid cell lines produced by fusing mouse myeloma cells with spleen cells from mice recovering from Friend virus-induced erythroleukemia. Of the 17 clones characterized, two appeared to have the Friend, Moloney, Rauscher pattern of specificity. One of these was specific for the envelope protein, gp70, and the other reacted with a core protein, p15. Seven other anti-gp70 clones and one anti-p15 clone were restricted in reactivity to cells infected with Friend or Rauscher viruses only. One clone reacted with p15E and recognized this protein on many ecotropic and dual-tropic viruses. In addition, six IgM antibodies were obtained which appeared to recognize nonviral antigens present only on leukemia cells of the erythroid lineage. Six monoclonal antibodies of complement-fixing immunoglobulin classes with specificity for gp70 or p15 were compared for their ability to bind or lyse erythroleukemia cells in the presence of complement. Individual antibodies to the same viral protein appeared to differ markedly in their ability to mediate cytolysis.

Endoplasmic Reticulum Stress Is a Determinant of Retrovirus-Induced Spongiform Neurodegeneration

ABSTRACT FrCasE is a mouse retrovirus that causes a fatal noninflammatory spongiform neurodegenerative disease with pathological features strikingly similar to those induced by transmissible spongiform encephalopathy (TSE) agents. Neurovirulence is determined by the sequence of the viral envelope protein, though the specific role of this protein in disease pathogenesis is not known. In the present study, we compared host gene expression in the brain stems of mice infected with either FrCasE or the avirulent virus F43, differing from FrCasE in the sequence of the envelope gene. Four of the 12 disease-specific transcripts up-regulated during the preclinical period represent responses linked to the accumulation of unfolded proteins in the endoplasmic reticulum (ER). Among these genes was CHOP/GADD153, which is induced in response to conditions that perturb endoplasmic reticulum function. In vitro studies with NIH 3T3 cells revealed up-regulation of CHOP as well as BiP, calreticulin, and Grp58/ERp57 in cells infected with FrCasE but not with F43. Immunoblot analysis of infected NIH 3T3 cells demonstrated the accumulation of uncleaved envelope precursor protein in FrCasE- but not F43-infected cells, consistent with ER retention. These results suggest that retrovirus-induced spongiform neurodegeneration represents a protein-folding disease and thus may provide a useful tool for exploring the causal link between protein misfolding and the cytopathology that it causes.

Murine retrovirus-induced spongiform encephalopathy: Productive infection of microglia and cerebellar neurons in accelerated CNS disease

We have examined the pathological lesions and sites of infection in mice inoculated with a highly neurovirulent recombinant wild mouse ecotropic retrovirus (FrCasE). The spongiform lesions appeared initially as swollen postsynaptic neuronal processes, progressing to swelling in neuronal cell bodies, all in the absence of detectable gliosis. Infection of neurons in regions of vacuolation was not detected. However, high level infection of cerebellar granule neurons was observed in the absence of cytopathology, wherein viral protein was found associated with both axons and dendrites. Infection of ramified and amoeboid microglial cells was associated with cytopathology in the brain stem, and endothelial cell-pericyte infection was found throughout the CNS. No evidence of defective retroviral expression was observed. These results are consistent with an indirect mechanism of retrovirus-induced neuropathology.

Differences in Cytokine and Chemokine Responses during Neurological Disease Induced by Polytropic Murine Retroviruses Map to Separate Regions of the Viral Envelope Gene

ABSTRACT Infection of the central nervous system (CNS) by several viruses can lead to upregulation of proinflammatory cytokines and chemokines. In immunocompetent adults, these molecules induce prominent inflammatory infiltrates. However, with immunosuppressive retroviruses, such as human immunodeficiency virus (HIV), little CNS inflammation is observed yet proinflammatory cytokines and chemokines are still upregulated in some patients and may mediate pathogenesis. The present study examined expression of cytokines and chemokines in brain tissue of neonatal mice infected with virulent (Fr98) and avirulent (Fr54) polytropic murine retroviruses. While both viruses infect microglia and endothelia primarily in the white matter areas of the CNS, only Fr98 induces clinical CNS disease. The pathology consists of gliosis with minimal morphological changes and no inflammation, similar to HIV. In the present experiments, mice infected with Fr98 had increased cerebellar mRNA levels of proinflammatory cytokines tumor necrosis factor alpha (TNF-α), TNF-β, and interleukin-1α and chemokines macrophage inflammatory protein-1α (MIP-1α), MIP-1β, monocyte chemoattractant protein 1 (MCP-1), gamma-interferon-inducible protein 10 (IP-10), and RANTES compared to mice infected with Fr54 or mock-infected controls. The increased expression of these genes occurred prior to the development of clinical symptoms, suggesting that these cytokines and chemokines might be involved in induction of neuropathogenesis. Two separate regions of the Fr98 envelope gene are associated with neurovirulence. CNS disease associated with the N-terminal portion of the Fr98 envgene was preceded by upregulation of cytokines and chemokines. In contrast, disease associated with the central region of the Fr98env gene showed no upregulation of cytokines or chemokines and thus did not require increased expression of these genes for disease induction.

Effect of murine host genotype on MCF virus expression, latency, and leukemia cell type of leukemias induced by Friend murine leukemia helper virus

Leukemias induced by neonatal inoculations of several mouse strains with different strains of Friend murine leukemia helper virus (F-MuLV) were followed for time of disease onset, cytochemical analysis of predominant cell types in leukemic organs, and expression of infectious mink cell focus-inducing (MCF) viruses detected by mink cell foci or MCF-specific monoclonal antibodies. Most BALB.B and IRW mice had a rapidly appearing, severe anemia and hepatosplenomegaly consisting of erythroid cells. MCF viruses were usually isolated from enlarged spleens of IRW mice. In contrast, C57BL/10 mice had a lower incidence of disease and much slower course. Splenomegaly and lymphadenopathy with mild anemia were seen, and the predominant cell types were either myeloid (chloroleukemia) or lymphoid. MCF viruses were never isolated from this mouse strain. (C57BL/10 X IRW)F1 mice were intermediate in latency, but all mice had disease by 8 months. Myeloid, lymphoid, and some mixed leukemias with an erythroid component were observed, but in no case did we see the severe anemia or pure erythroid involvement typical of IRW and BALB.B mice. MCF viruses were, however, isolated from 22% of these mice regardless of leukemia cell type. DBA/2 mice had a disease pattern similar to the (C57BL/10 X IRW)F1 mice, and MCF viruses were isolated from three of six mice tested. Inoculation of IRW mice with the low virulence B3 strain of F-MuLV produced disease with a longer latency than F-MuLV 57, but similar cell types were transformed by both viruses. In vitro cell lines were derived from 14 mice, and most were tumorigenic in vivo. Three lines released infectious MCF virus, and three others expressed MCF-specific cell surface antigens but did not release virus. Eight lines expressed no MCF infectious virus or viral antigens. Several lines released infectious xenotropic viruses and/or expressed xenotropic MuLV cell surface antigens recognized by monoclonal antibodies reactive with xenotropic viruses. The lack of MCF expression in many primary leukemic tissues as well as in in vitro derived leukemia cell lines of C57BL/10 and (B10 X IRW)F1 mice suggested that MCF virus generation and expression may not be required for leukemogenesis in some mouse strains or in some hemopoietic lineages.

The Glycosylated Gag Protein of a Murine Leukemia Virus Inhibits the Antiretroviral Function of APOBEC3

ABSTRACT APOBEC proteins have evolved as innate defenses against retroviral infections. Human immunodeficiency virus (HIV) encodes the Vif protein to evade human APOBEC3G; however, mouse retroviruses do not encode a Vif homologue, and it has not been understood how they evade mouse APOBEC3. We report here a murine leukemia virus (MuLV) that utilizes its glycosylated Gag protein (gGag) to evade APOBEC3. gGag is critical for infection of in vitro cell lines in the presence of APOBEC3. Furthermore, a gGag-deficient virus restricted for replication in wild-type mice replicates efficiently in APOBEC3 knockout mice, implying a novel role of gGag in circumventing the action of APOBEC3 in vivo.

Monoclonal antibodies to xenotropic and mcf murine leukemia viruses derived during the graft-versus-host reaction.

Abstract The humoral immune response during adult graft-versus-host reaction (GVHR) was assessed by the recovery of antibody-forming host spleen cells using cell fusion techniques. Two parent → F 1 strain combinations were studied, (B6 × D2)F 1 and (NFS × AKR)F 1 injected with the respective parental spleen cells. Hybridomas derived from recipient spleens were found to produce antibody with predominant specificity for murine leukemia virus (MuLV) envelope (env) polypeptides. The recovery of anti-MuLV monoclonal antibodies was dependent on the donor strain. Thus, D2 → (B6 × D2)F 1 and AKR → (NFS × AKR) resulted in a high incidence of anti-MuLV antibody production among the primary fusion products whereas no anti-MuLV hybridomas were recovered when B6 or NFS, respectively, were used as donors. Hybridomas derived from D2 → (B6 × D2)F 1 produced anti-MuLV antibodies of two general specificities: (1) broadly reactive, detecting determinants expressed by ecotropic and xenotropic MuLV, and (2) xenotropic MuLV specific. The latter group included antibodies reacting with all xenotropic MuLV and antibodies with xenotropic MuLV strain specificity. Xenotropic MuLV-specific determinants tere expressed by gp70, p15(E), and the gp70-p15(E) complex (gp90). This is to our knowledge, the first report of monoclonal antibodies specific for xenotropic MuLV. In contrast, hybridomas derived from AKR → (NFS × AKR) produced antibodies which reacted predominantly with unique determinants of a subgroup of MCF viruses. These results suggested that the anti-MuLV antibody repertoire expressed during GVHR is influenced by the endogenous MuLV of the respective mouse strain combination.

Inflammation and Clearance of Chlamydia trachomatis in Enteric and Nonenteric Mucosae

ABSTRACT Immunization(s) fostering the induction of genital mucosa-targeted immune effectors is the goal of vaccines against sexually transmitted diseases. However, it is uncertain whether vaccine administration should be based on the current assumptions about the common mucosal immune system. We investigated the relationship between mucosal sites of infection, infection-induced inflammation, and immune-mediated bacterial clearance in mice using the epitheliotropic pathogenChlamydia trachomatis. Chlamydial infection of the conjunctival, pulmonary, or genital mucosae stimulated significant changes in tissue architecture with dramatic up-regulation of the vascular addressin, VCAM, a vigorous mixed-cell inflammatory response with an influx of α4β1+ T cells, and clearance of bacteria within 30 days. Conversely, intestinal mucosa infection was physiologically inapparent, with no change in expression of the local MAdCAM addressin, no VCAM induction, no histologically detectable inflammation, and no tissue pathology. Microbial clearance was complete within 60 days in the small intestine but bacterial titers remained at high levels for at least 8 months in the large intestine. These findings are compatible with the notion that VCAM plays a functional role in recruiting cells to inflammatory foci, and its absence from the intestinal mucosa contributes to immunologic homeostasis at that site. Also, expression of type 1 T cell-mediated immunity to intracellular Chlamydia may exhibit tissue-specific variation, with the rate and possibly the mechanism(s) of clearance differing between enteric and nonenteric mucosae. The implications of these data for the common mucosal immune system and the delivery of vaccines against mucosal pathogens are discussed.

Increased Expression of MIP-1α and MIP-1β mRNAs in the Brain Correlates Spatially and Temporally with the Spongiform Neurodegeneration Induced by a Murine Oncornavirus

ABSTRACT The chimeric murine oncornavirus FrCasE causes a rapidly progressive paralytic disease associated with spongiform neurodegeneration throughout the neuroaxis. Neurovirulence is determined by the sequence of the viral envelope gene and by the capacity of the virus to infect microglia. The neurocytopathic effect of this virus appears to be indirect, since the cells which degenerate are not infected. In the present study we have examined the possible role of inflammatory responses in this disease and have used as a control the virus F43. F43 is an highly neuroinvasive but avirulent virus which differs from FrCasE only in 3′pol and env sequences. Like FrCasE, F43 infects large numbers of microglial cells, but it does not induce spongiform neurodegeneration. RNAase protection assays were used to detect differential expression of genes encoding a variety of cytokines, chemokines, and inflammatory cell-specific markers. Tumor necrosis factor alpha (TNF-α) and TNF-β mRNAs were upregulated in advanced stages of disease but not early, even in regions with prominent spongiosis. Surprisingly there was no evidence for upregulation of the cytokines interleukin-1α (IL-1α), IL-1β, and IL-6 or of the microglial marker F4/80 at any stage of this disease. In contrast, increased levels of the β-chemokines MIP-1α and -β were seen early in the disease and were concentrated in regions of the brain rich in spongiosis, and the magnitude of responses was similar to that observed in the brains of mice injected with the glutamatergic neurotoxin ibotenic acid. MIP-1α and MIP-1β mRNAs were also upregulated in F43-inoculated mice, but the responses were three- to fivefold lower and occurred later in the course of infection than was observed in FrCasE-inoculated mice. These results suggest that the robust increase in expression of MIP-1α and MIP-1β in the brain represents a correlate of neurovirulence in this disease, whereas the TNF responses are likely secondary events.

Monoclonal antibodies derived during graft-versus-host reaction II. Antibodies detect unique determinants common to many MCF viruses

Hybridoma cell lines were recovered from the spleens of 6-week-old (B6 X D2)F1 mice undergoing graft-versus-host reaction induced by the transfer of 5-week-old B6 parental spleen cells. These cell lines produced antibodies reactive with envelope polypeptides of a variety of MuLV. The viral specificity assessed by membrane immunofluorescence and virus-binding radioimmunoassay indicated that the reactivity of these antibodies was distinctly different from monoclonal antibodies recovered from (B6 X D2)F1 recipients of D2 spleen cells reported previously (Portis et al., Virology 118, 181-190, 1982). Ten out of 17 monoclonal antibodies in the current study reacted exclusively with MCF viruses and three of these antibodies detected envelope determinants which were shared by a broad panel of MCF viruses of diverse origin. These common MCF determinants were expressed by the gp70 molecule as determined by Western blot analysis. The production of these antibodies by young mice in the absence of exogenous virus inoculation suggests that these antigens may be encoded by endogenous MCF-like sequences.