Ecotropic Virus Research Articles

Production of Replication-Defective Retrovirus by Transient Transfection of 293T cells

Our lab studies human myeloproliferative diseases induced by such oncogenes as Bcr-Abl or growth factor receptor-derived oncogenes (ZNF198-FGFR1, Bcr-PDGFRalpha, etc.). We are able to model and study a human-like disease in our mouse model, by transplanting bone marrow cells previously infected with a retrovirus expressing the oncogene of interest. Replication-defective retrovirus encoding a human oncogene and a marker (GFP, RFP, antibiotic resistance gene, etc.) is produced by a transient transfection protocol using 293T cells, a human renal epithelial cell line transformed by the adenovirus E1A gene product. 293 cells have the unusual property of being highly transfectable by calcium phosphate (CaPO4), with up to 50-80% transfection efficiency readily attainable. Here, we co-transfect 293 cells with a retroviral vector expressing the oncogene of interest and a plasmid that expresses the gag-pol-env packaging functions, such as the single-genome packaging constructs kat or pCL, in this case the EcoPak plasmid. The initial transfection is further improved by use of chloroquine. Stocks of ecotropic virus, collected as culture supernatant 48 hrs. post-transfection, can be stored at -80 degrees C and used for infection of cell-lines in view of transformation and in vitro studies, or primary cells such as mouse bone marrow cells, that can then be used for transplant in our mouse model.

Inhibition of infectious murine leukemia virus production by Fv-4 env gene products exerting dominant negative effect on viral envelope glycoprotein

Fv-4 is a mouse gene that confers resistance against ecotropic murine leukemia virus (MLV) infection on mice. While receptor interference by the Fv-4 env gene product, Fv-4 Env, that can bind to the ecotropic MLV receptor has been shown to play an important role in the resistance, other mechanisms have also been suggested because it confers extremely efficient, complete resistance in vivo. Here, we have examined the effect of Fv-4 Env on infectious MLV production. Infectious MLV titers in supernatants obtained after transfection with a Friend MLV (FMLV) Env-expressing plasmid from MLV gag– pol producer cells harboring a retroviral vector were largely reduced by coexpression of Fv-4 Env. Syncytia formation mediated by R-peptide-deleted FMLV Env in NIH 3T3 cells was impaired by Fv-4 Env coexpression. Similarly, Fv-4 Env inhibited infectious amphotropic MLV production and syncytia formation mediated by R-peptide-deleted amphotropic MLV Env. Immunoprecipitation analysis revealed interaction of Fv-4 Env with amphotropic MLV Env as well as FMLV Env. These results indicate that Fv-4 Env inhibits infectious ecotropic and amphotropic MLV production by exerting dominant negative effect on MLV Env, suggesting contribution of this inhibitory effect to the resistance against ecotropic MLV infection in Fv-4-expressing mice.

Characterization of R peptide of murine leukemia virus envelope glycoproteins in syncytium formation and entry

The C-terminal R peptide of ecotropic murine leukemia virus (MLV) envelope protein (Env) negatively controls membrane fusion activity. The R peptide cleavage during virion maturation activates its fusogenicity and is required for viral entry. We analyzed fusogenicity and transduction efficiency of mutant Env proteins of ecotropic, amphotropic, polytropic, and xenotropic MLVs. As the result, we found that the hydrophobic amino acid residues around the R peptide cleavage site are important for membrane fusion inhibition by the R peptide. In addition, we found that Env complexes with R peptide-truncated and -containing Env proteins have lower fusogenicity and transduction efficiency than those with the R-peptide-truncated Env alone, suggesting that efficient R peptide cleavage is required for efficient MLV vector transduction. The role of R peptide cleavage in amphotropic, polytropic, and xenotropic MLV infection has not been investigated. We found in this study that the R peptide cleavage is required for amphotropic, xenotropic, and polytropic MLV vector transduction, like with ecotropic MLV. The R-peptide-truncated Env proteins of the xenotropic and polytropic MLVs, however, had much lower fusogenicity than those of the ecotropic and amphotropic MLVs. These results provide valuable information for construction of efficient MLV vectors and for understanding the retroviral entry mechanism.

A Transient Three-plasmid Expression System for the Production of Hepatocytes Targeting Retroviral Vectors

Targeting of retroviral vectors to specific cells was attempted through modifying the surface protein of the murine leukemia viruses (MLVs), but in many cases the protein function was affected, and it is difficult to achieve the targeted delivery. In this study, we have tried to engineer ecotropic Moloney murine leukemia viruses (MoMLV)-based retroviral vectors to transduce hepatocytes. A chimeric envelope (Env) expression plasmid was constructed containing the hepatitis B virus PreS2 peptide fused to aa +1 at the N-terminus of Env. Following simultaneous transfection of pgag-pol, pLEGFP and chimeric env plasmids into 293T cells, helper-free retrovirus stocks with the titer of approximately 10(4) infectious units/ml were achieved at 48 h post-transfection. These pseudotype vectors showed the normal host range of retrovirus, infecting host NIH 3T3 cells, although the efficiency was reduced compared with that of virions carrying wild-type ecotropic MoMLV envelope. In addition, the resultant pseudotype viruses could transduce human hepatoma cells mediated by polymerized human serum albumin with relatively high titers in comparison with those transductions without polymerized human serum albumin. This approach can be used to target hepatocytes selectively.

Ecotropic murine leukemia virus envelope protein affects interaction of cationic amino acid transporter 1 with clathrin adaptor protein complexes, leading to receptor downregulation

Mouse cationic amino acid transporter 1 (mCAT1) serves as the receptor for ecotropic murine leukemia virus (eMuLV). It has been shown that mCAT1 is expressed on the basolateral surface of polarized epithelial MDCK cells. However, little is known about the mechanisms involved in the intracellular trafficking of mCAT1. Using the green fluorescent protein-tagged mCAT1 expressed in MDCK cells, we report here that mCAT1 is physically associated with clathrin adaptor protein complex 1 (AP-1) implicated in protein trafficking from trans-Golgi network (TGN) to the basolateral surface. When the cells were infected with eMuLV, reduction of cell surface mCAT1, as well as a concomitant decrease in mCAT1-AP-1 association, was observed while association of mCAT1 with AP-3 involved in the TGN-to-lysosome trafficking was increased. Similar results were obtained when eMuLV envelope protein alone was expressed. The results may provide useful insights into the mechanism by which a simple retrovirus downregulates its receptor.

Cryptococcus neoformans infection in mice previously infected with LP-BM5 MuLV, the agent of murine AIDS (MAIDS).

We studied susceptibility to experimental systemic cryptococcosis in mice previously infected with the retroviral complex LP-BM5 (responsible for murine AIDS). LP-BM5 was inoculated to C57B1/6 mice by intravenous (i.v.) injection 8 weeks before an i.v. challenge with 4 x 10(3) CFU of Cryptococcus neoformans. Uninfected and singly infected mice were used as controls. LP-BM5 infection did not result in a significant increase in fungal burdens in the lungs or brains of co-infected animals compared to mice infected with C. neoformans alone. However, mortality was enhanced in the co-infected animals. The kinetics of splenocyte subsets differed in co-infected mice and LP-BM5-infected mice; the increase in CD4+, CD8+ and Ly5+ cells was only moderate in the former. Cytokine production by concanavalin A (Con A)-stimulated splenocytes from co-infected mice showed a marked decrease in the Th1 response (IFN-gamma, IL-2) and an increase in the Th2 response (IL-4, IL-10). Furthermore, cryptococcosis altered the course of MAIDS, inhibiting splenomegaly. This effect was not related to a decrease in ecotropic virus titres in the spleen or to improved in vitro responsiveness of spleen cells to Con A. The marked decrease in IFN-gamma production in co-infected animals could partly explain the inhibition of LP-BM5-induced splenomegaly. This model of murine retroviral infection does not seem to be suitable for studying cryptococcosis in immunosuppressed animals, but remains valuable for investigating in vivo interactions between two pathogens.

Viral insertion in Evi12 causes expression of aberrant Grp94 mRNAs containing the viral gag myristylation motif

Ecotropic Virus Integration site 12 ( Evi12) is a common virus insertion site (cVIS) in retrovirally induced murine models of leukemia and lymphoma, suggesting an important role for this locus in these hematopoietic disorders. Evi12 is located near the promoter of the ER chaperone protein and Hsp90 family member Grp94. Here we show that viral insertion in Evi12 results in the expression of aberrant Grp94 transcripts in Cas-Br-MuLV as well as in AKXD induced hematopoietic tumors, demonstrating that Grp94 is a common viral target gene. While most transcripts encode for truncated forms of Grp94, transcripts containing viral gag sequences were detected in the leukemia cell line NFS107. Interestingly, these fusion transcripts encode for myristylated viral–Grp94 fusion proteins that localize to the plasma membrane. Combined with recent evidence that myristylated forms of Hsp90 transform cells, our data suggest that myristylation of target genes may be an important mechanism in retrovirally mediated oncogenesis. Since retroviral insertion in Evi12 also affects the expression of a recently identified novel gene Grp94 neighboring nucleotidase ( Gnn), located at the other side of Evi12, it appears that proviral insertion can lead to deregulation of two genes present in the same locus.

An interphase fluorescence in situ hybridisation assay for the detection of 3q26.2/EVI1 rearrangements in myeloid malignancies

Chromosome rearrangements involving band 3q26.2 are associated with myeloid malignancies, aberrant expression of the human ecotropic virus integration site-1 (EVI1) gene, an unfavourable prognosis and an aggressive clinical course. The 3q26.2 rearrangements are characteristically heterogeneous and typically difficult to detect in poor quality metaphases. To develop a dual-colour fluorescence in situ hybridisation (FISH) assay for the detection of 3q26.2/EVI1 aberrations, a series of 10 BAC clones corresponding to the EVI1 gene region were systematically evaluated and narrowed down to two probe sets; one probe set encompassed the EVI1 gene extending centromeric, while the second probe set covered the EVI1 gene and extends telomeric. Both probe sets were evaluated on 35 patient samples with cytogenetically defined 3q26.2 rearrangements collected at various treatment time points, the inv(3)(q21q26.2) Kasumi-4 cell line, and 10 known negative samples. The two-probe set strategy identified all samples, despite the vast breakpoint heterogeneity observed. In samples from acute myeloid leukaemia and myelodysplastic syndrome cases, the majority of inversion breakpoints were 3' to EVI1 whereas 3q26.2 translocation breakpoints frequently mapped 5' to EVI1. However, two 3q26.2 translocation samples had breakpoints 3' to EVI1. Most inv(3q) chronic myeloid leukaemia samples showed breakpoints within the EVI1 gene. This study demonstrated that, despite the extensive breakpoint heterogeneity observed with 3q26.2 aberrations, this FISH strategy is effective for the detection of 3q26.2 abnormalities in myeloid malignancies.

Complete genome sequences of the two viral variants of the Graffi MuLV: Phylogenetic relationship with other murine leukemia retroviruses

A detailed phylogenetic analysis of two variants of the Graffi murine retrovirus, GV-1.2 and GV-1.4, showed that they are closely related to SRS 19-6 and Moloney MuLVs. Two stretches of sequence testify to the divergence between Graffi and SRS 19-6 MuLVs, one corresponding to a recombination event of Graffi MuLV with a xenotropic virus. Moloney MuLV was found more distant, particularly in the GAG region. Our study encompasses every class of MuLVs (ecotropic, amphotropic, xenotropic, polytropic) with some focus on exogenous ecotropic viruses and further adds to previous phylogenetic studies. Graffi, SRS 19-6, Moloney, Friend and Rauscher MuLVs form a cluster that appears to share a common ancestor with the Casitas-amphotropic and -ecotropic MuLVs but are more distant to the Akv-type and xenotropic MuLVs. The analysis also revealed that the ENV region of HEMV, the prototype of the MuLV ancestor, was closely related to the corresponding region of Cas-Br-E.

A recessive genetic screen for host factors required for retroviral infection in a library of insertionally mutated Blm-deficient embryonic stem cells

A recessive genetic screen of an insertionally mutated Blm-/- ES cell library identifies host factors required for retroviral infection, and confirms that mCat-1 is the ecotropic murine leukaemia virus receptor in ES cells.

PCAF Modulates PTEN Activity

The PTEN protein has a single catalytic domain possessing both lipid phosphoinositol and protein phosphatase activities. The lipid phosphoinositol phosphatase activity is essential for PTEN to block the cell cycle in the G1 phase and thereby to suppress tumor formation and progression (Cantley, L. C., and Neel, B. G. (1999) Proc. Natl. Acad. Sci. U. S. A. 96, 4240-4245), although the mechanisms governing PTEN activity under normal and neoplastic growth conditions remain unclear. Here, we report that PTEN interacts physically and functionally with PCAF, a histone acetyltransferase that regulates gene transcription through interaction with p300/CBP and various sequence-specific transcription factors (Nakatani, Y. (2001) Genes Cells 6, 79-86). Expression of PCAF results in increased acetylation of lysine residues (Lys125 and Lys128) within the catalytic cleft of PTEN, a structure essential for phosphatidylinositol 3,4,5-trisphosphate specificity (Lee, J. O., Yang, H., Georgescu, M. M., Di Cristofano, A., Maehama, T., Shi, Y., Dixon, J. E., Pandolfi, P., and Pavletich, N. P. (1999) Cell 99, 323-334). The acetylation of PTEN caused by PCAF expression depends on the presence of growth factors. Reduction of endogenous PCAF activity using shRNA results in a loss of PTEN acetylation in response to growth factors and restores the ability of PTEN to down-regulate phosphatidylinositol 3-kinase signaling and to induce G1 cell cycle arrest. The retention of phosphatidylinositol 3-kinase/AKT signaling and cell cycle regulatory activities of acetylation-resistant PTEN K125R and K128R mutants in the presence of enforced PCAF expression suggest a causal relationship. Together, these findings indicate a mechanism of PTEN regulation that forges a link between distinct cancer-relevant pathways central to the control of growth factor signaling and gene expression.

Scrapie infection activates the replication of ecotropic, xenotropic, and polytropic murine leukemia virus (MuLV) in brains and spinal cords of senescence-accelerated mice: Implication of MuLV in progression of scrapie pathogenesis

Senescence-accelerated mice (SAMP8) have a short life span, whereas SAMR1 mice are resistant to accelerated senescence. Previously it has been reported that the Akv strain of ecotropic murine leukemia virus (E-MuLV) was detected in brains of SAMP8 mice but not in brains of SAMR1 mice. In order to determine the change of MuLV levels following scrapie infection, we analyzed the E-MuLV titer and the RNA expression levels of E-MuLV, xenotropic MuLV, and polytropic MuLV in brains and spinal cords of scrapie-infected SAM mice. The expression levels of the 3 types of MuLV were increased in scrapie-infected mice compared to control mice; E-MuLV expression was detected in infected SAMR1 mice, but only in the terminal stage of scrapie disease. We also examined incubation periods and the levels of PrP Sc in scrapie-infected SAMR1 (sR1) and SAMP8 (sP8) mice. We confirmed that the incubation period was shorter in sP8 (210 ± 5 days) compared to sR1 (235 ± 10 days) after intraperitoneal injection. The levels of PrP Sc in sP8 were significantly greater than sR1 at 210 ± 5 days, but levels of PrP Sc at the terminal stage of scrapie in both SAM strains were virtually identical. These results show the activation of MuLV expression by scrapie infection and suggest acceleration of the progression of scrapie pathogenesis by MuLV.

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.

The Early-Immediate Gene EGR-1 Is Induced by Transforming Growth Factor-β and Mediates Stimulation of Collagen Gene Expression

Transforming growth factor-beta (TGF-beta) stimulates collagen synthesis and accumulation, and aberrant TGF-beta signaling is implicated in pathological organ fibrosis. Regulation of type I procollagen gene (COL1A2) transcription by TGF-beta involves the canonical Smad signaling pathway as well as additional protein and lipid kinases, coactivators, and DNA-binding transcription factors that constitute alternate non-Smad pathways. By using Affymetrix microarrays to detect cellular genes whose expression is regulated by Smad3, we identified early growth response factor-1 (EGR-1) as a novel Smad3-inducible gene. Previous studies implicated Egr-1 in cell growth, differentiation, and survival. We found that TGF-beta induced rapid and transient accumulation of Egr-1 protein and mRNA in human skin fibroblasts. In transient transfection assays, TGF-beta stimulated the activity of the Egr-1 gene promoter, as well as that of a minimal Egr-1-responsive reporter construct. Furthermore, TGF-beta enhanced endogenous Egr-1 interaction with a consensus Egr-1-binding site element and with GC-rich DNA sequences of the human COL1A2 promoter in vitro and in vivo. Forced expression of Egr-1 by itself caused dose-dependent up-regulation of COL1A2 promoter activity and further enhanced the stimulation induced by TGF-beta. In contrast, the TGF-beta response was abrogated when the Egr-1-binding sites of the COL1A2 promoter were mutated or deleted. Furthermore, Egr-1-deficient embryonic mouse fibroblasts showed attenuated TGF-beta responses despite intact Smad activation, and forced expression of ectopic EGR-1 in these cells could restore COL1A2 stimulation in a dose-dependent manner. Taken together, these findings identify Egr-1 as a novel intracellular TGF-beta target that is necessary for maximal stimulation of collagen gene expression in fibroblasts. The results therefore implicate Egr-1 in the profibrotic responses elicited by TGF-beta and suggest that Egr-1 may play a new and important role in the pathogenesis of fibrosis.

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.

EphB4 controls blood vascular morphogenesis during postnatal angiogenesis

Guidance molecules have attracted interest by demonstration that they regulate patterning of the blood vascular system during development. However, their significance during postnatal angiogenesis has remained unknown. Here, we demonstrate that endothelial cells of human malignant brain tumors also express guidance molecules, such as EphB4 and its ligand ephrinB2. To study their function, EphB4 variants were overexpressed in blood vessels of tumor xenografts. Our studies revealed that EphB4 acts as a negative regulator of blood vessel branching and vascular network formation, switching the vascularization program from sprouting angiogenesis to circumferential vessel growth. In parallel, EphB4 reduces the permeability of the tumor vascular system via activation of the angiopoietin-1/Tie2 system at the endothelium/pericyte interface. Furthermore, overexpression of EphB4 variants in blood vessels during (i) vascularization of non-neoplastic cell grafts and (ii) retinal vascularization revealed that these functions of EphB4 apply to postnatal, non-neoplastic angiogenesis in general. This implies that both neoplastic and non-neoplastic vascularization is driven not only by a vascular initiation program but also by a vascular patterning program mediated by guidance molecules.

457. Reverse Transcriptase Deficient Retroviral Vectors for Transient Cell Modification

Short-term, reversible expression of foreign proteins could be useful to modify cell fate. We have previously shown that retroviral vector mutants that are unable to initiate reverse transcription of their plus-stranded mRNA genomes mediate transient and highly efficient delivery of the site specific recombinase Cre into human and mouse fibroblasts (Galla et al., Mol Cell 2004). Cre protein activity was detected in >95% of target cells with a single treatment. Cre activity was particle-mediated and required the retroviral mRNA packaging signal as well as the expression of both Gag and Env in the packaging cell. Furthermore we could exclude plasmid contamination and Cre protein transduction as the underlying mechanisms for this mode of Cre delivery. Our data thus suggest that retrovirally delivered mRNA serves as an immediate translation template if not being reverse transcribed. When delivering the Sleeping Beauty (SB) transposase (cooperation with Z. Ivics, Berlin) by this approach, RT-deficient retroviral vector mutants were sufficient to mediate transposition of transfected plasmids containing transposon DNA. However, the efficiency of stable integration was only 3-fold above background levels, suggesting that the timing and/or expression levels have to be improved. Side-by-side comparison with the delivery of episomal DNA by lentiviral vectors revealed that RT-deficient retroviral mutants express foreign proteins for a much shorter duration (max. 4-5 days in replicating cells as opposed to >7 days for episomal lentiviral vectors) and lower levels (one to two orders of magnitude below the activity provided by episomal lentiviral vectors). As many receptor molecules require relatively low levels of expression to confer biological effects, we tested RT-deficient retroviral vectors to deliver the sialomucin CD34 or the murine cationic acid transporter mCAT-1, which serves as the receptor for the ecotropic murine leukemia virus. More than 80% of the exposed cells could be transduced following a single exposure to RT-deficient vectors encoding these receptors, and mCAT-1 expression conferred susceptibility of Jurkat cells to transduction with retroviral or lentiviral vectors pseudotyped with the murine ecotropic envelope protein. We conclude that RT-deficient retroviral vectors hold great promise for applications where low and transient expression of proteins achieves striking biological effects.

Retrovirus molecular conjugates: a versatile and efficient gene transfer vector system for primitive human hematopoietic progenitor cells

In principle, transient nongenetic modification of a noninfectious gene transfer virus enabling a one time infection and transduction of human cells could eliminate the risk of formation of replication competent virus. Formation of a molecular conjugate vector by conjugation of noninfective ecotropic murine Moloney leukemia virus to polylysine (eMMLV-PL) enabled high-efficiency transduction of human HPC using in vitro and in vivo assays. Xenotransplanted NOD-SCID mice durably expressed the transgene in human leukocytes and human progenitor cells with eMMLV-PL achieving three-fold increased transduction efficiency when directly compared to optimized amphotropic MMLV (aMMLV) transduction. Both aMMLV and eMMLV assembled conjugate vectors showed similar transduction efficiency indicating predominant polylysine-mediated uptake. Integration of retroviral sequences was determined from individual human HPC recovered from eMMLV-PL-xenotransplanted animals. This simple and versatile concept of conjugate gene transfer vectors has the potential to enhance transduction efficiency as well as to improve certain safety aspects of human gene therapy. Moreover, because it permits effective cellular internalization of particles, this concept of molecular conjugates can be used as research tool to investigate the interactions of otherwise noninfectious viruses or modified viral particles at the genomic level.

Precise Identification of Endogenous Proviruses of NFS/N Mice Participating in Recombination with Moloney Ecotropic Murine Leukemia Virus (MuLV) To Generate Polytropic MuLVs

Polytropic murine leukemia viruses (MuLVs) are generated by recombination of ecotropic MuLVs with env genes of a family of endogenous proviruses in mice, resulting in viruses with an expanded host range and greater virulence. Inbred mouse strains contain numerous endogenous proviruses that are potential donors of the env gene sequences of polytropic MuLVs; however, the precise identification of those proviruses that participate in recombination has been elusive. Three different structural groups of proviruses in NFS/N mice have been described and different ecotropic MuLVs preferentially recombine with different groups of proviruses. In contrast to other ecotropic MuLVs such as Friend MuLV or Akv that recombine predominantly with a single group of proviruses, Moloney MuLV (M-MuLV) recombines with at least two distinct groups. In this study, we determined that only three endogenous proviruses, two of one group and one of another group, are major participants in recombination with M-MuLV. Furthermore, the distinction between the polytropic MuLVs generated by M-MuLV and other ecotropic MuLVs is the result of recombination with a single endogenous provirus. This provirus exhibits a frameshift mutation in the 3' region of the surface glycoprotein-encoding sequences that is excluded in recombinants with M-MuLV. The sites of recombination between the env genes of M-MuLV and endogenous proviruses were confined to a short region exhibiting maximum homology between the ecotropic and polytropic env sequences and maximum stability of predicted RNA secondary structure. These observations suggest a possible mechanism for the specificity of recombination observed for different ecotropic MuLVs.

A mouse model for study of systemic HIV-1 infection, antiviral immune responses, and neuroinvasiveness

We created a model of HIV-1 infection of conventional mice for investigation of viral replication, control, and pathogenesis. To target HIV-1 to mice, the coding region of gp120 in HIV-1/NL4-3 was replaced with that of gp80 from ecotropic murine leukemia virus, a retrovirus that infects only rodents. The resulting chimeric virus construct, EcoHIV, productively infected murine lymphocytes, but not human lymphocytes, in culture. Adult, immunocompetent mice were readily susceptible to infection by a single inoculation of EcoHIV as shown by detection of virus in splenic lymphocytes, peritoneal macrophages, and the brain. The virus produced in animals was infectious, as shown by passage in culture, and immunogenic, as shown by induction of antibodies to HIV-1 Gag and Tat. A second chimeric virus based on clade D HIV-1/NDK was also highly infectious in mice; it was detected in both spleen and brain 3 wk after tail vein inoculation, and it induced expression of infection response genes, MCP-1, STAT1, IL-1beta, and complement component C3, in brain tissue as determined by quantitative real-time PCR. EcoHIV infection of mice forms a useful model of HIV-1 infection of human beings for convenient and safe investigation of HIV-1 therapy, vaccines, and potentially pathogenesis.