Ectopic expression of murine CD163 enables cell-culture isolation of lactate dehydrogenase-elevating virus 63 years after its discovery.

IMPORTANCE: Mouse models of viral infection play an especially large role in virology. In 1960, a mouse virus, lactate dehydrogenase-elevating virus (LDV), was discovered and found to have the peculiar ability to evade clearance by the immune system, enabling it to persistently infect an individual mouse for its entire lifespan without causing overt disease. However, researchers were unable to grow LDV in culture, ultimately resulting in the demise of this system as a model of failed immunity. We solve this problem by identifying the cell-surface molecule CD163 as the critical missing component in cell-culture systems, enabling the growth of LDV in immortalized cell lines for the first time. This advance creates abundant opportunities for further characterizing LDV in order to study both failed immunity and the family of viruses to which LDV belongs, Arteriviridae (aka, arteriviruses).

Antibody-independent thrombocytopenia in lactate dehydrogenase-elevating virus-infected mice.

Previously we demonstrated that antibody-mediated thrombocytopenia is strongly enhanced by lactate dehydrogenase-elevating virus (LDV) infection. Here we report that mice infected with LDV develop a moderate thrombocytopenia, even in the absence of immunoglobulins or Fc receptors. A similar decrease of platelet counts was observed after mouse hepatitis virus infection. LDV-induced type I interferon-independent thrombocytopenia was partly suppressed by treatment with clodronate-containing liposomes. Therefore, we conclude that the thrombocytopenia results from increased phagocytosis of nonopsonized platelets by macrophages.

The lactate dehydrogenase-elevating virus capsid protein is a nuclear-cytoplasmic protein.

Arteriviruses replicate in the cytoplasm and do not require the nucleus function for virus multiplication in vitro. However, nucleocapsid (N) protein of two arteriviruses, porcine reproductive respiratory syndrome virus and equine arteritis virus, has been observed to localize in the nucleus and nucleolus of virus-infected and N-gene-transfected cells in addition to their normal cytoplasmic distribution. In the present study, the N protein of lactate dehydrogenase-elevating virus (LDV) of mice was examined for nuclear localization. The subcellular localization of LDV-N was determined by tagging N with enhanced green fluorescence protein (EGFP) at the N- and C-terminus. Both N-EGFP and EGFP-N fusion proteins localized to the nucleus and nucleolus of gene-transfected cells. Labeled N also accumulated in the perinuclear region, the site of virus replication. The LDV-N sequence contains a putative 'pat4'-type nuclear localization signal (NLS) consisting of 38-KKKK. To determine its functional significance, a series of deletion constructs of N were generated and individually expressed in cells. The results showed that the 'pat4' NLS was essential for nuclear translocation. In addition, the LDV-N interacted with the importin-alpha and -beta proteins, suggesting that the LDV-N nuclear localization may occur via the importin-mediated nuclear transport pathway. These results provide further evidence for the nuclear localization of N as a common feature within the arteriviruses.

Cell-mediated immunity in cervical cancer evolution.

Cell-mediated immunity (CMI) response to different antigens was examined in healthy women, in patients with cervical precancerous lesions, and in patients with cervical cancer. Cervical lesions were diagnosed by cytological (PAP) smears, from examination by colposcopy, and from "punch" biopsy material by histology. CMI response is related to specific processes in healthy and cancer cells. CMI was investigated by leukocyte adherence inhibition (LAI) assay using specific antigen (prepared from cervical carcinoma tissue) and non specific antigen (prepared from blood of mice infected by LDH--lactate dehydrogenase--virus). The CMI responses of healthy women and cancer patients to the antigens used are different: the majority of T lymphocytes display adherence and non adherence, respectively (but the CMI responses elicited by the antigens are not equal and small quantitative differences are observed). Regardless of the CIN (cervical intraepithelial neoplasia) grades, CMI responses correspond either to healthy women or to cervical carcinoma patients (at about similar ratio of cases in all the CIN groups). Effect of non specific antigen suggests that cervical carcinoma transformation may be connected with reduction of mitochondrial activity similar to processes in LDH virus infection.

Exacerbation of autoantibody-mediated thrombocytopenic purpura by infection with mouse viruses.

Antigenic mimicry has been proposed as a major mechanism by which viruses could trigger the development of immune thrombocytopenic purpura (ITP). However, because antigenic mimicry implies epitope similarities between viral and self antigens, it is difficult to understand how widely different viruses can be involved by this sole mechanism in the pathogenesis of ITP. Here, we report that in mice treated with antiplatelet antibodies at a dose insufficient to induce clinical disease by themselves, infection with lactate dehydrogenase-elevating virus (LDV) was followed by severe thrombocytopenia and by the appearance of petechiae similar to those observed in patients with ITP. A similar exacerbation of antiplatelet-mediated thrombocytopenia was induced by mouse hepatitis virus. This enhancement of antiplatelet antibody pathogenicity by LDV was not observed with F(ab')2 fragments, suggesting that phagocytosis was involved in platelet destruction. Treatment of mice with clodronate-containing liposomes and with total immunoglobulin G (IgG) indicated that platelets were cleared by macrophages. The increase of thrombocytopenia triggered by LDV after administration of antiplatelet antibodies was largely suppressed in animals deficient for gamma-interferon receptor. Together, these results suggest that viruses may exacerbate autoantibody-mediated ITP by activating macrophages through gamma-interferon production, a mechanism that may account for the pathogenic similarities of multiple infectious agents.

Analysis of protein expression by mammalian cell lines stably expressing lactate dehydrogenase-elevating virus ORF 5 and ORF 6 proteins.

Lactate dehydrogenase-elevating virus (LDV) has a strict species-specificity. Because only a subset of mouse primary macrophages have been identified that can support LDV replication in vitro, the precise molecular mechanism of viral entry and replication remains unclear. To analyze the LDV envelope proteins, which probably mediate viral attachment to the host cell, we developed a mammalian system for stable co-expression of LDV open reading frame (ORF) 5- and ORF 6-encoded proteins (ORF 5 and ORF 6 proteins), which correspond to envelope VP-3 and M/VP-2, respectively, and compared these expressed proteins to the native ones. Western blotting analysis combined with N-glycanase digestion revealed that ORF 5 and ORF 6 proteins were similar in size to native VP-3 and M/VP-2, and that ORF 5 protein was N-glycosylated, like the native VP-3. Immunofluorescence microscopy revealed that both ORF 5 and ORF 6 proteins were distributed throughout the cytoplasm and were colocalized in most cells. Moreover, ORF 5 protein was localized both in the perinuclear region and the Golgi complex and transported to the cell surface. This mammalian expression system in which the exogenously expressed proteins closely resemble the native proteins will provide the experimental basis for further studies of the interactions between LDV envelope proteins and host cells.

Mouse hepatitis virus infection of mice causes long-term depletion of lactate dehydrogenase-elevating virus-permissive macrophages and T lymphocyte alterations.

Intraperitoneal injection of pathogen-free B10.A mice with mouse hepatitis virus (MHV)-A59 resulted in a short subclinical infection which was terminated by a rapid antiviral immune response. The infection resulted in a rapid, but transient, about 10-fold increase in the number of macrophages and total cells in the peritoneum of the mice. This increase was preceded by a complete depletion of the peritoneum of the subpopulation of macrophages that supports a productive infection by lactate dehydrogenase-elevating virus (LDV). The depletion of LDV-permissive macrophages was a long-term effect; at 50 days post-infection with MHV, the proportion of LDV-permissive macrophages in the peritoneum had reached only 20% of that observed in the peritoneum of uninfected mice, whereas the total number of macrophages in the peritoneum had returned to normal. Furthermore, MHV infection resulted in a long-term alteration in the proliferative response of spleen T cells to concanavalin A (ConA) and in their ability to produce interferon gamma; several times higher concentrations of ConA were required to induce a maximum proliferative response in spleen T cell populations from 5-week MHV-infected B10.A mice than in spleen T cell populations from infected companion mice but the former produced 5 times more interferon gamma than the T cells from uninfected mice.

Disulfide bonds between two envelope proteins of lactate dehydrogenase-elevating virus are essential for viral infectivity.

Disulfide bonds were found to link the nonglycosylated envelope protein VP-2/M (19 kDa), encoded by open reading frame 6, and the major envelope glycoprotein VP-3 (25 to 42 kDa), encoded by open reading frame 5, of lactate dehydrogenase-elevating virus (LDV). The two proteins comigrated in a complex of 45 to 55 kDa when the virion proteins were electrophoresed under nonreducing conditions but dissociated under reducing conditions. Furthermore, VP-2/M was quantitatively precipitated along with VP-3 in this complex by three neutralizing monoclonal antibodies to VP-3. The infectivity of LDV was rapidly and irreversibly lost during incubation with 5 to 10 mM dithiothreitol (> 99% in 6 h at room temperature), which is known to reduce disulfide bonds. LDV inactivation correlated with dissociation of VP-2/M and VP-3. The results suggest that disulfide bonds between VP-2/M and VP-3 are important for LDV infectivity. Hydrophobic moment analyses of the predicted proteins suggest that VP-2/M and VP-3 both possess three adjacent transmembrane segments and only very short ectodomains (10 and 32 amino acids, respectively) with one and two cysteines, respectively. Inactivation of LDV by dithiothreitol and dissociation of the two envelope proteins were not associated with alterations in LDV's density or sedimentation coefficient.

Ia antigens and Fc receptors of mouse peritoneal macrophages as determinants of susceptibility to lactic dehydrogenase virus.

The relationship between susceptibility of mouse peritoneal macrophages to lactic dehydrogenase-elevating virus (LDV) infection and expression of I region-coded antigens (Ia) on these cells was investigated. The proportion of Ia-positive cells in resident peritoneal macrophages from adult and suckling mice were 4 to 10% and 50 to 70% respectively. Approximately the same percentage of the cells were susceptible to LDV, as detected by fluorescent antibody staining. In adult mice, double-labelling experiments showed that most of the Ia-positive cells were LDV-infected. When the cells were cultured for more than 24 h in vitro, Ia-positive cells rapidly disappeared and the culture became resistant to LDV. Removal of Ia-positive cells by treatment with anti-Ia plus complement or enrichment using an anti-Ia-coated Petri dish simultaneously removed or enriched for LDV-susceptible cells. Treatment of cells with trypsin (1 mg/ml) removed their I-A and I-E antigens and simultaneously abolished susceptibility for LDV. When LDV was preincubated with subneutralizing amounts of antibody, infectivity for macrophages was enhanced and the proportion of LDV-infected cells was higher than that of Ia-positive cells. This suggests that Fc receptors on macrophages can act as receptors for LDV coated with antiviral IgG.

Cell surface receptors for lactate dehydrogenase-elevating virus on subpopulation of macrophages.

We examined the binding and internalization of unlabeled and 125I-labeled, purified lactate dehydrogenase-elevating virus (LDV) by peritoneal macrophages cultured in vitro. Upon incubation of the cells at 4 degrees C with greater than 100 ID50/cell, the virus was surface-bound on a small subpopulation of macrophages (about 5% of the total cells) as determined by electron microscopy, fluorescent antibody staining, and autoradiography of cells incubated with 125I-labeled LDV. At 37 degrees C, LDV particles were seen in intracellular endocytic vesicles also in about 5% of the cells, and the proportion of cells with virus-containing vesicles correlated with the proportion of cells which became productively infected with LDV as assessed by determining LDV RNA synthesis in individual cells and by fluorescent antibody staining. Pretreatment of the resident peritoneal macrophages with trypsin inhibited the binding of 125I-labeled LDV and the productive infection of the cells with the virus. After removal of the trypsin and incubation in complete medium, permissiveness for LDV reappeared after an 8-12 h lag, whereas Fc and C3 receptors reappeared more rapidly after trypsin treatment. Populations of resident peritoneal macrophages, starch-elicited peritoneal macrophages, splenic macrophages, and bone marrow macrophages contained a similar proportion of cells that could be productively infected with LDV. Little, if any, LDV replication was detected in cultures of lung, liver and peripheral blood macrophages as well as in thioglycollate-elicited and BCG-activated macrophages. We conclude that the permissiveness for LDV of resident peritoneal macrophages correlates with the presence of a trypsin-sensitive receptor present on a subpopulation of these cells. The identity of the receptor has not been definitively established. Treatment of macrophages with neuraminidase or various sugars had no significant effect on LDV replication. Lysis of I-A-positive macrophages with a monoclonal antibody and complement reduced the number of macrophages which could be productively infected by 50%, which suggests that macrophages lacking surface Ia can be productively infected with LDV in vitro.

Immune response to lactate dehydrogenase-elevating virus: isolation of infectious virus-immunoglobulin G complexes and quantitation of specific antiviral immunoglobulin G response in wild-type and nude mice.

Lactate dehydrogenase-elevating virus (LDV) causes a normally benign persistent infection of mice, resulting in a life-long viremia characterized by the presence of circulating infectious immune complexes, impaired clearance of certain enzymes from the blood, and modification of the host immune response to various heterologous antigens. In this study, we isolated infectious immunoglobulin G (IgG)-LDV complexes in the plasma of persistently infected mice by adsorption to and elution from protein A-Sepharose CL-4B. We found that practically all infectious LDV in the plasma of persistently infected mice is complexed to IgG. LDV infectivity in these complexes was partially neutralized, but could be reactivated by treatment with 2-mercaptoethanol. We also quantitated total plasma IgG and anti-LDV IgG in wild-type and nude Swiss and BALB/c mice as a function of the time after infection with LDV by radial immunodiffusion and an enzyme-linked immunosorbent assay, respectively. Total plasma IgG levels nearly doubled in BALB/c mice during 150 days of infection. IgG levels in uninfected nude mice were only 20% of those in uninfected BALB/c mice, but during infection with LDV increased to approximately those found in uninfected BALB/c mice. Anti-LDV IgG levels were almost as high in nude mice as in normal BALB/c mice. Isoelectric focusing of purified IgG from BALB/c mice showed that LDV infection resulted in the enhanced synthesis of all 16 normal IgG fractions that we could separate by this method, which suggests that LDV infection results in polyclonal activation of IgG-producing lymphocytes.

Actinomycin D cytotoxicity for mouse peritoneal macrophages and effect on lactate dehydrogenase-elevating virus replication.

Treatment of lactate dehydrogenase-elevating virus(LDV)-infected mouse peritoneal macrophage cultures with actinomycin D (1 microgram/ml) resulted in a progressive reduction in the formation of LDV-specific RNA and mature virus as the time of incubation with actinomycin D increased beyond 2-3 h. This effect, however, seemed to reflect an unusual sensitivity of macrophages to toxic effects of actinomycin D. Macrophage cytotoxicity and lysis became apparent 3-4 h after addition of actinomycin D; the initiation of synthesis of Sindbis virus RNA, which is insensitive to inhibition by actinomycin D in other cell culture systems, was also reduced in actinomycin-D-treated macrophages. Macrophages propagated in L-cell-conditioned medium were found to be less sensitive to actinomycin D cytotoxicity and, correspondingly, the initiation and synthesis of LDV RNA were less affected.

Autoradiographic method for detection of lactate dehydrogenase-elevating virus-infected cells in primary mouse macrophage cultures.

Peritoneal cells from starch-injected Swiss mice were propagated in plastic petri dishes and on cover slips in a mouse L-cell-conditioned medium for 12 to 24 h and then infected with various multiplicities of lactate dehydrogenase-elevating virus (LDV). Over 95% of the cells in these cultures phagocytosed latex particles and were, therefore, considered macrophages. Infected and mock infected macrophage cultures were supplemented with [3H]uridine at various times after infection and with actinomycin D 30 min before addition of the [3H]uridine. After 1 or 2 h of further incubation, plate cultures were analyzed for LDV-specific RNA, and cover slip cultures were investigated by autoradiography. Other cultures were labeled in the absence of actinomycin D, and the culture fluid was analyzed for labeled LDV. There was a good correlation between the production of LDV-specific RNA and LDV and the number of heavily labeled cells in these cultures. The labeled cells in these cultures. The labeled cells, therefore, were equated with productively infected cells. Only a maximum of about 20% of the macrophages, however, became heavily labeled regardless of the multiplicity of infection or the time, after infection, at which the cells were exposed to [3H]uridine. Only background labeling was observed in the remainder of the cells and in mock-infected cells treated with actinomycin D. The highest proportion of labeled cells was observed when the cells were infected with a multiplicity of infection of about 2,000 mouse infectious units per cell and labeled from 6 to 8 h after infection. Thereafter, the proportion of productively infected cells decreased progressively, concomitant with a decrease in the amounts of viral specific RNA and of LDV produced by the cultures. The results indicate that the majority of the macrophages in primary macrophage cultures do not support LDV replication. Their nonpermissiveness may depend on the physiological state of the cells or reflect the presence of subpopulations of macrophages, but no morphological differences between productively infected an uninfected cells were detectable.

Distribution along the axon and into various subcellular fractions of molecules labeled with (3H)leucine and rapidly transported in the garfish olfactory nerve.

The distribution of molecules labeled with [3H]leucine by fast axoplasmic transport in vivo has been studied in the garfish olfactory nerve after incorporation of the amino acid by the olfactory mucosa. Owing to the size of the nerve, it has been possible to follow the fate of the labeled molecules in 10 different subcellular fractions of 6 consecutive nerve segments. Each segment represents a different part of the profile developed by the transported radioactive molecules. In order to determine the influence of the perikaryon (rate of protein synthesis and rate of protein release into the axon) transport was studied under 3 different conditions: (1) intact nerves (simply labeled with [3H]leucine); (2) nerves cut from the cell bodies 6 h after application of [3H]leucine; and (3) nerves pulse-chase labeled for 1 h. Several conclusions can be drawn. (1) The bulk of the rapidly transported molecules are membranous axonal proteins, as determined by enzyme markers. Most are found in subcellular fractions representing 17% of the total axonal protein. They are synthesized very rapidly in the cell bodies (less than 1 h after isotope deposition) and exhibit the highest specific activities measured. These high specific activities were found in the same axonal membrane fractions in both plateau and crest, suggesting that the membrane precursors are transported as particles rather than as subunits. (2) The majority of these proteins are released into the axon immediately after synthesis; however, at least 30% of the labeled axonal membranous proteins are not released with the fast wave itself but progressively over a long period of time. (3) The majority of the moving material, particularly in membranous fractions, is left behind the fast wave and is deposited in the axon. When the front base of the fast wve has covered 70% of the total nerve length, only 19% of the labeled material of the main axonal membranous fraction appears still to be moving. (4) Proteins with high specific activities are found near the cell bodies and may be the result of early axonal transport of amino acids, diffusing later into the surrounding cells and being incorporated into proteins. Some free amino acids are also transported along the axon.