Structure and chemical-physical characteristics of lactate dehydrogenase-elevating virus and its RNA.

Lactate dehydrogenase-elevating virus (LDV) was purified from culture fluid of infected primary cultures of various mouse tissues (peritoneal macrophage, bone marrow, spleen, and embryo) and from plasma of infected mice. Electron microscopy of negatively stained virus and positively stained sections of LDV revealed spherical particles of uniform size with a diameter of about 55 nm, containing an electron-dense core with a diameter of about 30 nm. During sample preparation the envelope had a tendency to slough off and disintegrate to form aggregates of various sizes and small hollow particles with a diameter of 8 to 14 nm. Two strains of LDV exhibited a density of 1.13 g/cm3 in isopycnic sucrose density gradient centrifugation whether propagated in primary cultures of the various mouse tissues or isolated from plasma of infected mice. A brief incubation of LDV in a solution containing 0.01% Nonidet P-40 or Triton X was sufficient to release the viral nucleocapsid, whereas a similar treatment had no effect on Sindbis virus. The nucleocapdis of LDV exhibited a density of 1.17 g/cm3, was devoid of phosphatidylcholine, and contained only the smallest of the viral proteins, VP-1, which had a molecular weight of about 15,000. The envelope contained two proteins. VP-2 with a molecular weight of 18,000 and a glycoprotein, VP-3, which migrated heterogenously (24,000 to 44,000 daltons) during polyacrylamide gel electrophoresis. When compared to the sedimentation rate of 29S rRNA, the RNAs of LDV and Sindbis virus sedimented at 48 and 45S, respectively, whether analyzed by zone sedimentation in sucrose density gradients containing low or high salt concentrations or denatured by treatment with formaldehyde. Our results indicate that LDV should be classified as a togavirus, but that LDV is sufficiently different from alpha and flaviviruses to be excluded from these groups.     

High-throughput, library-based selection of a murine leukemia virus variant to infect nondividing cells

Gammaretroviruses, such as murine leukemia virus (MLV), are functionally distinguished from lentiviruses, such as human immunodeficiency virus, by their inability to infect nondividing cells. Attempts to engineer this property into MLV have been hindered by an incomplete understanding of early events in the viral life cycle. We utilized a transposon-based method to generate saturated peptide insertion libraries of MLV gag-pol variants with nuclear localization signals randomly incorporated throughout these overlapping genes. High-throughput selection of the libraries via iterative retroviral infection of nondividing cells led to the identification of a novel variant that successfully transduced growth-arrested cells. Vector packaging by cotransfection of the gag-pol.NLS variant with wild-type gag-pol produced high-titer virions capable of infecting neurons in vitro and in vivo. The capacity of mutant virions to transduce nondividing cells could help to elucidate incompletely understood mechanisms of the viral life cycle and greatly broaden the gene therapy applications of retroviral vectors. Furthermore, the ability to engineer key intracellular viral infection steps has potential implications for the understanding, design, and control of other post-entry events. Finally, this method of library generation and selection for a desired phenotype directly in a mammalian system can be readily expanded to address other challenges in protein engineering.     

Proliferation and differentiation of Abelson virus-infected murine myeloid leukemia cell lines does not involve an autocrine mechanism

Culture in agar of cloned promonocytic leukemia cell lines derived from Abelson virus-infected mice produced colonies of both a compact and diffuse morphology. Diffuse colonies contained fewer cells capable of forming colonies when recultured in agar than did compact colonies. Serial subcloning of cells from diffuse, but not compact, colonies ultimately led to the complete loss of colony-forming cells, i.e., to clonal extinction. The production of both compact and diffuse agar colonies was independent of the cell density of either the static liquid culture from which cells were taken for culture in agar, or the number of cells per agar culture. Furthermore, bioassays of culture supernatants indicated the leukemia cells did not secrete hemopoietic growth factors active on normal hemopoietic cells, transforming growth factors active on adherent cell lines, or factors that influenced the growth of the leukemic cells themselves. Collectively, these data suggest neither growth-factor independent replication nor the spontaneous differentiation of Abelson virus-infected myeloid cells involves autocrine secretion of growth regulators.     

Highly inducible cell lines derived from mice genetically transmitting the Moloney murine leukemia virus genome.

Permanent, non-virus-producing cell lines have been established from a mouse embryo carrying an endogenous, genetically transmitted Moloney murine leukemia virus (M-MuLV) genome. These cells carry the M-MuLV genome, as demonstrated by hybridization of cellular DNA to M-MuLV complementary DNA, but do not express it at the levels of virus production, accumulation of intracellular viral p30, or M-MuLV-specific RNA. Treatment with bromodeoxyuridine (50 microgram/ml for 24 h) resulted in induction of XC-positive NB-tropic virus, although only a small fraction of the cells released virus (less than 0.1% after 48 h). Immunofluorescent staining and flow microfluorometry indicated that a wave of p30 accumulation occurs in the induced cells, with a maximum at 24 to 48 h after the addition of bromodeoxyuridine. Furthermore, most, if not all, cells were induced to produce p30 protein. Similar kinetics were found for the accumulation of M-MuLV-specific RNA in the cytoplasm of induced cells. This rapid induction of virus expression in a majority of cells was dependent on the presence of the M-MuLV genome and probably represents primarily the expression of this endogenous virus since induction was not observed in cells similarly derived from a sibling embryo lacking the M-MuLV genome.     

Physical and biological features of polyoma virus mutants able to infect embryonal carcinoma cell lines

Three new polyoma mutants were selected for their ability to grow on the embryonal carcinoma cell line F9. These mutants share in common an insertion of two nucleotides, a thymine and an adenine, in the noncoding region located on the late side of the origin of replication. We have found that these insertions exist in all of the other polyoma virus mutants able to grow on F9 cells (Fujimura et al., Cell 23:809-814, 1981; Katinka et al., Nature (London) 290:720-722, 1981; K. Sekikawa and A. J. Levine, Proc. Natl. Acad. Sci. U.S.A. 78:1100-1104, 1981). The region containing these insertions could be folded into a stable secondary structure which included a guanine plus cytosine (G + C)-rich stem. The adenine and thymine were inserted in such a way that they maintained the palindrome in the G + C-rich stem and were complementary in the putative secondary structure that we present here. Another class of polyoma virus mutants selected on a multipotential carcinoma cell line (PCC4-Aza) were characterized by a more complex rearrangement (deletion and duplication) which occurred in the same region. This arrangement preserved the G + C-rich palindrome and also yielded a sequence which still allowed the folding of another type of stable secondary structure. The significance of these findings is discussed.     

Sequence homology between Moloney murine sarcoma virus and Moloney leukemia virus RNA.

The Moloney murine sarcoma-leukemia virus [M-MSV (MuLV)], propagated at high multiplicity of infection (MOI), was demonstrated previously to contain a native genome mass of 4 X 10(6) daltons as contrasted to a mass of 7 X 10(6) daltons for Moloney murine leukemia virus (M-MuLV). The 4 X 10(6)-dalton classof RNA from M-MSV (MuLV) was examined for base sequence homology with DNA complementary to the 7 X 10(6)-dalton M-MuLV RNA genome. Approximately 86% of the M-MSV (MuLV) was protected from RNase digestion by hybridization, whereas 95% of M-MuLV was protected under identical conditions. These results indicate that the small RNA class of high-MOI M-MSV (MuLV) contains little (perhaps 10%) genetic information not present in M-MuLV. Virtually all of the 1.8 X 10(6)-dalton subunits of M-MSV (MuLV) RNA contained regions of poly(A) since 94% of the RNA bound to oligo(dT) cellulose in 0.5 M KCl. This suggests that the formation of the 1.8 X 10(6)-dalton subunits occurs before their packaging into virions and does not result from hydrolysis of intact 3.5 X 10(6)-dalton subunits by a virion-associated nuclease.     

Identification of the Gross cell surface antigen associated with murine leukemia virus-infected cells.

The Gross cell surface antigen (GCSA) is produced by cells that are either exogenously infected with murine leukemia virus (MuLV) or are expressing endogenous MuLV genomes. In immune precipitation assays, GCSA was resolved into two serologically distinct 85,000- and 95,000-dalton viral proteins. These antigenic components are glycosylated forms of the polyprotein precursors of the MuLV internal structural proteins.     

Infection of central nervous system cells by ecotropic murine leukemia virus in C58 and AKR mice and in in utero-infected CE/J mice predisposes mice to paralytic infection by lactate dehydrogenase-elevating virus.

Certain mouse strains, such as AKR and C58, which possess N-tropic, ecotropic murine leukemia virus (MuLV) proviruses and are homozygous at the Fv-1n locus are specifically susceptible to paralytic infection (age-dependent poliomyelitis [ADPM]) by lactate dehydrogenase-elevating virus (LDV). Our results provide an explanation for this genetic linkage and directly prove that ecotropic MuLV infection of spinal cord cells is responsible for rendering anterior horn neurons susceptible to cytocidal LDV infection, which is the cause of the paralytic disease. Northern (RNA) blot hybridization of total tissue RNA and in situ hybridization of tissue sections demonstrated that only mice harboring central nervous system (CNS) cells that expressed ecotropic MuLV were susceptible to ADPM. Our evidence indicates that the ecotropic MuLV RNA is transcribed in CNS cells from ecotropic MuLV proviruses that have been acquired by infection with exogenous ecotropic MuLV, probably during embryogenesis, the time when germ line proviruses in AKR and C58 mice first become activated. In young mice, MuLV RNA-containing cells were found exclusively in white-matter tracts and therefore were glial cells. An increase in the ADPM susceptibility of the mice with advancing age correlated with the presence of an increased number of ecotropic MuLV RNA-containing cells in the spinal cords which, in turn, correlated with an increase in the number of unmethylated proviruses in the DNA extracted from spinal cords. Studies with AKXD recombinant inbred strains showed that possession of a single replication-competent ecotropic MuLV provirus (emv-11) by Fv-1n/n mice was sufficient to result in ecotropic MuLV infection of CNS cells and ADPM susceptibility. In contrast, no ecotropic MuLV RNA-positive cells were present in the CNSs of mice carrying defective ecotropic MuLV proviruses (emv-3 or emv-13) or in which ecotropic MuLV replication was blocked by the Fv-1n/b or Fv-1b/b phenotype. Such mice were resistant to paralytic LDV infection. In utero infection of CE/J mice, which are devoid of any endogenous ecotropic MuLVs, with the infectious clone of emv-11 (AKR-623) resulted in the infection of CNS cells, and the mice became ADPM susceptible, whereas littermates that had not become infected with ecotropic MuLV remained ADPM resistant.     

Expression of the gag-pol fusion protein of Moloney murine leukemia virus without gag protein does not induce virion formation or proteolytic processing.

A mutant of Moloney murine leukemia virus was generated in which the UAG termination codon at the 3' end of the gag gene was changed to a CAG codon encoding glutamine. Cells carrying the mutant provirus constitutively express the gag-pol fusion protein and no detectable gag protein. The precursor is stable, is not processed by the protease domain within the precursor, and does not induce assembly and release of virion particles.     

RNA metabolism of murine leukemia virus II. Endogenous virus-specific RNA in the uninfected BALB/c cell line JLS-V9.

Type C virus-specific RNA sequences of BALB/c endogenous virus were detected in JLS-V9 cells (an uninfected BALB/c derived line) by annealing cell RNA with 3-H-labeled virus-specific DNA. Endogenous viruses used in preparing the 3-H-labeled DNA (mostly xenotropic) was prepared from JLS-V9 cells induced to produce virus with iododeoxyuridine. In whole-cell extracts, two virus-specific RNA species, 38S and 27S, were detected. No 60 to 70S virus-specific RNA was found. The same two species of virus-specific RNA were observed in isolated cytoplasmic RNA and in cytoplasmic RNA selected for polyadenylic acid-containing species by binding and elution from oligo(dT) cellulose. Very little, if any, of the virus-specific RNA was active as messenger RNA on polyribosomes. No virus-specific RNA transcribed from genes coding for the BALB/c endogenous N-tropic virus was detected, since 3-H-labeled DNA prepared from endogenous N-tropic virus did not hybridize measurably with JLS-V9 RNA.     

Differential sensitivity of murine leukemia virus to APOBEC3-mediated inhibition is governed by virion exclusion

While members of the APOBEC3 family of human intrinsic resistance factors are able to restrict the replication of Vif-deficient forms of human immunodeficiency virus type 1 (HIV-1), they are unable to block replication of wild-type HIV-1 due to the action of Vif, which induces their degradation. In contrast, HIV-1 Vif is unable to block inhibition mediated by APOBEC3 proteins expressed by several heterologous species, including mice. Here, we have asked whether the simple retrovirus murine leukemia virus (MLV) is sensitive to restriction by the cognate murine or heterologous, human APOBEC3 proteins. We demonstrate that MLV is highly sensitive to inhibition by human APOBEC3G and APOBEC3B but resistant to inhibition by murine APOBEC3 or by other human APOBEC3 proteins, including APOBEC3F. This sensitivity fully correlates with the ability of these proteins to be packaged into MLV virion particles: i.e., human APOBEC3G and APOBEC3B are packaged while murine APOBEC3 and human APOBEC3F are excluded. Moreover, this packaging in turn correlates with the differential ability of these APOBEC3 proteins to bind MLV Gag. Together, these data suggest that MLV Gag has evolved to avoid binding, and hence virion packaging, of the cognate murine APOBEC3 protein but that MLV infectivity is still restricted by certain heterologous APOBEC3 proteins that retain this ability. Moreover, these results suggest that APOBEC3 proteins may help prevent the zoonotic infection of humans by simple retroviruses and provide a mechanism for how simple retroviruses can avoid inhibition by APOBEC3 family members.     

Identification of an FMR cell surface antigen associated with murine leukemia virus-infected cells.

FMR antigens are found on the surface of cells infected with Friend, Moloney, and Rauscher murine leukemia viruses (MuLV). These antigens are serologically distinct from the G cell surface antigens that are found on cells infected with endogenous MuLV (AKR and Gross virus). Cell surface antigens of both virus groups are immunogenic in mice, and immunization with appropriate virus-infected cells leads to the production of cytotoxic antisera. The cytotoxic activity of FMR antisera can be absorbed by disrupted preparations of Rauscher MuLV, but not by AKR MuLV. FMR antisera precipitate the viral envelope proteins gp70, pl5(E), and p12(E) from detergent-disrupted preparations of [3H]leucine-labeled MuLV. The reaction of these antisera with p15(E) and p12(E) proteins is directed against group-specific antigens and can be absorbed with AKR MuLV; in contrast, the reaction of these antisera with gp70 is directed against type-specific antigens and is absorbed only by viruses of the FMR group. In immune precipitation assays with detergent-disrupted 125I surface-labeled cells, FMR antisera react only with type-specific antigens of the viral envelpe protein. On the basis of these findings we conclude that the FMR cell surface antigen is a determinant on the MuLV env gene product.     

Polyadenylate in the virion RNA of mouse hepatitis virus.

Mouse hepatitis (MH) virus was grown in SR-CDF1-DBT, a mouse cell line, and purified by ammonium sulfate precipitation and by density gradient centrifugation. Extraction of RNA from purified virions with 1% SDS and sedimentation analysis of the RNA revealed a major 50S component and two minor components. Treatment of virions with phenol/chloroform also produced the 50S component, although its yield was lower. MH virion RNA can bind to a poly(U)-fiberglass filter, indicating that MH virion RNA contains poly(A). A poly(A)-like fragment was isolated by digestion with ribonuclease A [EC 3.1.4.22] and T1 [EC 3.1.4.8] and by DEAE-Sephadex column chromatography. Analysis of the fragment for base composition showed it to be an adenine-rich material. Its chain length was about 90 nucleotides, as determined by ion-exchange chromatography and gel electrophoresis.     

Monoclonal antibody protection from age-dependent poliomyelitis: implications regarding the pathogenesis of lactate dehydrogenase-elevating virus.

Over 90% of cyclophosphamide-treated, 6- to 7-month-old C58/M mice developed fatal paralytic disease after infection with a virulent strain of lactate dehydrogenase-elevating virus (LDV), with a mean onset of paralysis of about 16 days. Passive immunization with polyclonal antibodies or with a group of anti-LDV monoclonal antibodies (MAbs) with single-epitope specificity 1 day before or at the time of LDV infection prevented the development of paralytic disease without interfering with the replication of LDV in permissive macrophages, the primary host cells of LDV. In situ hybridization of spinal cord sections with an LDV-specific cDNA probe indicated that the MAb specifically prevented the cytocidal infection of motor neurons by LDV without blocking the infection of smaller nonneuronal cells in the spinal cord. The protective antibodies recognize at least two different epitopes on the glycoprotein of LDV, VP-3. Passive immunizations with other anti-LDV MAbs, which recognize at least three other epitopes on VP-3 of LDV, afforded no protection. In contrast to the protective effect of anti-LDV MAb injection before or at the time of LDV infection, their administration postinfection exerted relatively little protection, though it delayed the appearance of paralytic symptoms. However, repeated injections of MAbs until at least 7 days postinfection also afforded a high degree of protection. The results indicate that protective MAbs may interfere with two stages in the development of LDV-induced paralytic disease. When administered at the time of LDV infection, they prevent the initial infection of spinal cord motor neurons. After this initial event, repeated injections of MAb are required to inhibit the spread of LDV between neurons until the endogenous production of protective anti-LDV antibodies in these mice.     

Investigation of xenotropic murine leukemia virus-related virus (XMRV) in human and other cell lines

Xenotropic murine leukemia virus-related virus (XMRV) was discovered in human prostate tumors and later in some chronic fatigue syndrome (CFS) patients. However, subsequent studies have identified various sources of potential contamination with XMRV and other murine leukemia virus (MLV)-related sequences in test samples. Biological and nucleotide sequence analysis indicates that XMRV is distinct from known xenotropic MLVs and has a broad host range and cell tropism including human cells. Therefore, it is prudent to minimize the risk of human exposure to infection by evaluating XMRV contamination in cell lines handled in laboratory research and particularly those used in the manufacture of biological products. Nested DNA PCR assays were optimized for investigating XMRV gag and env sequences in various cell lines, which included MRC-5, Vero, HEK-293, MDCK, HeLa, and A549, that may be used in the development of some vaccines and other cell lines broadly used in research. The sensitivity of the DNA PCR assays was <10 copies in approximately 1.8 x 10⁵ cells equivalent of human DNA. The results indicated the absence of XMRV in the cell lines tested; although in some cases DNA fragments identified as cellular sequences were seen following the first round of PCR amplification with the env primer pair.