Effect of murine host genotype on MCF virus expression, latency, and leukemia cell type of leukemias induced by Friend murine leukemia helper virus

Leukemias induced by neonatal inoculations of several mouse strains with different strains of Friend murine leukemia helper virus (F-MuLV) were followed for time of disease onset, cytochemical analysis of predominant cell types in leukemic organs, and expression of infectious mink cell focus-inducing (MCF) viruses detected by mink cell foci or MCF-specific monoclonal antibodies. Most BALB.B and IRW mice had a rapidly appearing, severe anemia and hepatosplenomegaly consisting of erythroid cells. MCF viruses were usually isolated from enlarged spleens of IRW mice. In contrast, C57BL/10 mice had a lower incidence of disease and much slower course. Splenomegaly and lymphadenopathy with mild anemia were seen, and the predominant cell types were either myeloid (chloroleukemia) or lymphoid. MCF viruses were never isolated from this mouse strain. (C57BL/10 X IRW)F1 mice were intermediate in latency, but all mice had disease by 8 months. Myeloid, lymphoid, and some mixed leukemias with an erythroid component were observed, but in no case did we see the severe anemia or pure erythroid involvement typical of IRW and BALB.B mice. MCF viruses were, however, isolated from 22% of these mice regardless of leukemia cell type. DBA/2 mice had a disease pattern similar to the (C57BL/10 X IRW)F1 mice, and MCF viruses were isolated from three of six mice tested. Inoculation of IRW mice with the low virulence B3 strain of F-MuLV produced disease with a longer latency than F-MuLV 57, but similar cell types were transformed by both viruses. In vitro cell lines were derived from 14 mice, and most were tumorigenic in vivo. Three lines released infectious MCF virus, and three others expressed MCF-specific cell surface antigens but did not release virus. Eight lines expressed no MCF infectious virus or viral antigens. Several lines released infectious xenotropic viruses and/or expressed xenotropic MuLV cell surface antigens recognized by monoclonal antibodies reactive with xenotropic viruses. The lack of MCF expression in many primary leukemic tissues as well as in in vitro derived leukemia cell lines of C57BL/10 and (B10 X IRW)F1 mice suggested that MCF virus generation and expression may not be required for leukemogenesis in some mouse strains or in some hemopoietic lineages.     

Characterization of a murine immunoglobulin VH gene segment in subgroup III: a new member of the 7183 gene family.

A new gene for the variable region of the immunoglobulin heavy chain (VH gene) has been isolated from BALB/c adult liver DNA using a cDNA plasmid probe containing a mouse VH sequence. The complete nucleotide sequence of this germline gene (VH10-19), shows that it belongs to the 7183 gene family. The VH gene appears to contain an intervening 104-base-long sequence and displays the same recombination signal sequences that those observed in the germline 81X. The presence of an internal heptamer at the 3' end of the VH10-19 coding region let an alternative recombination event that could increase the representation of this gene in the immature repertoire.     

Factors determining the susceptibility of NIH swiss mice to erythroleukemia induced by Friend murine leukemia virus

The phage φ80, an Escherichia coli temperate phage, has similarities in sequence and physiology with bacteriophage λ. The isolation of new λ derivatives which carry recombination genes of φ80 (red₈₀ genes) under the control of the λ immunity region is described. Study of these hybrids suggests that the recombination system of φ80 is analogous to the recE system of Escherichia coli. Expression of φ80 recombination in lysogens corrects, like recE, the recombination defect presented by recB^? mutants. The φ80 recombination system also promotes phage recombination in recA hosts, which is also a characteristic of the recE system. The study of recombination defective red₈₀ phages suggests that the φ80 recombination system also determines φ80 growth on P2 lysogens.     

Generation of thymotropic envelope gene recombinant virus and induction of lymphoma by ecotropic Moloney murine leukemia virus

Biologically cloned pure ecotropic Moloney MuLV was used to infect Balb/c and AKR mice to determine the replication of ecotropic virus, the possible generation of recombinant viruses, and the induction of disease. Infectious cell center (ICC) experiments carried out with lymphoid cells of individual Balb/c mice showed that e-M-MuLV rapidly infected up to 30% of lymphoid cells in liver, spleen, and especially in the thymus. No recombinant virus was seen until about Day 35 when a burst of RM-MuLV was observed only in the thymus. New RM-MuLV was found in all 32 preleukemic and leukemic mice tested and persisted at low levels until death. The RM-MuLV recovered early in the preleukemic phase had an env-related M-MuLV but grew very poorly. Cells from a late tumor which grew and cloned readily were examined to see whether the new RM-MuLV was present in every clone. Overtly, most tumor cells did not seem to contain RM-MuLV, but when "unmasking" was performed, every tumor cell contained identical RM-MuLV. In AKR mice, both e-M-MuLV and recombinant M-MuLV caused an acceleration of lymphoma. De novo appearance of a thymotropic RM-MuLV, which was of the Moloney RM-MuLV type and the absence of early detectable endogenous AKR-MCF-type recombinants, suggested that the early lymphoma was due to the induction of a new disease. Several theoretical approaches dealing with viral env-gene permutations are discussed.     

Identification of an epitope encoded in the env gene of Friend murine leukemia virus recognized by anti-Friend virus cytotoxic T lymphocytes.

We have previously shown that strong epitopes recognized by anti-Friend virus (FV) cytotoxic T lymphocytes (CTL) in H-2b mice are encoded in both the env and gag/pol regions of the helper friend leukemia virus genome. Two approaches have been used to identify these epitopes. At the nucleic acid level, we have constructed env genes with either of two in-frame deletions: pKR2, an env gene with a 681-bp deletion in the gp70 region and inserted into the pSV2-gpt-1 expression vector; and pKR1, an env gene with an 81-bp deletion in the p15E region and inserted into pSV2-gpt-1. Cell clones were established by transfecting Fisher rat embryo cells with pDb (the H-2Db restriction element), pNEO (for G418 selection) and either pKR1 or pKR2. Db and env gene expression was monitored by immunoprecipitation with polyclonal antibodies or by detection of viral RNA on Northern blots. Expressor cell clones were tested for susceptibility to lysis by polyclonal anti-FV/Db CTL in 51Cr-release assays. Whereas cells expressing pKR1 were lysed to the same extent as cells expressing the intact env gene, cells expressing pKR2 were resistant to lysis, suggesting that all detectable env epitopes are encoded within the 681-bp deletion. Polypeptides representing the two most likely candidate epitopes encoded in this segment were synthesized and tested for their abilities to sensitize FRE cells expressing Db alone for lysis by the CTL. One 17-mer polypeptide, AGTGDRLLNLVQGAYQA [corrected], functioned as a strong CTL epitope in this assay, but the other 18-mer polypeptide was inactive. Studies of the role of this epitope in the immune response to candidate viral vaccines are in progress.     

Wrch-1, a novel member of the Rho gene family that is regulated by Wnt-1

We report the isolation and cloning of the Wrch-1 (Wnt-1 responsive Cdc42 homolog) cDNA. Wrch-1 is a novel gene whose mRNA level increases in response to Wnt-1 signaling in Wnt-1 transformed cells, Wnt-1 transgene induced mouse mammary tumors, and Wnt-1 retrovirus infected cells. Wrch-1 encodes a homolog of the Rho family of GTPases. It shares 57% amino acid sequence identity with Cdc42, but possesses a unique N-terminal domain that contains several putative PXXP SH3-binding motifs. Like Cdc42, Wrch-1 can activate PAK-1 and JNK-1, and induce filopodium formation and stress fiber dissolution. Active Wrch-1 stimulates quiescent cells to reenter the cell cycle. Moreover, overexpression of Wrch-1 phenocopies Wnt-1 in morphological transformation of mouse mammary epithelial cells. Taken together, Wrch-1 could mediate the effects of Wnt-1 signaling in the regulation of cell morphology, cytoskeletal organization, and cell proliferation.     

Cloning and expression of the murine homologue of a putative human X-linked nuclear protein gene closely linked to PGK1 in Xq13.3

Several human inherited diseases have been localized to theXq13.3 region of the human X chromosome (X-linked dystonia withParkinsonism, sideroblasmic anemia, SCID, Menkes disease andX-linked mental retardation loci). Genes involved in the phenotypeshave been isolated for only two of them (Menkes and SCIDX).It was therefore interesting to isolate and characterize newgenes from the region. In a previous work (12 and Consalez etal,in preparation) we isolated a gene (XNP), located 350 Kb proximalto PGK1, potentially coding for a nuclear protein. We describehere the cloning and characterization of the murine homologue.The pattern of expression of the gene in the newborn mouse (especiallythe expression in particular reglons of the brain: optical lobe,frontal cortex, hippocampus and cerebellum), as well as theexpression in human tissues, suggests that this gene might beinvolved in neuronal differentiation. Among the different morbidphenotypes assigned to the region, X-linked mental retardationwould be the best candidate to be associated with this gene.     

Rapid reversion of a deletion mutation in Moloney murine leukemia virus by recombination with a closely related endogenous provirus

During abortive infection of mouse cells, defective retroviruses carrying deletions in essential functions can recombine with endogenous retroviral sequences to form viable, replication-competent viruses. We have examined the reversion of a mutant Moloney murine leukemia virus with a deletion in the protease domain of the pol gene after infection of NIH/3T3 cells. In this system revertants arise quickly, only 2 weeks after infection. Analysis of DNA clones of the revertant viral genomes showed that they were derived by recombination with a long sequence of gag and pol exhibiting 95% sequence identity to Moloney virus. One such cloned recombinant was fully infectious, indicating that the repertoire of viral sequences in the NIH/3T3 genome must include substantial stretches of functional viral genes. Examination of the viral DNAs very early in the infection revealed the presence of defective genomes, formed by nonhomologous crossovers between the two parental sequences. We suggest that these may serve as intermediates in the eventual formation of the viable revertant genomes.     

Complete nucleotide sequence of rose yellow leaf virus,a new member of the family Tombusviridae

The genome of the rose yellow leaf virus (RYLV) has been determined to be 3918 nucleotides long and to contain seven open reading frames (ORFs). ORF1 encodes a 27-kDa peptide (p27). ORF2 shares a common start codon with ORF1 and continues through the amber stop codon of p27 to encode an 87-kDa (p87) protein that has amino acid similarity to the RNA-dependent RNA polymerase (RdRp) of members of the family Tombusviridae. ORFs 3 and 4 have no significant amino acid similarity to known functional viral ORFs. ORF5 encodes a 6-kDa (p6) protein that has similarity to movement proteins of members of the Tombusviridae. ORF5A has no conventional start codon and overlaps with p6. A putative +1 frameshift mechanism allows p6 translation to continue through the stop codon and results in a 12-kDa protein that has high homology to the carmovirus p13 movement protein. The 37-kDa protein encoded by ORF6 has amino acid sequence similarity to coat proteins (CP) of members of the Tombusviridae. ORF7 has no significant amino acid similarity to known viral ORFs. Phylogenetic analysis of the RdRp amino acid sequences grouped RYLV together with the unclassified Rosa rugosa leaf distortion virus (RrLDV), pelargonium line pattern virus (PLPV), and pelargonium chlorotic ring pattern virus (PCRPV) in a distinct subgroup of the family Tombusviridae.     

Genetic and serologic re-evaluation of LyM-1 antigen specified by a gene closely linked to Mls: establishment of LyM-1 antigen of the mouse

An alloantiserum prepared against LyM-1 antigen of the mouse has been analysed in detail. This antiserum, C3H/HeJ anti-CBA/J, has turned out to define two antigens which are specified by independently segregating loci. One of these is Lyb-2.1 or similar antigen specified by a gene located on chromosome 4. The other is identified as LyM-1.2 based on the result that a gene determining alloantigenicity in the Lyb-2- strain is closely linked to Mls with recombination frequency of 0.066 +/- 0.028. Since both antigens are expressed preferentially on B lymphocytes and their strain distribution is significantly overlapped, there has been much confusion about LyM-1 antigen. This is overcome by re-evaluating anti-LyM-1.2 serum under the condition in which Lyb-2.1 is not involved in analysis. The strain distribution of LyM-1 antigen, its preferential expression on B lymphocytes and close genetic linkage of LyM-1 to Mls have been established.     

Cloning and expression of a gamma-interferon-inducible gene in monocytes: a new member of a cytokine gene family

To define activation-specific sequences in human peripheral blood lymphocytes (PBL), a cDNA library was constructed by subtractive hybridization using resting and stimulated PBL pairs. Stimulation of PBL was achieved by triggering with mitogenic anti-CD2 (T11) monoclonal antibodies. Differential library screening with cDNA probes derived from stimulated versus resting PBL led to identification of two novel sequences, termed HC11 and HC14. The predicted primary and secondary structure of HC11 deduced from the translated nucleotide sequence suggests that the gene encodes a secreted protein of 99 amino acids (aa), including a 23 aa residue leader sequence. Surprisingly, Northern blot analysis demonstrated that HC11 mRNA is induced predominantly in peripheral blood non-T cells. Subsequently, we observed that the HC11 mRNA is induced in macrophages and the monocytic line U-937 by gamma-IFN, raising the possibility that T cell-derived gamma-IFN induced upon anti-CD2 stimulation activated monocytes to express HC11 RNA. In support of this notion, neutralizing anti-gamma-IFN monoclonal antibody inhibits the induction of HC11 mRNA in PBL activated through anti-CD2 antibodies. These findings suggest that there is a molecular cascade involving T cell-produced lymphokines and monokines which serve as a means for intercellular communication. Transient expression of HC11 cDNA results in a readily detectable specific set of protein bands in SDS-PAGE analysis of supernatants from radio-labeled COS cells, consistent with HC11 encoding a secreted product(s). Protein sequence comparison reveals homology with other members of a recently described inducible cytokine family whose functions are yet to be defined.     

Identification of a novel human DDX40 gene, a new member of the DEAH-box protein family

 The DExH/D-box superfamily of RNA helicases seems to play key roles during RNA metabolism, such as pre-mRNA splicing, ribosome biogenesis, and others. We have cloned a new gene of the DEAH-box protein subgroup, designated DDX40 (DEAD/H-box polypeptide 40 gene). DDX40 contains 3656 nucleotides and codes for a putative 779-amino-acid protein. Sequence analysis of the cDNA product revealed that it contained a DEAH (Asp-Glu-Ala-His) sequence motif and other conserved motifs. The DDX40 protein shared 53% and 43% amino acid identity with human DDX8 and yeast Drh1, respectively, in the conserved region. Northern blot analysis showed that DDX40 was expressed ubiquitously in the eight tissues examined, implying a general physiological function of the protein. We speculate that, like other members of the DExH/D-box superfamily, DDX40 may play roles in pre-mRNA splicing, ribosome biogenesis and other RNA processing functions. Received: August 7, 2002 / Accepted: September 12, 2002     

Identification of a novel human DDX40gene,a new member of the DEAH-box protein family

The DExH/D-box superfamily of RNA helicases seems to play key roles during RNA metabolism, such as pre-mRNA splicing, ribosome biogenesis, and others. We have cloned a new gene of the DEAH-box protein subgroup, designated DDX40 (DEAD/H-box polypeptide 40 gene). DDX40 contains 3656 nucleotides and codes for a putative 779-amino-acid protein. Sequence analysis of the cDNA product revealed that it contained a DEAH (Asp-Glu-Ala-His) sequence motif and other conserved motifs. The DDX40 protein shared 53% and 43% amino acid identity with human DDX8 and yeast Drh1, respectively, in the conserved region. Northern blot analysis showed that DDX40 was expressed ubiquitously in the eight tissues examined, implying a general physiological function of the protein. We speculate that, like other members of the DExH/D-box superfamily, DDX40 may play roles in pre-mRNA splicing, ribosome biogenesis and other RNA processing functions.     

Complete nucleotide sequence of Rosa rugosa leaf distortion virus, a new member of the family Tombusviridae

This report describes the complete nucleotide sequence and genome organization of Rosa rugosa leaf distortion virus (RrLDV), the causal agent of a previously undescribed virus disease of Rosa rugosa. The RrLDV genome is a positive-sense ssRNA, 3971 nucleotides in length, containing five open reading frames (ORFs). ORF1 encodes a 27-kDa peptide (p27). ORF2 shares a common start codon with ORF1 and continues through the amber stop codon of p27 to produce an 87-kDa protein (p87) with amino acid sequence similarity to the RNA-dependent RNA polymerases (RdRp) of members of the family Tombusviridae. ORF3 encodes a protein of 8 kDa with no significant similarity to known viral sequences. ORF4 encodes a 6-kDa protein (p6) with similarity to the p13 movement proteins of members of the family Tombusviridae. ORF5 has no conventional start codon and overlaps with p6. A putative +1 frame shift mechanism allows p6 translation to continue through the stop codon and results in a 12-kDa protein with high homology to the carmovirus p13 movement protein. The 37-kDa protein encoded by ORF6 has amino acid sequence similarity to coat proteins (CPs) of members of the family Tombusviridae. Phylogenetic analyses of the RdRp and CP amino acid sequences placed RrLDV in a subgroup close to members of the genus Carmovirus of the family Tombusviridae.     

Cellular tropism and localization in the rodent nervous system of a neuropathogenic variant of Friend murine leukemia virus.

BACKGROUND: We studied PVC-211 murine leukemia virus (MuLV) (1), a neuropathogenic variant of Friend MuLV, to determine its cellular tropism and distribution in the nervous system of infected rats and the factors that affected disease expression. EXPERIMENTAL DESIGN: Rats from five different strains and mice from 3 strains were inoculated intracerebrally or intraperitoneally from birth to 10 days of age and observed for signs of neurologic disease and tumors for 24 weeks. Nervous system pathology, MuLV gp70 expression, and virus production were evaluated weekly for 4 weeks after perinatal infection of Fisher (F344) rats. Blood-brain-barrier integrity and ultrastructure were evaluated in 21-day-old symptomatic infected rats. Microvessel and mixed glial cell cultures were prepared from brains of infected and uninfected 21-day-old F344 rats and evaluated for virus production, MuLV gp70 expression, and the presence of PVC-211 MuLV DNA. RESULTS: Tremor, ataxia, spasticity, and hindlimb weakness occurred in rats and mice as early as 3 weeks after neonatal infection. Severity, latency, and progression varied among mouse and rat strains but exposure to PVC-211 MuLV before 6 days of age was required for disease expression. Rapid PVC-211 MuLV replication in brain capillary endothelial cells (BCEC) early in the perinatal period was followed by widespread astrogliosis, neuropil vacuolation, and finally, neuronal degeneration in the spinal cord, brainstem, cerebellum, and subcortex. MuLV gp70 expression in vivo increased during infection, was restricted to BCEC, but was not associated with perivascular inflammatory infiltrates. BCEC cultured from microvessel preparations but not astrocytes or microglia in mixed glial cell cultures isolated from infected rats contained PVC-211 MuLV DNA, expressed MuLV gp70, and produced infectious virus. CONCLUSIONS: The rapid replication of PVC-211 MuLV that occurs in the nervous system of infected rodents is restricted to BCEC. These infected BCEC appear to play a critical role in initiating the astroglial response in this neurodegenerative process through mechanisms that remain to be defined.     

Sensenbrenner syndrome: a new member of the hepatorenal fibrocystic family

Cranioectodermal dysplasia (CED, Sensenbrenner syndrome; OMIM #218330) is an autosomal recessive disorder reported only in 15 cases, which is characterized by dolichocephaly, rhizomelic dwarfism, dental and nail dysplasia, and progressive tubulo-interstitial nephritis (TIN) leading to end-stage renal failure. Herein, we describe a new patient with cranio-ectodermal dysplasia. Unlike previously reported cases, this 4-year-old child presented with tubulo-interstitial nephropathy associated with liver cystic disease and elevated liver enzymes. The liver biopsy demonstrated congenital hepatic fibrosis secondary to ductal plate malformation. The coexistence of a chronic tubulo-interstitial renal disease with lesions associated to malformations of the hepatic ductal plate indicates that CED as a new member of the congenital hepatorenal fibrocystic syndromes.