Linkage of reduced receptor affinity and superinfection to pathogenesis of TR1.3 murine leukemia virus

TR1.3 is a Friend murine leukemia virus (MLV) that induces selective syncytium induction (SI) of brain capillary endothelial cells (BCEC), intracerebral hemorrhage, and death. Syncytium induction by TR1.3 has been mapped to a single tryptophan-to-glycine conversion at position 102 of the envelope glycoprotein (Env102). The mechanism of SI by TR1.3 was examined here in comparison to the non-syncytium-inducing, nonpathogenic MLV FB29, which displays an identical BCEC tropism. Envelope protein expression and stability on both infected cells and viral particles were not statistically different for TR1.3 and FB29. However, affinity measurements derived using purified envelope receptor binding domain (RBD) revealed a reduction of >1 log in the K(D) of TR1.3 RBD relative to FB29 RBD. Whole-virus particles pseudotyped with TR1.3 Env similarly displayed a markedly reduced binding avidity compared to FB29-pseudotyped viral particles. Lastly, decreased receptor affinity of TR1.3 Env correlated with the failure to block superinfection following acute and chronic infection by TR1.3. These results definitively show that acquisition of a SI phenotype can be directly linked to amino acid changes in retroviral Env that decrease receptor affinity, thereby emphasizing the importance of events downstream of receptor binding in the cell fusion process and pathology.     

Disparate regions of envelope protein regulate syncytium formation versus spongiform encephalopathy in neurological disease induced by murine leukemia virus TR

The murine leukemia virus (MLV) TR1.3 provides an excellent model to study the wide range of retrovirus-induced central nervous system (CNS) pathology and disease. TR1.3 rapidly induces thrombotic events in brain microvessels and causes cell-specific syncytium formation of brain capillary endothelial cells (BCEC). A single amino acid substitution, W102G, in the MLV envelope protein (Env) regulates the pathogenic effects. The role of Env in determining this disease phenotype compared to the induction of spongiform encephalomyelitis with a longer latency, as seen in several other MLV and in human retroviruses, was determined by studying in vitro-attenuated TR1.3. Virus cloned from this selection, termed TRM, induced progressive neurological disease characterized by ataxia and paralysis and the appearance of spongiform neurodegeneration throughout the brain stem and spinal cord. This disease was associated with virus replication in both BCEC and highly ramified glial cells. TRM did not induce syncytium formation, either in vivo or in vitro. Sequence and mutational analyses demonstrated that TRM contained a reversion of Env G102W but that neurological disease mapped to the single amino acid substitution Env S159P. The results demonstrate that single nucleotide changes within disparate regions of Env control dramatically different CNS disease patterns.     

TR1.3 viral pathogenesis and syncytium formation are linked to Env-Gag cooperation

Infection with murine leukemia virus (MLV) TR1.3 or the related molecular construct W102G causes severe neuropathology in vivo. Infection is causally linked to the development of extensive syncytia in brain capillary endothelial cells (BCEC). These viruses also induce cell fusion of murine cell lines, such as SC-1 and NIH 3T3, which are otherwise resistant to MLV-induced syncytium formation. Although the virulence of these viruses maps within the env gene, the mechanism of fusion enhancement is not fully determined. To this end, we examined the capacity of the syncytium-inducing (SI) TR1.3 and W102G MLVs to overcome the fusion inhibitory activity inherent in the full-length Env cytoplasmic tail. These studies showed that the TR1.3 and W102G Envs did not induce premature cleavage of p2E, nor did they override p2E fusion inhibition. Indeed, in the presence of mutations that disrupt p2E function, the TR1.3 and W102G Envs significantly increased the extent of cell fusion compared to that with the non-syncytium-inducing MLV FB29. Surprisingly, we also observed that TR1.3 and W102G Envs failed to elicit syncytium formation in these in vitro assays. Coexpression of gag-pol with env restored syncytium formation, and accordingly, mutations within gag-pol were used to examine the minimal functional requirements for the SI phenotype. The results indicate that both gag-dependent particle budding and cleavage of p2E are required to activate the SI phenotype of TR1.3 and W102G viruses. Collectively, these data suggest that the TR1.3 and W102G viruses induce cell fusion by the fusion-from-without pathway.     

A point mutation in the env gene of a murine leukemia virus induces syncytium formation and neurologic disease.

TR1.3 is a Friend-related murine leukemia virus that has been shown to cause intracerebral hemorrhages and neurologic disease due to infection and subsequent cytopathology of cerebral vessel endothelium. A striking feature of this pathology is the formation of endothelial cell syncytia. The pathogenesis of this disease has now been mapped to a single amino acid substitution of tryptophan to glycine in the variable region of the envelope protein. This same mutation enabled TR1.3 to form syncytia and retard cell proliferation in vitro in the SC-1 mouse embryoblast line but did not affect the pH dependence of viral entry. These results demonstrate that subtle molecular changes in retroviral env genes can induce both syncytium formation and overt clinical disease.     

Induction of Syncytia by Neuropathogenic Murine Leukemia Viruses Depends on Receptor Density, Host Cell Determinants, and the Intrinsic Fusion Potential of Envelope Protein

Infection by the neuropathogenic murine leukemia virus (MLV) TR1.3 results in hemorrhagic disease that correlates directly to in vivo syncytium formation of brain capillary endothelial cells (BCEC). This phenotype maps to amino acid 102 in the envelope (Env) protein of TR1.3. Substitution of glycine (G) for tryptophan (W) at this position (W102G Env) in the nonpathogenic MLV FB29 induces both syncytium formation and neurologic disease in vivo. Using an in vitro gene reporter cell fusion assay, we showed that fusion either with murine NIH 3T3 cells or with nonmurine target cells that expressed receptors at or below endogenous murine levels mirrored that seen in BCEC in vivo. In these instances only TR1.3 and W102G Env induced cell fusion. In contrast, when receptor levels on nonmurine cells were raised above endogenous murine levels, FB29 Env was as fusogenic as the neuropathogenic TR1.3 and W102G Env. These results indicate that TR1.3 Env and W102G Env are intrinsically more fusogenic than FB29 Env, that the induction of fusion requires a threshold number of receptors that is greater for FB29 Env than for TR1.3 or W102G Env, and that receptor density on murine NIH 3T3 cells and BCEC is below the threshold for FB29-dependent fusion. Surprisingly, receptor density on NIH 3T3 cells could not be increased by stable expression of exogenous receptors, and FB29-dependent fusion was not observed in NIH 3T3 cells that transiently expressed elevated receptor numbers. These results suggest that an additional undefined host cell factor(s) may limit both receptor expression and fusion potential in murine cells.     

Quinolinic Acid Levels in a Murine Retrovirus-Induced Immunodeficiency Syndrome

Abstract: Mice infected with the retrovirus mixture designated LP-BM5 murine leukemia virus (MuLV) develop an immunosuppressive disease. Quinolinic acid (QUIN) is an endogenous neurotoxic N -methyl- d -aspartate agonist that may contribute to the pathogenesis of HIV-associated neurologic disease. In the present study, the levels of QUIN in brain and blood were measured in mice infected with LP-BM5 MuLV and compared with those in uninfected mice and mice infected with the nonpathogenic strain of ecotropic MuLV (helper component of LP-BM5 MuLV). Infection with LP-BM5 MuLV resulted in progressive increases in blood QUIN levels beginning 2 weeks after inoculation that peaked by 16 weeks postinfection. QUIN levels were also increased in cerebral cortex, hippocampus, and striatum. In systemic tissues, QUIN levels were increased in lung, liver, and spleen. In contrast, infection with the ecotropic viral component of the LP-BM5 MuLV mixture was not associated with any changes in brain, blood, or systemic tissue QUIN levels, even though helper virus burdens were comparable to those in mice infected with LP-BM5 MuLV. Treatment of LP-BM5 MuLV-infected mice with the antiretroviral agent zidovudine (azidothymidine) significantly reduced blood and brain QUIN levels in association with reductions in viral load in brain and spleen. These observations suggest that elevated QUIN production is not attributable to productive infection with retrovirus per se but occurs in response to an agent or agents, such as cytokines, that are produced by the host in response to virus infection.     

Role of immunity in age-related resistance to paralysis after murine leukemia virus infection.

Resistance to the paralytic effects of a wild mouse (Cas-Br-M) murine leukemia virus infection develops with age and is complete by 10 days of age in susceptible NFS mice. The possibility that cell-mediated immunity plays a significant role in this resistance was suggested by the observation that treatment of 10-day-old mice with antithymocyte serum rendered them susceptible to paralysis. By comparison, mice rendered incapable of generating a humoral immune response by treatment from birth to 1 month of age with anti-immunoglobulin M serum did not develop paralysis after challenge with virus at day 10. Transfer of unseparated and T-cell-enriched populations of Cas-Br-M murine leukemia virus-immune spleen cells protected neonatally infected NFS recipients from paralysis; transfer of Cas-Br-M murine leukemia virus-immune populations enriched for B cells delayed the onset but did not ultimately protect neonatally infected NFS mice from paralysis. Transfer of naive adult spleen cells had no protective effect in neonatally infected NFS mice. High-level virus replication occurred in the spleens and brains of all mice that developed paralysis regardless of treatment; low-level virus replication in spleen and barely detectable replication in brain occurred in mice that remained clinically normal. These studies suggest that the age-acquired resistance to the paralytic effect of Cas-Br-M murine leukemia virus infection is immunologically mediated and that T cells may play a major role.     

The major virus-producing cell type during murine cytomegalovirus infection, the hepatocyte, is not the source of virus dissemination in the host

The course of systemic viral infections is determined by the virus productivity of infected cell types and the efficiency of virus dissemination throughout the host. Here, we used a cell-type-specific virus labeling system to quantitatively track virus progeny during murine cytomegalovirus infection. We infected mice that expressed Cre recombinase selectively in vascular endothelial cells or hepatocytes with a murine cytomegalovirus for which Cre-mediated recombination would generate a fluorescently labeled virus. We showed that endothelial cells and hepatocytes produced virus after direct infection. However, in the liver, the main contributor to viral load in the mouse, most viruses were produced by directly infected hepatocytes. Remarkably, although virus produced in hepatocytes spread to hepatic endothelial cells (and vice versa), there was no significant spread from the liver to other organs. Thus, the cell type producing the most viruses was not necessarily the one responsible for virus dissemination within the host.     

Effects of an epitope-specific CD8+ T-cell response on murine coronavirus central nervous system disease: protection from virus replication and antigen spread and selection of epitope escape mutants

Both CD4(+) and CD8(+) T cells are required for clearance of the murine coronavirus mouse hepatitis virus (MHV) during acute infection. We investigated the effects of an epitope-specific CD8(+) T-cell response on acute infection of MHV, strain A59, in the murine CNS. Mice with CD8(+) T cells specific for gp33-41 (an H-2D(b)-restricted CD8(+) T-cell epitope derived from lymphocytic choriomeningitis glycoprotein) were infected with a recombinant MHV-A59, also expressing gp33-41, as a fusion protein with enhanced green fluorescent protein (EGFP). By 5 days postinfection, these mice showed significantly (approximately 20-fold) lower titers of infectious virus in the brain compared to control mice. Furthermore mice with gp33-41-specific CD8(+) cells exhibited much reduced levels of viral antigen in the brain as measured by immunohistochemistry using an antibody directed against viral nucleocapsid. More than 90% of the viruses recovered from brain lysates of such protected mice, at 5 days postinfection, had lost the ability to express EGFP and had deletions in their genomes encompassing EGFP and gp33-41. In addition, genomes of viruses from about half the plaques that retained the EGFP gene had mutations within the gp33-41 epitope. On the other hand, gp33-41-specific cells failed to protect perforin-deficient mice from infection by the recombinant MHV expressing gp33, indicating that perforin-mediated mechanisms were needed. Virus recovered from perforin-deficient mice did not exhibit loss of EGFP expression and the gp33-41 epitope. These observations suggest that the cytotoxic T-cell response to gp33-41 exerts a strong immune pressure that quickly selects epitope escape mutants to gp33-41.     

In vivo interactions of ecotropic and polytropic murine leukemia viruses in mixed retrovirus infections

Mixed retrovirus infections are the rule rather than the exception in mice and other species, including humans. Interactions of retroviruses in mixed infections and their effects on disease induction are poorly understood. Upon infection of mice, ecotropic retroviruses recombine with endogenous proviruses to generate polytropic viruses that utilize different cellular receptors. Interactions among the retroviruses of this mixed infection facilitate disease induction. Using mice infected with defined mixtures of the ecotropic Friend murine leukemia virus (F-MuLV) and different polytropic viruses, we demonstrate several dramatic effects of mixed infections. Remarkably, inoculation of F-MuLV with polytropic MuLVs completely suppressed the generation of new recombinant viruses and dramatically altered disease induction. Co-inoculation of F-MuLV with one polytropic virus significantly lengthened survival times, while inoculation with another polytropic MuLV induced a rapid and severe neurological disease. In both instances, the level of the polytropic MuLV was increased 100- to 1,000-fold, whereas the ecotropic MuLV level remained unchanged. Surprisingly, nearly all of the polytropic MuLV genomes were packaged within F-MuLV virions (pseudotyped) very soon after infection. At this time, only a fractional percentage of cells in the mouse were infected by either virus, indicating that the co-inoculated viruses had infected the same small subpopulation of susceptible cells. The profound amplification of polytropic MuLVs in coinfected mice may be facilitated by pseudotyping or, alternatively, by transactivation of the polytropic virus in the coinfected cells. This study illustrates the complexity of the interactions between components of mixed retrovirus infections and the dramatic effects of these interactions on disease processes.     

Leukemogenicity and cell transformation mechanisms in vitro by Gross murine leukemia virus: analysis of virus subpopulations.

The leukemogenic activity of Gross murine leukemia virus adapted to rats was tested in W/Fu rats and NIH/Swiss mice. All animals infected with this virus developed thymic and nonthymic T-cell leukemia with a short latency period. It was observed that cell-free extracts from thymic lymphoma tissue of mice and rats, induced by either Gross murine leukemia virus or Gross murine leukemia virus adapted to rats, consisted of both small-plaque-forming and large-plaque-forming viruses, as determined by the XC plaque test. MCF-type virus was found in these virus complexes. Transformed cell foci were induced in SC-1 cell layers by double infection of the cloned MCF-type virus and an ecotropic virus. SC-1 cells containing transformed cell foci were shown to be tumorigenic upon inoculation into nude mice. The formation of transformed cell foci in mink lung cells was also observed after double infection with the cloned MCF-type virus and a xenotropic virus. The possible mechanism of leukemogenesis by endogenous viruses is discussed.     

Retrovirus-mediated single-cell gene knockout technique in adult newborn neurons in vivo

Single-cell genetic manipulation in an intact brain environment is an informative approach to study molecular mechanism of adult neurogenesis. Here, we describe a protocol for retrovirus-mediated single-cell gene knockout in adult new neurons in vivo. A gene of interest is disrupted in adult floxed mice by a vector based on the Moloney murine leukemia retrovirus, expressing Cre recombinase. High-titer retrovirus is prepared by transfecting plasmids into the HEK293T cells and by concentrating the supernatant containing virus. The retrovirus is stereotaxically injected into the dentate gyrus. Cre recombinase is transduced and expressed in a small fraction of adult new neurons in an intact environment, and the gene knockout is highly efficient within the transduced neurons. Virus preparation takes 7 days, but virus injections take less than 1 h per mouse. By changing the survival time of the mice after the injection, one can analyze the effects on new neurons at different ages.     

Infection of cardiomyocytes and induction of left ventricle dysfunction by neurovirulent polytropic murine retrovirus

Viral infections of the heart are a causative factor of myocarditis as well as of sudden, unexpected deaths of children, yet the mechanisms of pathogenesis remain unclear, in part due to the relatively few animal models of virus-induced myocarditis. In the current study, we examined the ability of polytropic murine retroviruses to infect the heart and induce cardiac dysfunction. In situ hybridization and immunohistochemistry analysis detected virus-infected cardiomyocytes and macrophages in the heart. A significant decrease in left ventricle function, as measured by fractional shortening, was detected in mice infected with the neurovirulent retrovirus Fr98 but not in mice infected with the nonneurovirulent retrovirus Fr54. Virus infection was not associated with consistent findings of fibrosis or substantial cellular infiltrate. Fr98-induced left ventricle dysfunction was associated with a higher virus load, increased mRNA expression of the macrophage marker F4/80, increased chemokine production, and a small number of apoptotic cells in the heart.     

Changes in hepatic lipid composition after infection by LP-BM5 murine leukemia virus causing murine AIDS.

The severe hepatic disorders in patients with acquired immune deficiency syndrome (AIDS) is often attributed to a variety of other factors which could affect hepatic function. To evaluate the mechanism of liver damage in murine AIDS-induced immune-suppressed animal, a murine model of AIDS (MAIDS), caused by infection with LP-BM5 murine leukemia virus was used at a late stage of the disease. Retroviral infection significantly increased hepatic cholesterol, triacylgycerol and the cholesterol/phospholipid ratio. Similarly, the proportions of palmitic, palmitoleic, linoleic, ratios of linoleic to arachidonic and saturated to unsaturated fatty acids were significantly lower while the proportion of oleic, docosatetraenoic and docosahexenoic fatty acids were significantly increased in the retrovirus infected mice. Hepatic dysfunction as evidence by increased serum transaminase levels were also observed in the retrovirus infected animals. The data suggest that the liver damage in murine AIDS is induced by retroviral infection and the desaturase enzymes system necessary to maintain regular balance of the fatty acids in the cells may be affected during retroviral infection.     

Inhibition of retrovirus-induced disease in mice by camptothecin.

We have previously shown that noncytotoxic doses of camptothecin (CPT), a topoisomerase I-specific antagonist, inhibit retrovirus replication in acutely and chronically infected cells. To evaluate the efficacy of CPT as an antiretroviral drug in vivo, we injected newborn BALB/c mice with Moloney murine leukemia virus and adult NFS mice with Friend spleen focus-forming virus. The Moloney murine leukemia virus-injected mice developed lymphoma, and the Friend spleen focus-forming virus-injected mice developed erythroleukemia. CPT, administrated together with the virus or 1 or 2 days after virus injection, prevented the onset of the disease in both cases. We showed that repeated CPT treatments increased the effectiveness of the drug when administrated 3 days after virus injection. This ability of CPT to inhibit retrovirus-induced disease in vivo without causing any apparent toxic side effects suggests its application as a legitimate remedy for the treatment of retroviral diseases.     

Enhancement by tumor necrosis factor alpha of dengue virus-induced endothelial cell production of reactive nitrogen and oxygen species is key to hemorrhage development

Hemorrhage is a severe manifestation of dengue disease. Virus strain and host immune response have been implicated as the risk factors for hemorrhage development. To delineate the complex interplay between the virus and the host, we established a dengue hemorrhage model in immune-competent mice. Mice inoculated intradermally with dengue virus develop hemorrhage within 3 days. In the present study, we showed by the presence of NS1 antigen and viral nuclei acid that dengue virus actively infects the endothelium at 12 h and 24 h after inoculation. Temporal studies showed that beginning at day 2, there was macrophage infiltration into the vicinity of the endothelium, increased tumor necrosis factor alpha (TNF-alpha) production, and endothelial cell apoptosis in the tissues. In the meantime, endothelial cells in the hemorrhage tissues expressed inducible nitric oxide synthase (iNOS) and nitrotyrosine. In vitro studies showed that primary mouse and human endothelial cells were productively infected by dengue virus. Infection by dengue virus induced endothelial cell production of reactive nitrogen and oxygen species and apoptotic cell death, which was greatly enhanced by TNF-alpha. N(G)-nitro-L-arginine methyl ester and N-acetyl cysteine reversed the effects of dengue virus and TNF-alpha on endothelial cells. Importantly, hemorrhage development and the severity of hemorrhage were greatly reduced in mice lacking iNOS or p47(phox) or treatment with oxidase inhibitor, pointing to the critical roles of reactive nitrogen and oxygen species in dengue hemorrhage.     

CD8+ T lymphocytes control murine cytomegalovirus replication in the central nervous system of newborn animals

Human CMV infection of the neonatal CNS results in long-term neurologic sequelae. To define the pathogenesis of fetal human CMV CNS infections, we investigated mechanisms of virus clearance from the CNS of neonatal BALB/c mice infected with murine CMV (MCMV). Virus titers peaked in the CNS between postnatal days 10-14 and infectious virus was undetectable by postnatal day 21. Congruent with virus clearance was the recruitment of CD8(+) T cells into the CNS. Depletion of CD8(+) T cells resulted in death by postnatal day 15 in MCMV-infected animals and increased viral loads in the liver, spleen, and the CNS, suggesting an important role for these cells in the control of MCMV replication in the newborn brain. Examination of brain mononuclear cells revealed that CD8(+) T cell infiltrates expressed high levels of CD69, CD44, and CD49d. IE1(168)-specific CD8(+) T cells accumulated in the CNS and produced IFN-gamma and TNF-alpha but not IL-2 following peptide stimulation. Moreover, adoptive transfer of brain mononuclear cells resulted in decreased virus burden in immunodepleted MCMV-infected syngeneic mice. Depletion of the CD8(+) cell population following transfer eliminated control of virus replication. In summary, these results show that functionally mature virus-specific CD8(+) T cells are recruited to the CNS in mice infected with MCMV as neonates.     

Productive measles virus brain infection and apoptosis in CD46 transgenic mice

Measles virus (MV) infection causes acute childhood disease, associated in certain cases with infection of the central nervous system (CNS) and development of neurological disease. To develop a murine model of MV-induced pathology, we generated several lines of transgenic mice ubiquitously expressing as the MV receptor a human CD46 molecule with either a Cyt1 or Cyt2 cytoplasmic tail. All transgenic lines expressed CD46 protein in the brain. Newborn transgenic mice, in contrast to nontransgenic controls, were highly sensitive to intracerebral infection by the MV Edmonston strain. Signs of clinical illness (lack of mobility, tremors, and weight loss) appeared within 5 to 7 days after infection, followed by seizures, paralysis, and death of the infected animals. Virus replication was detected in neurons from infected mice, and virus was reproducibly isolated from transgenic brain tissue. MV-induced apoptosis observed in different brain regions preceded the death of infected animals. Similar results were obtained with mice expressing either a Cyt1 or Cyt2 cytoplasmic tail, demonstrating the ability of different isoforms of CD46 to function as MV receptors in vivo. In addition, maternally transferred immunity delayed death of offspring given a lethal dose of MV. These results document a novel CD46 transgenic murine model where MV neuronal infection is associated with the production of infectious virus, similarly to progressive infectious measles encephalitis seen in immunocompromised patients, and provide a new means to study pathogenesis of MV infection in the CNS.     

Characteristics and contributions of defective, ecotropic, and mink cell focus-inducing viruses involved in a retrovirus-induced immunodeficiency syndrome of mice.

LP-BM5 murine leukemia virus, a derivative of Duplan-Laterjet virus, contains a mixture of replication-competent B-tropic ecotropic and mink cell focus-inducing (MCF) viruses and a defective genome that is the proximal cause of a syndrome, murine AIDS (MAIDS), characterized by lymphoproliferation and immunodeficiency. The defective (BM5d) and ecotropic components of this mixture were molecularly cloned, and complete (BM5d) or partial (ecotropic) nucleotide sequences were determined. BM5d closely resembled the Du5H genome cloned from the Duplan virus, featuring a highly divergent p12 sequence in the gag open reading frame. In MAIDS-sensitive C57BL/6 mice, BM5d was detected in tissues within 2 weeks of infection but was absent from tissues of the MAIDS-resistant strain, A/J, 12 weeks after infection. B-cell-lineage tumors from mice with MAIDS contained and expressed BM5d, and clonal integrations of this genome were variably associated with clonal expansions of B cells in infected mice. Finally, mRNA crosshybridizing with a probe for BM5d was present in spleen but not kidney cells of uninfected B6 mice.