Molecular characterization of the Akvr-1 restriction gene: a defective endogenous retrovirus-borne gene identical to Fv-4r.

A dominant restriction allele, Akvr-1r, from California wild mice (Mus musculus domesticus) confers resistance to exogenous ecotropic murine leukemia virus (MuLV) infection. The presence of an ecotropic MuLV envelope-related glycoprotein in uninfected virus-resistant cells suggests that viral interference is a possible mechanism for this resistance. We molecularly cloned the ecotropic MuLV envelope-related sequence from the genomic DNA of a wild mouse homozygous for the Akvr-1r locus. The cloned provirus was defective and contained a C-terminal end of the pol gene, a complete envelope gene, and a 3' long terminal repeat. The presence of this provirus was directly correlated with Akvr-1r-mediated virus resistance in cell cultures and hybrid mice. The Akvr-1r provirus restriction map and partial DNA sequence were identical to those of the Fv-4r allele, an ecotropic MuLV resistance locus from Japanese feral mice (M. musculus molossinus), which was previously shown to be allelic with the Akvr-1r gene. The 3' host flanking sequences of Fv-4r and Akvr-1r also had identical restriction maps. These findings indicate that Akvr-1r and Fv-4r are the same gene. It was probably acquired by interbreeding of these feral species in recent times. Conservation of this locus might be favored by the useful function that it performs in protection against ecotropic MuLV infection endemic in both populations of wild mice.     

Genetic studies of the Fv-1 locus of mice: linkage with Gpd-1 in recombinant inbred lines.

Multiple recombinant inbred lines, derived from crosses between strains permissive to N-tropic murine leukemia viruses (Fv-1n) and strains permissive to B-tropic murine leukemia viruses (Fv-1b), have been characterized as to Fv-1 genotype and other chromosome 4 markers, including the closely linked hexose-6-phosphate dehydrogenase isozyme locus (Gpd-1). Only one recombinant between Fv-1 and Gpd-1 was found among 45 lines tested. On this basis, the distance between Fv-1 and Gpd-1 is estimated to be 0.6 centimorgans. None of the lines was either resistant or susceptible to both N- and B-tropic viruses. Nineteen other inbred strains, previously untested, were characterized as either Fv-1n or Fv-1b.     

Fv-1 host cell restriction of friend leukemia virus: microinjection of unintegrated viral DNA

The murine gene Fv-1 has been shown to exert a major influence over the replication of ecotropic murine leukemia viruses. Studies of the replication of Friend murine leukemia virus have shown that the restriction of viral replication occurs intracellularly after the initiation of viral DNA synthesis. The precise mechanism of the block imposed by the Fv-1 gene product is not completely understood. Our studies of Fv-1 restrictive infection have shown a variable decrease in the accumulation of intracellular unintegrated form I viral DNA. Analysis by microinjection of the viral DNA formed in nonpermissively infected BALB/c cells indicates that this DNA is infectious. These studies indicate that the form I DNA accumulated in nonpermissively infected BALB/c cells contains the complete viral sequences necessary for the production of viral progeny, and therefore, they suggest that the Fv-1 host restrictive mechanism recognizes viral factors other than form I DNA alone. These results support the possibility that Fv-1 host restriction occurs after formation of infectious viral DNA, perhaps at the integration step itself.     

Fv-1 host restriction of Friend leukemia virus: analysis of unintegrated proviral DNA.

The murine gene Fv-1 predominantly controls the outcome of infection by murine ecotropic retroviruses. The inhibition of virus replication by the Fv-1 gene product has been determined to be at an early stage in virus replication. Mechanistically, its effect appears to be on the accumulation of unintegrated proviral DNA or its integration or both. We investigated the synthesis of unintegrated proviral DNA, using several clones of B-, N-, or NB-tropic Friend murine leukemia virus. Our results indicate that the accumulation of B-tropic proviral DNA in NIH cells may be inhibited at either the level of linear (form III) or covalently closed circular DNA (form I), depending upon the degree of restriction of the clone of virus used. We confirmed that there is an effect of the Fv-1 gene on the accumulation of form I DNA of either B- or N-tropic Friend murine leukemia virus. However, the decrease in infectious centers effected by the Fv-1 gene did not correlate quantitatively with the effect on form I proviral DNA produced by N-tropic Friend murine leukemia virus in nonpermissive cells. Lastly, we demonstrated in nonpermissively infected NIH cells that a rapidly migrating doublet of viral DNA is formed.     

Fv-1 determinants in xenotropic murine leukemia viruses studied with biological assay systems: Isolation of xenotropic virus with N-tropic Fv-1 activity in the cryptic form.

By a biological assay system using phenotypically mixed ecotropic and xenotropic murine leukemia viruses, we investigated whether in the virions of a xenotropic virus there is N- or B-tropic Fv-1 determinant in active form. The existence of N-tropic Fv-1 determinant was demonstrated in SL-XT-1 xenotropic virus isolated from the spleen of a 3-month-old SL mouse, and the N-tropic Fv-1 tropism was confirmed by analysis of the phenotypically mixed viruses harvested from clonal SC-1 cells doubly infected with the SL-XT-1 and B-tropic ecotropic viruses. However, neither N- nor B-tropic Fv-1 determinant was demonstrated in any xenotropic viruses isolated from embryo cells of BALB/c, NZB, or DBA/2 mice, or Cas E #1-IU, and xenotropic-like virus isolated from a wild mouse.     

Unstable resistance of G mouse fibroblasts to ecotropic murine leukemia virus infection.

G mouse cells were resistant to N- and NB-tropic Friend leukemia viruses and to B-tropic WN 1802B. Though the cells were resistant to focus formation by the Moloney isolate of murine sarcoma virus, they were relatively sensitive to helper component murine leukemia virus. To amphotropic murine leukemia virus and to focus formation by amphotropic murine sarcoma virus, G mouse cells were fully permissive. When the cell lines were established starting from the individual embryos, most cell lines were not resistant to the murine leukemia viruses. Only one resistant line was established. Cloning of this cell line indicated that the resistant cells constantly segregated sensitive cells during the culture; i.e., the G mouse cell cultures were probably always mixtures of sensitive and resistant cells. Among the sensitive cell clones, some were devoid of Fv-1 restriction. Such dually permissive cells, and also feral mouse-derived SC-1 cells, retained glucose-6-phosphate dehydrogenase-1 and apparently normal number 4 chromosomes. The loss of Fv-1 restriction in these mouse cells was not brought about by any gross structural changes in the vicinity of Fv-1 on number 4 chromosomes.     

Susceptibility of wild mouse cells to exogenous infection with xenotropic leukemia viruses: control by a single dominant locus on chromosome 1.

Although xenotropic murine leukemia viruses cannot productively infect cells of laboratory mice, cells from various wild-derived mice can support replication of these viruses. Although the virus-sensitive wild mice generally lack all or most of the xenotropic proviral genes characteristic of inbred strains, susceptibility to exogenous infection is unrelated to inheritance of these sequences. Instead, susceptibility is controlled by a single dominant gene, designated Sxv, which maps to chromosome 1. Sxv is closely linked to, but distinct from Bxv-1, the major locus for induction of xenotropic murine leukemia viruses in laboratory mice. Genetic experiments designed to characterize Sxv show that this gene also controls sensitivity to a wild mouse virus with the interference properties of mink cell focus-forming murine leukemia viruses, and that Sxv-mediated susceptibility to xenotropic murine leukemia viruses is restricted by the mink cell focus-forming virus resistance gene Rmcf. These data, together with genetic mapping of the mink cell focus-forming virus cell surface receptor locus to this same region of chromosome 1, suggest that Sxv may encode a wild mouse variant of the mink cell focus-forming virus receptor that allows penetration by xenotropic murine leukemia viruses.     

Abrogation of Fv-1 restriction by genome-deficient virions produced by a retrovirus packaging cell line.

The Fv-1b-mediated restriction of N-tropic retrovirus vector infection of BALB/3T3 cells was partially abrogated by prior infection with N-tropic murine leukemia virus. Likewise, abrogation of the Fv-1b restriction of N-tropic murine leukemia virus replication was accomplished by prior infection with genome-deficient virions produced by an N-tropic murine leukemia virus packaging cell line. The latter observation suggests that the Fv-1 target in genome-deficient virions abrogates Fv-1 restriction in the absence of any viral genome-directed processes.     

Inherited resistance to N- and B-tropic murine leukemia viruses in vitro: titration patterns in strains SIM and SIM.R congenic at the Fv-1 locus.

We have investigated the titration patterns of murine leukemia viruses on mouse embryo cultures derived from a pair of congenic strains differing at the Fv-1 locus. XC plaque and infectious center assays carried out with N- and B-tropic viruses on both SIM (Fv-1nn) and SIM.R(Fv-1bb) host cells yielded results that were best approximated by Poisson one-hit curves. Titration curves of N-tropic virus by direct XC plaque assay were linear and parallel on the different hosts, with titers 1.8 to 2.7 log10 lower on SIM.R and on (SIM X SIM.R)F1 than on SIM cells; similar linear and parallel curves were found for B-tropic virus, with titers 1.4 to 2.0 log10 lower on SIM and (SIM XSIM-R)F1 than on SIM-R cells. In the infectious center assays, the proportion of infected cells was linearly related to multiplicity of infection on both permissive (N- on SIM and B- on SIM.R) restrictive (B- on SIM and N- on SIM.R) genotypes at multiplicities of infection below 0.5; the line relating the variables was about 1 log10 lower in the restrictive than in the permissive situations. At multiplicities of infection where the proportion of infected cells reached a plateau, differences between the results on permissive and restrictive genotypes were considerably reduced. This appeared to be due to the action of non-Fv-1 factors in permissive host. We conclude that the major action of the restrictive allele at the Fv-1 locus in this system is to reduce the probability of successful murine leukemia virus infection without a change in hitness.     

Host restriction of friend leukemia virus; fate of input virion RNA

Host restriction of oncogenesis by RNA tumor viruses may be studied in vitro by measuring the replication of the lymphatic leukemia component of the Friend Virus Complex (LLV-F) in either NIH-Swiss or Balb/C mouse embryo cells. These cells derive from mice differing at the Fv-1 locus, which controls the replication of all murine RNA leukemia viruses. Studies of early events in the replication of LLV-F were carried out by following the infection of permissive and restrictive mouse embryo cells by ³²P labeled LLV-F. ³²P labeled viral genome RNA rapidly becomes associated with cell nuclei and may be found integrated to the same extent with high molecular weight host DNA of either permissive or restrictive cells. These results suggest that Fv-1 mediated host restriction of LLV-F occurs at a step following integration of viral genome RNA into host DNA.Two other conclusions are suggested by these data. The nucleus appears to be the site of activation and synthesis of DNA of the infecting virus; and the “provirus”, at least transiently, is represented as an RNA-DNA hybrid molecule covalently integrated with host cell DNA.     

Fv-1 restriction and its effects on murine leukemia virus integration in vivo and in vitro.

We have investigated the mechanisms by which alleles at the mouse Fv-1 locus restrict replication of murine leukemia viruses. Inhibition of productive infection is closely paralleled by reduced accumulation of integrated proviral DNA as well as by reduced levels of linear viral DNA in a cytoplasmic fraction. Nevertheless, viral DNA is present at nearly normal levels in a nuclear fraction, and total amounts of viral DNA are only mildly affected in restrictive infections, suggesting a block in integration to account for reduced levels of proviral DNA. However, integrase (IN)-dependent trimming of 3' ends of viral DNA occurs normally in vivo during restrictive infections, demonstrating that not all IN-mediated events are prevented in vivo. Furthermore, viral integration complexes present in nuclear extracts of infected restrictive cells are fully competent to integrate their DNA into a heterologous target in vitro. Thus, the Fv-1-dependent activity that restricts integration in vivo may be lost in vitro; alternatively, Fv-1 restriction may prevent a step required for integration in vivo that is bypassed in vitro.     

Murine retroviral restriction genes Fv-4 and Akvr-1 are alleles of a single locus.

The two murine retroviral restriction genes, Fv-4 and Akvr-1, are very similar in their effects, distributions, ranges of action, and phenotypes. Akvr-1 has been shown to segregate independently in backcrosses with a variety of retroviral restriction loci, including Fv-1, Fv-2, Ril-1, and Ril-2. An allelism test cross of FRG (Fv-4R) X LCRR (Akvr-1R) hybrids mated to AKR mice failed to produce any viremic offspring. These results suggested that Akvr-1R and Fv-4R are alleles of a single locus, Fv-4, on mouse chromosome 12.     

Lymphocyte Deficiencies Increase Susceptibility to Friend Virus-Induced Erythroleukemia in Fv-2 Genetically Resistant Mice

The study of genetic resistance to retroviral diseases provides insights into the mechanisms by which organisms overcome potentially lethal infections. Fv-2 resistance to Friend virus-induced erythroleukemia acts through nonimmunological mechanisms to prevent early virus spread, but it does not completely block infection. The current experiments were done to determine whether Fv-2 alone could provide resistance or whether immunological mechanisms were also required to bring infection under control. Fv-2-resistant mice that were CD4(+) T-cell deficient were able to restrict early virus replication and spread as well as normal Fv-2-resistant mice, but they could not maintain control and developed severe Friend virus-induced splenomegaly and erythroleukemia by 6 to 8 weeks postinfection. Mice deficient in CD8(+) T cells and, to a lesser extent, B cells were also susceptible to late Friend virus-induced disease. Thus, Fv-2 resistance does not independently prevent FV-induced erythroleukemia but works in concert with the immune system by limiting early infection long enough to allow virus-specific immunity time to develop and facilitate recovery.     

Genetic evidence for a product of the Fv-1 locus that transfers resistance to mouse leukemia viruses.

Extracts of mouse cells have been shown to transfer to N- or B-trophic host range types of mouse leukemia viruses. The genetic specificity of the inhibition was tested in two ways: (i) by correlating the Fv-1 genotype of a number of mouse strains with the restriction-transferring activity of extracts of the respective embryo cell cultures, and (ii) by correlating the Fv-1 genotype of BLC3F2 (C57BL/6 female [Fv-1bb] by C3H male [Fv-1nn] parental strains) mouse embryos, which segregate the Fv-1 alleles in a 12:1 ratio, with the inhibitor activity of extracts of the cells from each embryo. Five independent matings, totaling 45 individual embryos, were tested. Each embryo was cultured, and the Fv-1 genotype was determined independently by titration of N- and B-tropic viruses; the extracts of replicate secondary cultures were tested for their effect on infection of permissive cells by N- and B-tropic viruses. The specific-restriction-transferring activity of the embryos was found to segregate with the appropriate Fv-1 genotype. These res-lts confirm the suggestion that the inhibitor of the leukemia virus host range types in the cellular extracts is a product of the Fv-1 locus.     

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 performance and soleus muscle fiber composition in wild-derived and laboratory inbred mouse strains.

We compared four inbred mouse strains in their physical performance, measured as a maximal treadmill running time, characteristics of soleus muscle, anatomic character, and growth. The strains used were Mus musculus domesticus [C57BL/6 (B6) and BALB/c], Mus musculus molossinus (MSM/Ms), and Mus spretus. Maximal running time was significantly different among these four mouse strains. Running time until exhaustion was highest in MSM/Ms and lowest in M. spretus. Maximal times for the laboratory mouse strains were nearly identical. Soleus muscle fiber type and cross-sectional area also differed significantly among the species. In particular, M. spretus was significantly different from the other inbred mouse strains. Growth in the wild-derived inbred mice appeared to be complete earlier than in the laboratory mice, and the body size of the wild strains was about half that of the laboratory strains. From these results, we propose that wild-derived inbred mouse strains are useful models for enhancing phenotypic variation in physical performance and adaptability.     

Loss of Fv-1 restriction in Balb/3T3 cells following infection with a single N tropic murine leukemia virus particle.

The ability of various murine leukemia viruses (MuLVs) to replicate in mouse cells exhibiting Fv-1 restriction was analyzed by quantitative dose-response assays. In particular, the effect of infection with N, B, or NB tropic MuLVs on Fv-1b restriction in Balb/3T3 cells was measured with an infection center technique in which pseudotypes of murine sarcoma virus (MSV), which have been shown to exhibit Fv-1 dependence of expression, were used to quantitate the degree of restriction. The resulting dose-response curves indicate that productive infection of a single Balb/3T3 cell with N tropic MSV requires co-infection with two MuLV particles. These two MuLV particles are functionally distinguishable. One of them must be N tropic and must be added less than 18 hr after infection with N tropic MSV. The second MuLV particle, on the other hand, need not be N tropic and may be added at any time. Balb/3T3 cultures infected with sufficient N tropic MuLV become fully permissive to transformation by N tropic MSV and to productive infection by N tropic MuLV. This effect, termed "abrogation" of Fv-1 restriction, results from infection of a Balb/3T3 cell with a single N tropic MuLV particle, but apparently occurs without viral replication. It seems probable that a requirement for abrogation of Fv-1b restriction by a single infectious particle of N tropic MuLV, which does not itself replicate, is responsible for the two-hit dose-response relationship observed in infectivity titrations of N tropic MuLV in Balb/3T3 cells. The requirements that N tropic MuLV be added within a specified time period with regard to N tropic MSV in order for abrogation to occur suggests that in the absence of N tropic MuLV, the cellular Fv-1b restriction mechanism inactivates N tropic MSV by 9 hr after infection.     

Studies with aphidicolin on the Fv-1 host restriction of Friend murine leukemia virus.

The murine gene Fv-1 exerts a major control over the replication of Friend murine leukemia virus (F-MuLV). An effect of the gene product has been determined to be at the level of accumulation and integration of viral DNA. Aphidicolin, an inhibitor of eucaryotic DNA polymerase alpha, was studied in murine cells infected either permissively or nonpermissively with regard to the Fv-1 genotype. Results indicated that inhibition of DNA polymerase alpha did not affect the accumulation of form III viral DNA in either permissive or nonpermissive cells. However, the normal accumulation of circular form I DNA in permissive cells was inhibited. The block in the accumulation of form I DNA resembled that occurring in some F-MuLV Fv-1-nonpermissive infections. Additionally, aphidicolin treatment resulted in the accumulation of novel low-molecular-weight viral DNA species, normally detectable in a nonpermissive infection of NIH cells with B-tropic F-MuLV. These data suggest that the Fv-1 gene product may interact with host DNA polymerase alpha to prevent viral replication.     

Transgenic Fv-4 mice resistant to Friend virus.

Fv-4 is a mouse gene that confers resistance to infection with ecotropic retroviruses. A candidate Fv-4 gene was cloned previously and found to resemble the 3' half of a murine leukemia virus (MuLV). To study the effect of this gene in vivo, we generated two transgenic mouse strains carrying the Fv-4 env gene under control of its presumed natural promoter, a cellular sequence unrelated to retroviruses. Transgenic progeny expressed a 3-kb Fv-4 env RNA in all of the organs and tissues examined, as well as an Fv-4 envelope antigen on the surface of thymocytes and spleen cells, similar to mice carrying the natural Fv-4 gene. One of the two transgenic strains (designated Fv4-2) expressed three to nine times as much transgene RNA and protein as the other strain (Fv4-11). When challenged with a Friend virus complex containing up to 10(4) XC PFU of Friend MuLV, Fv4-2 mice were completely resistant to development of splenomegaly and had no detectable ecotropic virus in the spleen or blood, confirming that the cloned Fv-4 gene is responsible for resistance to ecotropic MuLV in vivo. In contrast, Fv4-11 mice were only partially resistant, developing viremia and splenomegaly at the highest inoculum dose but recovering from viremia several weeks after inoculation with 10-fold less virus. The phenotype of recovery from viremia in Fv4-11 mice was unexpected and suggests that low levels of expression of the Fv-4 gene enhance the effectiveness of the immune response.