Infectious Murine Leukemia Virus Research Articles

The tandem direct repeats within the long terminal repeat of murine leukemia viruses are the primary determinant of their leukemogenic potential.

To map the viral sequences encoding the leukemogenic determinant(s) of nondefective murine leukemia viruses (MuLVs), we constructed chimeric viral genomes in vitro between cloned viral DNAs from the highly leukemogenic Gross passage A (Gross A) MuLV and from the related nonleukemogenic BALB/c N-tropic MuLV. Infectious chimeric MuLVs, recovered from murine cells microinjected with these DNAs, were inoculated into newborn mice to test the leukemogenic potential of these viruses. We found that the U3 long terminal repeat region from Gross A genomes was sufficient to confer an intermediate leukemogenic potential to chimeric MuLVs. Sequencing data indicated that the U3 tandem direct repeat was responsible for this effect. Adding most of the Gross A p15E-coding sequences to the Gross A U3 long terminal repeat enhanced the leukemogenic potential of chimeric viruses significantly. Adding a larger 3'-end env region (all p15E-coding sequences and 345 base pairs of the carboxy terminus of gp70) to the Gross A U3 long terminal repeat restored the full leukemogenic potential of Gross A MuLV. Chimeric viruses harboring only the Gross A 3'-end env region were, however, nonleukemogenic. Similar chimeric MuLVs, constructed with genomes from the parental weakly leukemogenic BALB/c B-tropic MuLVs and nonleukemogenic BALB/c N-tropic MuLVs, were also studied. Our data indicate that the U3 tandem direct repeat sequences appear to be necessary and sufficient to confer some leukemogenic potential to MuLV. However, env 3'-end sequences, mostly the p15E-encoding sequences, are required for the expression of fully leukemic phenotypes.

Expression of differentiation and murine leukemia virus antigens on cells of primary tumors and cell lines derived from chemically induced lymphomas of RF/J mice.

3-Methylcholanthrene (MCA) skin painting rapidly induced a 100% incidence of thymic lymphomas in RF/J mice. Tumors developed exclusively in cells of the T-lymphocyte lineage and displayed the surface phenotype: Thy-1+, Lyt-1+, Lyt-2+, Qa-1+, H-2K+, H-2D+, TL-, Ig-, Ia-. This phenotype resembled that of cortisone-resistant, medullary thymocytes, whereas the phenotype of spontaneous AKR thymomas resembles that of cortisone-sensitive, cortical thymocytes. The phenotype differed very little from one tumor to another and was maintained during syngeneic passage in vivo and adaptation to growth in culture. Infectious ecotropic murine leukemia virus (MuLV) was produced in a minority of primary tumors and cell lines, but no xenotropic or mink cell focus-inducing virus (MCF) MuLV production was reliably detected. MuLV-related antigen expression on normal thymocytes increased in an age-dependent manner, and levels on thymoma cells were similar to those on thymocytes from age-matched normal controls. MuLV nonproducer tumor cells expressed few or no epitopes of AKR ecotropic gp70, but they were positive when tested with an antiserum prepared against MCFenv glycoprotein.

Physical mapping of the paralysis-inducing determinant of a wild mouse ecotropic neurotropic retrovirus.

We have recently shown that a molecularly cloned ecotropic retrovirus, initially isolated from the brain of a paralyzed wild mouse, retained the ability to induce hind limb paralysis when inoculated into susceptible mice (Jolicoeur et al., J. Virol. 45:1159-1163, 1983). To map the viral DNA sequences encoding the determinant of paralysis, we constructed chimeric viral DNA genomes in vitro between parental cloned infectious viral DNA genomes from this neurotropic murine leukemia virus (MuLV) and from nonneurotropic amphotropic 4070-A MuLV. Infectious chimeric MuLVs, recovered after microinjection of NIH 3T3 cells with these recombinant DNAs, were inoculated into newborn SIM.S and SWR/J mice to test the paralysis-inducing potential. We found that the 3.9-kilobase-pair SalI-ClaI fragment of the neurotropic MuLV comprising the 3' end of pol and all env sequences was sufficient to confer the paralysis-inducing potential to chimeric viruses. Therefore, this region of the neurotropic MuLV genome most likely harbors the primary determinant of paralysis.

Laboratory and wild-derived mice with multiple loci for production of xenotropic murine leukemia virus.

Mendelian segregation analysis was used to define genetic loci for the induction of infectious xenotropic murine leukemia virus in several laboratory and wild-derived mice. MA/My mice contain two loci for xenotropic virus inducibility, one of which, Bxv -1, is the only induction locus carried by five other inbred strains. The second, novel MA/My locus, designated Mxv -1, is unlinked to Bxv -1 and shows a lower efficiency of virus induction. The NZB mouse carries two induction loci; both are distinct from Bxv -1 since neither is linked to the Pep-3 locus on chromosome 1. Finally, one partially inbred strain derived from the wild Japanese mouse, Mus musculus molossinus, carries multiple (at least three) unlinked loci for induction of xenotropic virus. Although it is probable that inbred strains inherited xenotropic virus inducibility from Japanese mice, our data suggest that none of the induction loci carried by this particular M. m. molossinus strain are allelic with Bxv -1.

Physical mapping of the Fv-1 tropism host range determinant of BALB/c murine leukemia viruses.

The murine leukemia viruses (MuLVs) have different host ranges and were originally designated N-tropic and B-tropic if they replicated preferentially in vitro on NIH and BALB/c fibroblasts, respectively. It was later found that N-tropic MuLVs were in fact restricted in BALB/c cells, that B-tropic MuLVs were restricted in NIH cells, and that both viruses were restricted in (BALB X NIH) F1 cells. A single gene, Fv-1, with two alleles, Fv-1b and Fv-1n, determines this dominant restriction. A virus-encoded protein seems to carry the viral host range determinant which is recognized by the Fv-1 gene product. To map the viral DNA sequences encoding this determinant, we constructed viral DNA recombinants in vitro between the cloned infectious viral DNA genomes from BALB/c N-tropic and B-tropic MuLVs. Infectious recombinant MuLVs were recovered by microinjecting these recombinant DNAs into murine Fv-1- SC-1 cells and were subsequently tested in vitro for their host ranges (N- or B-tropic). We found that a short 302-base pair 5'-end fragment was necessary and sufficient to confer a specific host range to a recombinant. Our sequencing data revealed that this fragment codes for amino acid sequences in gag p30. They also showed that only two consecutive amino acid differences, Gln-ArgN- and Thr-GluB-, in p30 are responsible for the N- and B-tropic host ranges of the BALB/c MuLVs, respectively. Therefore, it appears that the Fv-1b and Fv-1n gene products can discriminate between these two p30 amino acid sequences.

Thymotropism of murine leukemia virus is conferred by its long terminal repeat.

Several murine leukemia viruses (MuLV) replicate efficiently in the thymus (T+) of the mouse, whereas others are unable to replicate (T-) in this organ. To map the region of the viral genome harboring the sequences responsible for this thymotropic phenotype, we constructed viral DNA recombinants in vitro between cloned infectious viral DNAs from T- BALB/c N-tropic MuLV and from T+ BALB/c B-tropic MuLV or AKR Gross passage A MuLV. (N- and B-tropic refer to the Fv-1 host range of MuLV.) Infectious recombinant MuLVs, recovered from murine cells microinjected with these recombinant DNAs, were injected into newborn mice to test their ability to replicate in the thymus. We found that the long terminal repeat from the T+ BALB/c B-tropic or AKR Gross passage A MuLV genome was sufficient to allow replication of recombinant MuLVs in the thymus. Our sequence data suggested that the U3 tandem direct repeat was responsible for this effect. These results suggest a new role for the U3 long terminal repeat in the replication of MuLV in specific differentiated target cells.

Molecular cloning of infectious viral DNA from ecotropic neurotropic wild mouse retrovirus.

Among a mixture of amphotropic and ecotropic murine leukemia viruses (MuLVs) isolated from paralyzed wild mice, only N-tropic ecotropic MuLV, cloned by cell culture techniques, has been shown to induce paralysis after reinjection into susceptible mice (M. B. Gardner, Curr. Top. Microbiol. Immunol. 79:215-239, 1978). The viral DNA genome of one of these neurotropic MuLVs (Cas-Br-E) has been cloned in Charon 21A at the SalI site. One clone, designated NE-8, was studied in more detail. A restriction endonuclease map of this cloned DNA was derived. Cloned viral DNA microinjected into NIH 3T3 cells produced infectious MuLV which was characterized as XC+, ecotropic, and N-tropic. The virus that was recovered after the microinjection of NE-8 DNA was also injected into susceptible SIM.S and NIH Swiss mice and was found to induce lower limb paralysis in these animals. These results make it highly unlikely that other agents (which might have escaped detection and separation from ecotropic MuLV by the techniques previously used) play a role in the etiology of this disease and clearly indicate that the ecotropic MuLV genome harbors sequences responsible for this paralysis. The availability of this clone DNA would now allow us to map these sequences on the genome.

A novel sequence segment and other nncleotide structural features in the long terminal repeat of a BALB/c mouse genomic leukemia virus-related DNA clone

A recombinant DNA clone, named AL10, that contains murine leukemia virus (MuLV) related sequences was isolated from BALB/c mouse chromosomal DNA and examined in detail. Restriction endonuclease mapping revealed that the 10.5 kbp EcoRI insert consists of a 3.6 kbp left flanking cellular DNA region and a 6.9 kbp MuLV-related region that has a typical proviral LTR-gag-pol-env structure up to the EcoRI site in the env gene region. Comparison of the AL10 map with ecotropic and xenotropic virus isolates revealed many common restriction sites in the LTR and pol gene regions, but much fewer in the leader and gag regions. A stretch of 1,700 nucleotides containing the cellprovirus junctional region was sequenced and revealed transcriptional consensus signals and other structural features characteristic of MuLV LTRs, as well as two distinctive features: (a) a sequence of approximately 170 bp with direct and inverted terminal repeats not seen in infectious MuLV LTRs was identified in the U3 region between the "enhancer" region and the "CAT" box. This novel segment or its homologous sequences appear to be present in most of the endogenous MuLV-related LTRs and in other chromosomal locations of the mouse (b) The tRNA primer binding site is not complementary to proline tRNA, the primer for all known MuLVs, but is a 17/18 match with rat glutamine tRNA. The integration site of AL10 provirus was in a unique DNA region but contained an "Alu"-like short interdispersed repeat in the 5' adjacent cellular region. The AL10 proviral integration found in BALB/c was also apparent in RFM, AKR and SENCAR mouse cells but not in cells of NFS/N, C3H, HRS/J, SC-1, and a California Lake Casitas wild mouse.

Neoplastic transformation of rabbit cells by murine sarcoma viruses.

Neoplastic transformation of rabbit cells by Kirsten murine sarcoma virus (Ki-MSV), the Ki-MSV pseudotype of baboon endogenous virus (Ki-MSV[BaEV]) and the Moloney-MSV pseudotype of feline leukemia virus (M-MSV[FeLV]) is reported. Rabbit cells can be readily transformed by Ki-MSV, Ki-MSV(BaEV) and M-MSV(FeLV). Rabbit cells transformed by Ki-MSV and M-MSV(FeLV) were found to be virus producers, whereas those transformed by Ki-MSV(BaEV) were nonproducers (NP). The NP cells were obtained by simply infection rabbit cells with Ki-MSV(BaEV) and subculturing the infected cells. Although the morphologically altered NP cells did not produce infectious virus or murine leukemia virus antigen, they did contain a rescuable MSV genome. All of the transformed cells formed colonies in soft agar, grew to high saturation densities and produced tumors when transplanted into nude mice. The Ki-MSV and M-MSV(FeLV)-transformed cells produced tumors in newborn WH/J rabbits, thus providing an important tool for studying tumor immunity in the rabbit.

Internal organization of endogenous proviral DNAs of xenotropic murine leukemia viruses.

The internal organization of endogenous xenotropic murine leukemia virus proviruses was determined in a series of blot hybridization experiments in which DNA from several different inbred mouse strains, digested with restriction enzymes known to cleave xenotropic proviral DNAs at least twice, was annealed to generalized murine leukemia virus or xenotropic env-specific DNA probes. Comigrating bands of variable intensity which hybridized to the xenotropic env probe were identified in all inbred mouse DNA preparations. At least seven classes of endogenous xenotropic proviral DNA with respect to SacI cleavage maps were detected in mouse DNA. Two of the seven classes were indistinguishable from proviruses associated with known infectious xenotropic murine leukemia viruses. These results are consistent with the existence of related but organizationally distinct families of endogenous xenotropic proviral DNA that are present in different relative abundances in mouse genomic DNA.

Properties of mouse leukemia viruses XVIII. Effective treatment of AKR leukemia with antibody to gp7l eliminates the neonatal burst of ecotropic AKR virus producing cells

To suppress spontaneous leukemia, treatment of AKR mice with antibody to the murine leukemia virus (MuLV) major glycoprotein (gp71) must commence during a narrow “window” period between birth and Day 3. To examine the effect of treatment on virus expression, infectious MuLV cell centers (ICCs), free MuLV, and viral antigen presence were examined in various organs of control and antibody-treated neonatal AKR mice. Ecotropic MuLV ICCs and viral antigens were found to increase 100-fold between Days 3 and 8 in thymuses, spleens, and livers in untreated mice. Quantitatively, spleens and livers had about 100-fold more viral ICCs than the thymus. After Day 8 viral ICCs persisted at plateau levels in all organs, with thymic ICCs 100-fold below spleens and livers. Maximal plateau levels of free ecotropic virus were reached only by Day 16. Xenotropic MuLV was occasionally isolated in ICC assays, but no recombinant MuLV was ever detected in any organ early in life. Viral p30 and gp7l antigen expression detected by immunofluorescent assays (IFA) closely corresponded to ICCs. In contrast, in AKR mice treated three or more times with gp7l antibody, MuLV ICCs were essentially undetectable during the first 3 weeks of life. In IFA, anti-gp7l-treated mice also had no virus antigen-positive cells in the organs tested. Spleen cells from control antibody-treated AKR mice were also treated in vitro with immune antibody and tested for virus- and antigen-positive cells. Anti-gp7l antibody did bind in vitro to virus-positive cells as measured by IFA but was neither cytotoxic nor reduced viral ICCs. However, the addition of complement to anti-gp7l antibody-coated cells resulted in a highly specific, effective killing of those cells scoring as viral ICCs. To determine the role of induction and/or clonal expansion of virus-positive cells relative to virus spread, perinatal F 1 (NIH × AKR) (by convention, maternal parent listed first in F 1 designations) and (BALB/c × AKR) mouse cells were assayed for ecotropic ICCs. Fv-1 n permissive (NIH × AKR) mice had only slightly fewer ICCs, and the timing and distribution of virus release was similar to AKR mice. Fv-1 nb restrictive (BALB/c × AKR) mice had different kinetics of ICC formation. At plateau, the ICCs were severalfold lower than that in Fv-1 n permissive mice, suggesting that an increase of virus-positive cells occurs during the neonatal period by means other than virus spread. However, virus spread may account for up to a 10-fold-increase in ICCs in AKR mice. The above data graphically demonstrated that the treatment “window” corresponded to a time just prior to the burst of ecotropic virus ICCs. The nature of ecotropic virus-positive cells and their elimination with treatment are discussed relative to later events in AKR leukemogenesis.

In Vivo Antigenic Modification of Tumor Cells. I. Introduction of Murine Leukemia Virus Antigens on Non-Virus-Producing Murine Sarcomas23

Murine oncovirus antigens represent excellent targets for immune recognition, and virus-associated tumors are generally susceptible to various immunotherapy protocols. Virus-negative tumors, however, are nonimmunogenic and refractory to immunologic control. Therefore, the feasibility of the introduction of antigens onto non-virus-expressing tumors in situ in inbred C57BL/6J mice by systemic administration of nononcogenic murine retroviruses was investigated. Two classes of murine fibrosarcomas were studied: a 3-methylcholanthrene-induced fibrosarcoma syngeneic to C57BL/6 mice (MCA-FS) and a Harvey murine sarcoma virus-transformed, nonproducer fibrosarcoma syngeneic to C57BL/6 mice (H-NP). Both were found to be devoid of infectious ecotropic murine leukemia virus (MuLV) or MuLV antigens. A single dose of Friend murine leukemia virus (F-MuLV) was used to superinfect MCA-FS- and H-NP-induced tumors in vivo and converted these tumors to a highly productive, virus-positive state. In vivo superinfected tumors were indistinguishable from their preinfected counterparts by competition radioimmunoassays for the virion's major envelope glycoprotein, gp71, and its group-specific antigen, p30, and by assays for infectious virus. Analysis of virus from tumor extracts proved that the antigenic specificity of the superinfected tumor was provided by F-MuLV administered systemically to the animals. Finally, an immunoperoxidase technique, applied to tumor cross sections, demonstrated the uniform appearance of viral antigens in the superinfected tumors.

Mutant cells that abnormally process plasma membrane glycoproteins encoded by murine leukemia virus

Wild-type normal rat kidney fibroblasts infected with the Friend strain of murine leukemia virus (MuLV) contain two virus-encoded glycoproteins on the outer surfaces of their plasma membranes: an envelope glycoprotein with an apparent molecular weight of 70,000 (gp70), and a glycoprotein that reacts with antisera to the major virion internal core proteins p30, p15, p12 and p10 and has an apparent molecular weight of 93,000 (gp93 gag). To analyze the functions of these glycoproteins and to develop a model system for studying genetics of membrane synthesis, we used an immunoselection method to isolate variant cell clones defective in processing these glycoproteins into their plasma membranes. Several lines of evidence, including complementation of glycoprotein processing defects by fusion with uninfected wild-type cells, indicate that the immunoselected variants have stably inherited membrane synthesis abnormalities that are encoded by cellular rather than by viral genes. The H4 cell line, which was selected by use of antiserum to gp70, has metabolic defects that interfere with processing of both gp70 and gp93 gag into its plasma membranes. Nevertheless, this cell line releases noninfectious MuLV. Furthermore, two cell lines (2 and 5), which were selected by use of antiserum to the virion core protein p30, specifically lack detectable cell surface or intracellular gp93 gag but contain cell surface gp70 and release infectious MuLV. These results suggest that MuLV particles can bud efficiently from cells that lack known virus-encoded plasma membrane constituents.

Genetic diversity in leukemia-prone feral house mice infected with murine leukemia virus.

The Lake Casitas (LC) mouse population located in south western Ventura county in California is unusual insofar as 85% of these mice are persistently viremic with congenitally transmitted murine leukemia virus (MuLV). The virus has been identified as the etiological agent responsible for lymphoma and neuromotor paralysis in large numbers of the mice. The majority of other wild mouse populations are generally free of infectious MuLV despite the presence of endogenous cellular DNA sequences homologous to infectious virus isolated from wild mice. Electrophoretic variation in 46 gene-enzyme systems was surveyed using mice from Lake Casitas and from a virus-negative population located in Bouquet Canyon (BC) approximately 40 miles from Lake Casitas. The LC and BC populations are genetically very similar to each other and to feral mouse populations previously studied in California and Europe. In the LC population 24% of the loci are polymorphic compared to 17% in the BC population. The average heterozygosities for the LC and Bc populations are 0.094 and 0.073, respectively. The large amount of genic variation in LC fails to support the concept of the derivation of the colony from a small number of founders. Tests for linkage disequilibrium and/or selective association of viremia and polymorphism at 15 loci located on nine mouse chromosomes did not reveal any nonrandom assortments. The viremic LC population, then, appears indistinguishable within the limits of experimental resolution from the virus-negative BC population in its population genetic structure.

Genetic and nongenetic factors in expression of infectious murine leukemia viruses in mice of the DBA/2 × RF cross

Mice of the RF and DBA/2 strains possess endogenous ecotropic murine leukemia virus (E-MuLV) genomes but express only low to undetectable levels of infectious virus in their lymphoid tissues. F1 mice of this cross showed high levels of infectious E-MuLV if DBA/2 was the maternal parent but very low levels if RF was the maternal parent. E-MuLV expression, if present, was always higher in the spleen than in the thymus. Studies of reciprocal backcross generations with both parental strains indicated that the presence of the virus was governed by a single dominant autosomal locus present in the RF strain, and that RF females, but neither RF males nor DBA/2 females or males, transmitted a non-Mendelian factor which powerfully suppressed virus expression in their progeny. Some but not all (DBA/2♀ × RF♂)F1 females also possessed the capacity to transmit this maternal suppression to their progeny. Xenotropic murine leukemia virus (X-MuLV) showed a different pattern of expression in this cross. In the thymus it was detected in a minority of DBA/2 and in no RF mice; in crosses the presence of X-MuLV in this organ was independent of the presence of E-MuLV. In the spleen, X-MuLV was detected only in a percentage of E-MuLV-positive mice. The maternal factor from RF mothers which suppressed E-MuLV did not suppress thymic expression of X-MuLV. Skin painting with 3-methylcholanthrene induced a high incidence of thymic lymphoma in mice of both parental strains and in F1 hybrids, all of which normally show only low incidences of the diseases; the treatment did not induce markedly increased expression of E-MuLV or X-MuLV in mice of either parental strain, although it did abrogate the diminution of E-MuLV titers seen with age in (DBA/2♀ × RF♂)F1 mice beyond the age of three months.

Association of endogenous retroviruses with radiation-induced leukemias of BALB/c mice.

X-irradiation of BALB/c mice in the second month of life induced a high incidence of generalized lymphatic leukemia of T-cell origin, beginning at 7 months of age. Infectious ecotropic murine leukemia virus (B-tropic predominant over N-tropic) was isolable from all tumor extracts but exhibited a wide titer range among individual leukemias. Detection of infectious xenotropic virus usually required extensive amplification on indicator cells. Dual-tropic (mink cell focus-forming) virus has not been found in the leukemias. Expression of ecotropic virus in tail extracts prepared at 6.5 months of age, although greatly enhanced compared with unirradiated controls, was not found to be prognostic of tumor development in individual mice. We conclude that leukemogenesis does not show a simple dependence on infectious murine leukemia virus expression in these mice.

Molecular cloning of infectious integrated murine leukemia virus DNA from infected mouse cells.

The lack of an endonuclease EcoRI site in the AKR murine leukemia virus (MuLV) DNA genome was utilized to molecularly clone, in Charon 4A lambda DNA, integrated infectious AKR MuLV DNA isolated from productively infected mouse cells. Three lambda-mouse recombinants (clones 614, 621, and 623) were selected by virtue of their reactivity with AKR MuLV [32P]cDNA. Clones 614 and 623 contained the complete AKR MuLV DNA flanked by nonviral cell sequences of which no more than 100 base pairs beyond the viral DNA appear to be shared. DNAs from both clones 614 and 623 were highly infectious for mouse cells and yielded N-tropic ecotropic MuLV; the specific infectivity of the DNA and the titer of the derived virus was more than 10-fold higher with 623. Clone 621 contained only some viral DNA and was not infectious under similar conditions.

In vitro induction of T-lymphocyte-mediated cytotoxicity by infectious murine type C oncornaviruses.

We have developed a system to induce oncornavirus-specific secondary cytotoxic response in vitro. When Moloney strain of murine sarcoma virus-immune spleen cells were cultivated with purified infectious Moloney murine leukemia virus (M-MuLV) or with supernates of tissue culture cells containing infectious virus, a virus-specific secondary cytotoxic response directed against type-specific determinant(s) of M-MuLV was generated in vitro, as determined by a 4-h 51Cr-release assay. The effector cells were susceptible to the treatment with anti-Thyl.2 plus complement, but were unrelated to natural killer cells (NK), because they could not lyse some target cells specific for M-MuLV in both the induction phase and the interaction between effector cells and target cells. Furthermore, a product of the env gene of M-MuLV, perhaps gp70, appeared to be responsible for this response, because viruses with recombinations in the env gene between ecotropic M-MuLV and a xenotropic virus failed to induce a response. When infectious M-MuLV was exposed to UV-light at different doses, the ability of UV-treated M-MuLV to induce a secondary cytotoxic response decreased in parallel with infectivity, indicating that infectivity was necessary for the induction of this response.

Expression of endogenous xenotropic retrovirus by methylcholanthrene-induced squamous cell carcinoma of the mouse respiratory tract.

As a model for human lung cancer, squamous cell carcinomas were induced by 3-methylcholanthrene in mouse tracheas which had been explanted to a subcutaneous site. The tumors that developed were examined for both ecotropic and xenotropic infectious murine leukemia virus (MuLV). From all squamous carcinomas--six out of six--a xenotropic MuLV was isolated. From some of the fibrosarcomas that occurred incidentally in our induction system, ecotropic MuLV was isolated. However, in the fibrosarcomas, no xenotropic MuLV at all was found.

Target Cell Heterogeneity in Murine Leukemia Virus Infection

Abstract The susceptibility of mouse lymphocytes from various organs to infection by murine leukemia virus (MuLV) (Friend) was studied both in vivo and in vitro by using either Friend virus complex (FV) or lymphatic leukemic virus (LLV), the helper virus isolated from FV. Productively infected cells releasing infectious MuLV were identified as infectious centers (IC) on sarcoma+, leukemia- indicator fibroblasts. IC were detected by days 4 to 6 postinfection in spleen and bone marrow (BM); fewer IC were observed in lymph nodes (LN) and their appearance was delayed until day 8 in FV-infected mice and day 15 in LLV-infected mice. In both groups, the appearance of IC in LN correlated with the onset of splenomegaly (mean = 300 mg) and the development of infection in peripheral blood exceeding 1 × 106 focus-forming units/ml, suggesting that the IC observed in LN were due to dislodged, circulating infected spleen and BM cells rather than due to infection of LN cells. This possibility was tested directly by using a culture system whereby cells from spleen, BM, and LN from normal mice were infected with FV or LLV in vitro. The principal target for MuLV replication in the cultures appeared to be B lymphocytes because 1) stimulation with bacterial lipopolysaccharide (LPS) greatly enhanced the infection of unseparated BALB/c spleen and bone marrow cells, nylon wool-adherent BALB/c spleen cells, and spleen and BM cells from athymic (nude) mice, 2) pretreatment of spleen cells with a heterologous anti-mouse immunoglobulin antiserum plus complement reduced the infection, and 3) the majority of virus-permissive cells in BALB/c spleens were recovered in the pure population of Ig+ cells from an immunoadsorbent column. However, no IC were detectable in LPS-stimulated LN cells from either BALB/c or nude mice, confirming the fundamental difference in susceptibility to the MuLV component of FV between B cells from spleen, BM, and LN suggested by the in vivo studies.