Endogenous retrovirus Gag antigen and its gene variants are unique autoantigens expressed in the pancreatic islets of non-obese diabetic mice.

Endogenous retrovirus (ERV) are remnants of ancient retroviruses that have been incorporated into the genome and evidence suggests that they may play a role in the etiology of T1D. We previously identified a murine leukemia retrovirus-like ERV whose Env and Gag antigens are involved in autoimmune responses in non-obese diabetic (NOD) mice. In this study, we show that the Gag antigen is present in the islet stromal cells. Although Gag gene transcripts were present, Gag protein was not detected in diabetes-resistant mice. Cloning and sequencing analysis of individual Gag genes revealed that NOD islets express Gag gene variants with complete open-reading frames (ORFs), in contrast to the diabetes-resistant mice, whose islet Gag gene transcripts are mostly non-ORFs. Importantly, the ORFs obtained from the NOD islets are extremely heterogenous, coding for various mutants that are absence in the genome. We further show that Gag antigens are stimulatory for autoreactive T cells and identified one islet-expressing Gag variant that contains an altered peptide ligand capable of inducing IFN-gamma release by the T cells. The data highlight a unique retrovirus-like factor in the islets of the NOD mouse strain, which may participate in key events triggering autoimmunity and T1D.

Mouse APOBEC3 expression in NIH 3T3 cells mediates hypermutation of AKV murine leukemia virus.

Mouse APOBEC3 (mA3) is a cytidine deaminase that can act on the single-stranded DNA reverse transcripts of retroviruses resulting in G→A hypermutation of proviral DNA. Many mA3 studies have used NIH 3T3 cells assuming that endogenous mA3 production was negligible. We developed a monoclonal antibody specific for mA3 that reveals detectable mA3 in NIH 3T3 cells and we demonstrate that AKV released from the cells undergoes G→A hypermutation. Inactivation of the mA3 gene abolished the deamination confirming that AKV hypermutation was mediated by mA3. The G→A mutations in AKV viral transcripts deviated from a normal distribution with all the mutations contained within only 20% of the transcripts. Single cell analyses revealed that the expression of mA3 in NIH 3T3 cells was limited to 20% of the cells, which likely accounted for the abnormal distribution of mutations. Endogenous NIH 3T3 mA3 was found to restrict AKV replication.

Latent murine leukemia virus infection characterized by the release of non-infectious virions.

Clonal cell lines derived from cultures infected with a polytropic MuLV release vastly different levels of infectious virions ranging from undetectable to very high. Low producing clones release an overwhelming proportion of non-infectious virions containing retroviral RNA but deficient in the Env protein. Non-infectious virion production is not due to an inability of the cells to support infectious MuLV production or to an inherent replicative defectiveness of the proviruses. Reinfection of the lowest producing lines with the polytropic or an ecotropic MuLV results in enormous increases in the specific infectivity of the released virions. This indicates a reversible state of retroviral latency characterized by the release of non-infectious virions that is likely the result of insufficient levels of Env protein required for infectivity. The latency state described here may have important roles in in vivo retroviral infections including alterations of the immune response and the production of defective interfering particles.

Endogenous retroviruses mobilized during friend murine leukemia virus infection.

We have demonstrated in a mouse model that infection with a retrovirus can lead not only to the generation of recombinants between exogenous and endogenous gammaretrovirus, but also to the mobilization of endogenous proviruses by pseudotyping entire polytropic proviral transcripts and facilitating their infectious spread to new cells. However, the frequency of this occurrence, the kinetics, and the identity of mobilized endogenous proviruses was unclear. Here we find that these mobilized transcripts are detected after only one day of infection. They predominate over recombinant polytropic viruses early in infection, persist throughout the course of disease and are comprised of multiple different polytropic proviruses. Other endogenous retroviral elements such as intracisternal A particles (IAPs) were not detected. The integration of the endogenous transcripts into new cells could result in loss of transcriptional control and elevated expression which may facilitate pathogenesis, perhaps by contributing to the generation of polytropic recombinant viruses.

Incorporation of mouse APOBEC3 into murine leukemia virus virions decreases the activity and fidelity of reverse transcriptase.

APOBEC3 proteins are restriction factors that induce G→A hypermutation in retroviruses during replication as a result of cytidine deamination of minus-strand DNA transcripts. However, the mechanism of APOBEC inhibition of murine leukemia viruses (MuLVs) does not appear to be G→A hypermutation and is unclear. In this report, the incorporation of mA3 in virions resulted in a loss in virion reverse transcriptase (RT) activity and RT fidelity that correlated with the loss of virion-specific infectivity.

Analysis of two monoclonal antibodies reactive with envelope proteins of murine retroviruses: one pan specific antibody and one specific for Moloney leukemia virus.

Many monoclonal antibodies (MAbs) reactive with various proteins of murine leukemia viruses (MuLVs) have been developed. In this report two additional MAbs with differing and unusual specificities are described. MAb 573 is reactive with the envelope protein of all MuLVs tested including viruses in the ecotropic, xenotropic, polytropic and amphotropic classes. Notably, MAb 573 is one of only two reported MAbs that react with the envelope protein of amphotropic MuLVs. This MAb appears to recognize a conformational epitope within the envelope protein, as it reacts strongly with live virus and live infected cells, but does not react with formalin-fixed or alcohol-fixed infected cells or denatured viral envelope protein in immunoblots. In contrast, Mab 538 reacts only with an epitope unique to the envelope protein of the Moloney (Mo-) strain of MuLV, a prototypic ecotropic MuLV that is the basis for many retroviral tools used in molecular biology. MAb 538 can react with live cells and viruses, or detergent denatured or fixed envelope protein. The derivation of these antibodies as well as their characterization with regard to their isotype, range of reactivity with different MuLVs and utility in different immunological procedures are described in this study.

Three Sgp loci act independently as well as synergistically to elevate the expression of specific endogenous retroviruses implicated in murine lupus.

Endogenous retroviruses are implicated in murine lupus nephritis. They provide a source of nephritogenic retroviral gp70-anti-gp70 immune complexes through the production of serum gp70 protein and anti-gp70 autoantibodies as a result of the activation of TLR7. The Sgp (serum gp70 production) loci identified in lupus-prone mice play distinct roles for the expression of different classes of endogenous retroviruses, as Sgp3 regulates the transcription of xenotropic, polytropic and modified polytropic (mPT) viruses, and Sgp4 the transcription of only xenotropic viruses. In the present study, we extended these analyses to a third locus, Sgp5, using BALB/c mice congenic for the NZW-derived Sgp5 allele and also explored the possible interaction of Sgp3 and Sgp4 loci to promote the expression of endogenous retroviruses and serum gp70. The analysis of Sgp5 BALB/c congenic mice demonstrated that the Sgp5 locus enhanced the expression of xenotropic and mPT viruses, thereby upregulating the production of serum gp70. These data indicate a distinct action of the Sgp5 locus on the expression of endogenous retroviruses, as compared with two other Sgp loci. Moreover, comparative analysis of C57BL/6 double congenic mice for Sgp3 and Sgp4 loci with single congenic mice revealed that Sgp3 and Sgp4 acted synergistically to elevate the transcription of the potentially replication-competent Xmv18 provirus and the production of serum gp70. This indicates that the combined effect of three different Sgp loci markedly enhance the expression of endogenous retroviruses and their gene product, serum gp70, thereby contributing to the formation of nephritogenic gp70-anti-gp70 immune complexes in murine lupus.

Sgp3 and TLR7 stimulation differentially alter the expression profile of modified polytropic retroviruses implicated in murine systemic lupus.

The envelope glycoprotein, gp70, of endogenous retroviruses represents one of the major nephritogenic autoantigens implicated in murine systemic lupus erythematosus. Among different endogenous retroviruses (ecotropic, xenotropic and polytropic), lupus-prone mice express remarkably high levels of modified polytropic (mPT) retroviruses, which are controlled by the Sgp3 (serum gp70 production) locus. To define the contribution of the Sgp3 locus derived from lupus-prone mice to the expression of the specific mPT proviruses, the genetic origin of different mPT viruses expressed in livers and thymi of wild-type and Sgp3 congenic C57BL/6 mice was determined through clonal analysis of their transcripts. Among 13 mPT proviruses present in the C57BL/6 genome, only 3 proviruses (Mpmv6, Mpmv10 and Mpmv13) were selectively but differentially expressed in livers and thymi. This was likely a result of co-regulated expression with host genes because of their integration in the same transcriptional direction. In contrast, Sgp3 induced the steady-state expression of an additional select group of mPT proviruses and, after stimulation of TLR7, the highly upregulated expression of a potentially replication-competent mPT virus Mpmv4. These results indicated that the expression of distinct subpopulations of mPT retroviruses was regulated by Sgp3- and TLR7-dependent mechanisms. The induction of potentially replication-competent mPT viruses and the upregulation of one such virus after stimulation with TLR7 in Sgp3 congenic mice further highlight the implication of Sgp3 in autoimmune responses against nephritogenic serum gp70 through the activation of TLR7.

Profound amplification of pathogenic murine polytropic retrovirus release from coinfected cells.

Previous studies indicate that mice infected with mixtures of mouse retroviruses (murine leukemia viruses [MuLVs]) exhibit dramatically altered pathology compared to mice infected with individual viruses of the mixture. Coinoculation of the ecotropic virus Friend MuLV (F-MuLV) with Fr98, a polytropic MuLV, induced a rapidly fatal neurological disease that was not observed in infections with either virus alone. The polytropic virus load in coinoculated mice was markedly enhanced, while the ecotropic F-MuLV load was unchanged. Furthermore, pseudotyping of the polytropic MuLV genome within ecotropic virions was nearly complete in coinoculated mice. In an effort to better understand these phenomena, we examined mixed retrovirus infections by utilizing in vitro cell lines. Similar to in vivo mixed infections, the polytropic MuLV genome was extensively pseudotyped within ecotropic virions; polytropic virus release was profoundly elevated in coinfected cells, and the ecotropic virus release was unchanged. A reduced level of polytropic SU protein on the surfaces of coinfected cells was observed and correlated with a reduced level of nonpseudotyped polytropic virion release. Marked amplification and pseudotyping of the polytropic MuLV were also observed in mixed Fr98-F-MuLV infections of cell lines derived from the central nervous system (CNS), the target for Fr98 pathogenesis. Additional experiments indicated that pseudotyping contributed to the elevated polytropic virus titer by increasing the efficiency of packaging and release of the polytropic genomes within ecotropic virions. Mixed infections are the rule rather than the exception in retroviral infection, and the ability to examine them in vitro should facilitate a more thorough understanding of retroviral interactions in general.

Sgp3 and Sgp4 control expression of distinct and restricted sets of xenotropic retroviruses encoding serum gp70 implicated in murine lupus nephritis.

The envelope glycoprotein gp70 of endogenous retroviruses implicated in murine lupus nephritis is secreted by hepatocytes and its expression is controlled by Sgp3 (serum gp70 production 3) and Sgp4 loci derived from lupus-prone mice. Among three different endogenous retroviruses (ecotropic, xenotropic and polytropic), xenotropic viruses are considered to be the major source of serum gp70. Although the abundance of xenotropic viral gp70 RNA in livers was up-regulated by the presence of these two Sgp loci, it has not yet been clear whether Sgp3 and Sgp4 regulate the expression of a fraction or multiple xenotropic viruses present in mouse genome. To address this question, we determined the genetic origin of xenotropic viral sequences expressed in wild-type and two different Sgp congenic C57BL/6 mice. Among 14 xenotropic proviruses present in the C57BL/6 genome, only two proviruses (Xmv10 and Xmv14) were actively transcribed in wild-type C57BL/6 mice. In contrast, Sgp3 enhanced the transcription of Xmv10 and induced the transcription of three additional xenotropic viruses (Xmv15, Xmv17 and Xmv18), while Sgp4 induced the expression of a different xenotropic virus (Xmv13). Notably, stimulation of TLR7 in Sgp3 congenic C57BL/6 mice led to a highly enhanced expression of potentially replication-competent Xmv18. These results indicated that Sgp3 and Sgp4 independently regulated the transcription of distinct and restricted sets of xenotropic viruses in trans, thereby promoting the production of nephritogenic gp70 autoantigens. Furthermore, the induced expression of potentially replication-competent xenotropic viruses by Sgp3 may contribute to the development of autoimmune responses against gp70 through the activation of TLR7.

Noninfectious retrovirus particles drive the APOBEC3/Rfv3 dependent neutralizing antibody response.

Members of the APOBEC3 family of deoxycytidine deaminases counteract a broad range of retroviruses in vitro through an indirect mechanism that requires virion incorporation and inhibition of reverse transcription and/or hypermutation of minus strand transcripts in the next target cell. The selective advantage to the host of this indirect restriction mechanism remains unclear, but valuable insights may be gained by studying APOBEC3 function in vivo. Apobec3 was previously shown to encode Rfv3, a classical resistance gene that controls the recovery of mice from pathogenic Friend retrovirus (FV) infection by promoting a more potent neutralizing antibody (NAb) response. The underlying mechanism does not involve a direct effect of Apobec3 on B cell function. Here we show that while Apobec3 decreased titers of infectious virus during acute FV infection, plasma viral RNA loads were maintained, indicating substantial release of noninfectious particles in vivo. The lack of plasma virion infectivity was associated with a significant post-entry block during early reverse transcription rather than G-to-A hypermutation. The Apobec3-dependent NAb response correlated with IgG binding titers against native, but not detergent-lysed virions. These findings indicate that innate Apobec3 restriction promotes NAb responses by maintaining high concentrations of virions with native B cell epitopes, but in the context of low virion infectivity. Finally, Apobec3 restriction was found to be saturable in vivo, since increasing FV inoculum doses resulted in decreased Apobec3 inhibition. By analogy, maximizing the release of noninfectious particles by modulating APOBEC3 expression may improve humoral immunity against pathogenic human retroviral infections.

The glycosylated Gag protein of a murine leukemia virus inhibits the antiretroviral function of APOBEC3.

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.

TLR-mediated up-regulation of serum retroviral gp70 is controlled by the Sgp loci of lupus-prone mice.

The endogenous retroviral envelope glycoprotein, gp70, implicated in murine systemic lupus erythematosus (SLE), has been considered to be a product of xenotropic, polytropic (PT) and modified PT (mPT) endogenous retroviruses. It is secreted by hepatocytes like an acute phase protein, but its response is under a genetic control. Given critical roles of TLR7 and TLR9 in the pathogenesis of SLE, we assessed their contribution to the acute phase expression of serum gp70, and defined a pivotal role of the Sgp3 (serum gp70 production 3) and Sgp4 loci in this response. Our results demonstrated that serum levels of gp70 were up-regulated in lupus-prone NZB mice injected with TLR7 or TLR9 agonist at levels comparable to those induced by injection of IL-1, IL-6 or TNF. In addition, studies of C57BL/6 Sgp3 and/or Sgp4 congenic mice defined the major roles of these two loci in up-regulated production of serum gp70 during acute phase responses. Finally, the analysis of Sgp3 congenic mice strongly suggests the presence of at least two distinct genetic factors in the Sgp3 interval, one of which controlled the basal-level expression of xenotropic, PT and mPT gp70 and the other which controlled the up-regulated production of xenotropic and mPT gp70 during acute phase responses. Our results uncovered an additional pathogenic role of TLR7 and TLR9 in murine lupus nephritis by promoting the expression of nephritogenic gp70 autoantigen. Furthermore, they revealed the involvement of multiple regulatory genes for the expression of gp70 autoantigen under steady-state and inflammatory conditions in lupus-prone mice.

Selective up-regulation of intact, but not defective env RNAs of endogenous modified polytropic retrovirus by the Sgp3 locus of lupus-prone mice.

Endogenous retroviruses are implicated in the pathogenesis of systemic lupus erythematosus (SLE). Because four different classes of endogenous retroviruses, i.e., ecotropic, xenotropic, polytropic, or modified polytropic (mPT), are expressed in mice, we investigated the possibility that a particular class of endogenous retroviruses is associated with the development of murine SLE. We observed >15-fold increased expression of mPT env (envelope) RNA in livers of all four lupus-prone mice, as compared with those of nine nonautoimmune strains of mice. This was not the case for the three other classes of retroviruses. Furthermore, we found that in addition to intact mPT transcripts, many strains of mice expressed two defective mPT env transcripts which carry a deletion in the env sequence of the 3' portion of the gp70 surface protein and the 5' portion of the p15E transmembrane protein, respectively. Remarkably, in contrast to nonautoimmune strains of mice, all four lupus-prone mice expressed abundant levels of intact mPT env transcripts, but only low or nondetectable levels of the mutant env transcripts. The Sgp3 (serum gp70 production 3) locus derived from lupus-prone mice was responsible for the selective up-regulation of the intact mPT env RNA. Finally, we observed that single-stranded RNA-specific TLR7 played a critical role in the production of anti-gp70 autoantibodies. These data suggest that lupus-prone mice may possess a unique genetic mechanism responsible for the expression of mPT retroviruses, which could act as a triggering factor through activating TLR7 for the development of autoimmune responses in mice predisposed to SLE.

Mobilization of endogenous retroviruses in mice after infection with an exogenous retrovirus.

Mammalian genomes harbor a large number of retroviral elements acquired as germ line insertions during evolution. Although many of the endogenous retroviruses are defective, several contain one or more intact viral genes that are expressed under certain physiological or pathological conditions. This is true of the endogenous polytropic retroviruses that generate recombinant polytropic murine leukemia viruses (MuLVs). In these recombinants the env gene sequences of exogenous ecotropic MuLVs are replaced with env gene sequences from an endogenous polytropic retrovirus. Although replication-competent endogenous polytropic retroviruses have not been observed, the recombinant polytropic viruses are capable of replicating in numerous species. Recombination occurs during reverse transcription of a virion RNA heterodimer comprised of an RNA transcript from an endogenous polytropic virus and an RNA transcript from an exogenous ecotropic MuLV RNA. It is possible that homodimers corresponding to two full-length endogenous RNA genomes are also packaged. Thus, infection by an exogenous virus may result not only in recombination with endogenous sequences, but also in the mobilization of complete endogenous retrovirus genomes via pseudotyping within exogenous retroviral virions. We report that the infection of mice with an ecotropic virus results in pseudotyping of intact endogenous viruses that have not undergone recombination. The endogenous retroviruses infect and are integrated into target cell genomes and subsequently replicate and spread as pseudotyped viruses. The mobilization of endogenous retroviruses upon infection with an exogenous retrovirus may represent a major interaction of exogenous retroviruses with endogenous retroviruses and may have profound effects on the pathogenicity of retroviral infections.

Dissection of genetic mechanisms governing the expression of serum retroviral gp70 implicated in murine lupus nephritis.

The endogenous retroviral envelope glycoprotein, gp70, implicated in murine lupus nephritis is secreted by hepatocytes as an acute phase protein, and it has been thought to be a product of an endogenous xenotropic virus, NZB-X1. However, since endogenous polytropic (PT) and modified polytropic (mPT) viruses encode gp70s that are closely related to xenotropic gp70, these viruses can be additional sources of serum gp70. To better understand the genetic basis of the expression of serum gp70, we analyzed the abundance of xenotropic, PT, or mPT gp70 RNAs in livers and the genomic composition of corresponding proviruses in various strains of mice, including two different Sgp (serum gp70 production) congenic mice. Our results demonstrated that the expression of different viral gp70 RNAs was remarkably heterogeneous among various mouse strains and that the level of serum gp70 production was regulated by multiple structural and regulatory genes. Additionally, a significant contribution of PT and mPT gp70s to serum gp70 was revealed by the detection of PT and mPT, but not xenotropic transcripts in 129 mice, and by a closer correlation of serum levels of gp70 with the abundance of PT and mPT gp70 RNAs than with that of xenotropic gp70 RNA in Sgp3 congenic mice. Furthermore, the injection of lipopolysaccharides selectively up-regulated the expression of xenotropic and mPT gp70 RNAs, but not PT gp70 RNA. Our data indicate that the genetic origin of serum gp70 is more heterogeneous than previously thought, and that distinct retroviral gp70s are differentially regulated in physiological vs inflammatory conditions.

Expression of infectious murine leukemia viruses by RAW264.7 cells, a potential complication for studies with a widely used mouse macrophage cell line.

The mouse macrophage-like cell line RAW264.7, the most commonly used mouse macrophage cell line in medical research, was originally reported to be free of replication-competent murine leukemia virus (MuLV) despite its origin in a tumor induced by Abelson MuLV containing Moloney MuLV as helper virus. As currently available, however, we find that it produces significant levels of ecotropic MuLV with the biologic features of the Moloney isolate and also MuLV of the polytropic or MCF class. Newborn mice developed lymphoma following inoculation with the MuLV mixture expressed by these cells. These findings should be considered in interpretation of increasingly widespread use of these cells for propagation of other viruses, studies of biological responses to virus infection and use in RNA interference and cell signalling studies.

Suppression of acute anti-friend virus CD8+ T-cell responses by coinfection with lactate dehydrogenase-elevating virus.

Friend virus (FV) and lactate dehydrogenase-elevating virus (LDV) are endemic mouse viruses that can cause long-term chronic infections in mice. We found that numerous mouse-passaged FV isolates also contained LDV and that coinfection with LDV delayed FV-specific CD8(+) T-cell responses during acute infection. While LDV did not alter the type of acute pathology induced by FV, which was severe splenomegaly caused by erythroproliferation, the immunosuppression mediated by LDV increased both the severity and the duration of FV infection. Compared to mice infected with FV alone, those coinfected with both FV and LDV had delayed CD8(+) T-cell responses, as measured by FV-specific tetramers. This delayed response accounted for the prolonged and exacerbated acute phase of FV infection. Suppression of FV-specific CD8(+) T-cell responses occurred not only in mice infected concomitantly with LDV but also in mice chronically infected with LDV 8 weeks prior to infection with FV. The LDV-induced suppression was not mediated by T regulatory cells, and no inhibition of the CD4(+) T-cell or antibody responses was observed. Considering that most human adults are carriers of chronically infectious viruses at the time of new virus insults and that coinfections with viruses such as human immunodeficiency virus and hepatitis C virus are currently epidemic, it is of great interest to determine how infection with one virus may impact host responses to a second infection. Coinfection of mice with LDV and FV provides a well-defined, natural host model for such studies.

Increased proinflammatory cytokine and chemokine responses and microglial infection following inoculation with neural stem cells infected with polytropic murine retroviruses.

Proinflammatory cytokines and chemokines are often detected in brain tissue of patients with neurological diseases such as multiple sclerosis (MS), HIV-associated dementia (HAD) and Alzheimer's disease (AD). We have utilized a mouse model of retrovirus-induced neurological disease to examine how these proinflammatory responses contribute to neuropathogenesis. In previous studies with this model, a correlation was found between neurovirulence and cytokine and chemokine expression. However, it was unclear whether the induction of these cytokines and chemokines was in response to specific virus envelope determinants or was regulated by the level of virus infection in the brain. In the current study, we demonstrated that multiple polytropic retroviruses induced cytokine and chemokine mRNA expression following increased virus levels in the brain. Increased virus levels of polytropic viruses also correlated with increased neuropathogenesis. In contrast, the ecotropic retrovirus, FB29, did not induce cytokine or chemokine mRNA expression or neurological disease, despite virus levels either similar to or higher than the polytropic retroviruses. As polytropic and ecotropic viruses utilize different receptors for entry, these receptors may play a critical role in the induction of these innate immune responses in the brain.

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.