Domain structure of the Moloney murine leukemia virus reverse transcriptase: mutational analysis and separate expression of the DNA polymerase and RNase H activities

The reverse transcriptase of Moloney murine leukemia virus, like that of all retroviruses, exhibits a DNA polymerase activity capable of synthesis on RNA or DNA templates and an RNase H activity with specificity for RNA in the form of an RNA.DNA hybrid. We have generated a library of linker insertion mutants of the Moloney murine leukemia virus enzyme expressed in bacteria and assayed these mutants for both enzymatic activities. Those mutations affecting the DNA polymerase activity were clustered in the 5'-proximal two-thirds of the gene, and those affecting RNase H were in the remaining 3' one-third. Based on these maps, plasmids were made that expressed each one of the domains separately; assays of the proteins encoded by these plasmids showed that each domain exhibited only the expected activity

Characterization of endonuclease activities in Moloney murine leukemia virus and its replication-defective mutants

To study Moloney murine leukemia virus (M-MulV) proteins associated with the integration of proviral DNA into the host chromosome, we isolated endonuclease activities from purified virion preparations of the wild type and two of its replication mutants. A major endonuclease activity was identified in virions of M-MuLV; the enzyme catalyzed nicks in double-stranded DNA in the presence of either Mn2+ or Mg2+ and was stimulated by ATP. The endonuclease nicked DNA adjacent to all four nucleotides with some preference for G and C. The same enzyme, and in comparable amounts, was isolated from two virus replication mutants: dl2905, deficient in the processing of Pr65gag and Pr200gag-pol, and dl50401, deficient for the virus integration function. In the process of these experiments, the residual reverse transcriptase in mutant dl2905 was shown to be the mature size, implying that the uncleaved precursor lacks enzymatic activity. It appears that the major endonuclease activity found in virions of M-MuLV is not encoded by either the gag or pol genes

Intramolecular integration within Moloney murine leukemia virus DNA

By screening a library of unintegrated, circular Moloney murine leukemia virus (M-MuLV) DNA cloned in lambda phage, we found that approximately 20% of the M-MuLV DNA inserts contained internal sequence deletions or inversions. Restriction enzyme mapping demonstrated tht the deleted segments frequently abutted a long terminal repeat (LTR) sequence, whereas the inverted segments were usually flanked by LTR sequences, suggesting that many of the variants arose as a consequence of M-MuLV DNA molecules integrating within their own DNA. Nucleotide sequencing also suggested that most of the variant inserts were generated by autointegration. One of the recombinant M-MuLV DNA inserts contained a large inverted repeat of a unique M-MuLV sequence abutting an LTR. This molecule was shown by nucleotide sequencing to have arisen by an M-MuLV DNA Molecule integrating within a second M-MuLV DNA molecule before cloning. The autointegrated M-MuLV DNA had generally lost two base pairs from the LTR sequence at each junction with target site DNA, whereas a four-base-pair direct repeat of target site DNA flanked the integrated viral DNA. Nucleotide sequencing of preintegration target site DNA showed that this four-base-pair direct repeat was present only once before integration and was thus reiterated by the integration event. The results obtained from the autointegrated clones were supported by nucleotide sequencing of the host-virus junction of two cloned M-MuLV integrated proviruses obtained from infected rat cells. Detailed analysis of the different unique target site sequences revealed no obvious common features

Hachimoji DNA and RNA: A genetic system with eight building blocks

Reported here are DNA and RNA-like systems built from eight (hachi-) nucleotide letters (-moji) that form four orthogonal pairs. This synthetic genetic biopolymer meets the structural requirements needed to support Darwinism, including a polyelectrolyte backbone, predictable thermodynamic stability, and stereoregular building blocks that fit a Schrödinger aperiodic crystal. Measured thermodynamic parameters predict the stability of hachimoji duplexes, allowing hachimoji DNA to double the information density of natural terran DNA. Three crystal structures show that the synthetic building blocks do not perturb the aperiodic crystal seen in the DNA double helix. Hachimoji DNA was then transcribed to give hachimoji RNA in the form of a functioning fluorescent hachimoji aptamer. These results expand the scope of molecular structures that might support life, including life throughout the cosmos

Differential disease restriction of Moloney and Friend murine leukemia viruses by the mouse Rmcf gene is governed by the viral long terminal repeat.

Neonatal CxD2 (Rmcfr) and Balb/c (Rmcfs) mice inoculated with Moloney murine leukemia virus (M-MuLV) exhibited approximately equivalent time course and pathology for disease. CxD2 mice showed only slightly reduced presence of Moloney mink cell focus-forming virus (M-MCF) provirus as seen by Southern blot analysis compared to Balb/c mice. This lack of restriction for disease and spread of MCF was in sharp contrast to that seen for CxD2 mice inoculated with Friend murine leukemia virus (F-MuLV), where incidence of disease and propagation of MCFs were severely restricted, as previously reported. Inoculation of CxD2 mice with FM-MuLV, a recombinant F-MuLV virus containing M-MuLV LTR sequences (U3 and R), resulted in T cell disease of time course equal to that seen in Balb/c mice; there also was little restriction for propagation of MCFs. This indicated that presence of the M-MuLV long terminal repeat (LTR) was sufficient for propagation of MCFs in CxD2 mice. Differing restriction for F-MuLV vs. M-MuLV in CxD2 mice was explained on the basis of different "MCF propagator cells" for the two viruses. It was suggested that cells propagating F-MCF (e.g., erythroid progenitors) are blocked by endogenous MCF-like gp70env protein, whereas cells propagating M-MCF (e.g., lymphoid) do not express this protein on their surface. F-MuLV disease in CxD2 mice was greatly accelerated when neonates were inoculated with a F-MuLV/F-MCF pseudotypic mixture. However, F-MCF provirus was not detectable or only barely detectable in F-MuLV/F-MCF-induced tumors, suggesting that F-MCF acted indirectly in induction of these tumors

The effect of helper virus on Abelson virus-induced transformation of lymphoid cells

Abelson murine leukemia virus (A-MuLV)-transformed fibroblast nonproducer cells were used to prepare A-MuLV stocks containing a number of different helper viruses. The oncogenicity of the A-MuLV stocks was tested by animal inoculation and their ability to transform normal mouse bone marrow cells was measured in vitro. All of the A-MuLV stocks transformed fibroblast cells efficiently. However, only A-MuLV stocks prepared with helper viruses that are highly oncogenic were efficient in vivo and in vitro in hematopoietic cell transformation. In addition, inefficient helpers did not establish a stable infection in lymphoid nonproducer cells. Thus, helper virus has a more central role in lymphoid cell transformation than in fibroblast cell transformation

Murine leukemia virus RNA dimerization is coupled to transcription and splicing processes

Most of the cell biological aspects of retroviral genome dimerization remain unknown. Murine leukemia virus (MLV) constitutes a useful model to study when and where dimerization occurs within the cell. For instance, MLV produces a subgenomic RNA (called SD') that is co-packaged with the genomic RNA predominantly as FLSD' heterodimers. This SD' RNA is generated by splicing of the genomic RNA and also by direct transcription of a splice-associated retroelement of MLV (SDARE). We took advantage of these two SD' origins to study the effects of transcription and splicing events on RNA dimerization. Using genetic approaches coupled to capture of RNA heterodimer in virions, we determined heterodimerization frequencies in different cellular contexts. Several cell lines were stably established in which SD' RNA was produced by either splicing or transcription from SDARE. Moreover, SDARE was integrated into the host chromosome either concomitantly or sequentially with the genomic provirus. Our results showed that transcribed genomic and SD' RNAs preferentially formed heterodimers when their respective proviruses were integrated together. In contrast, heterodimerization was strongly affected when the two proviruses were integrated independently. Finally, dimerization was enhanced when the transcription sites were expected to be physically close. For the first time, we report that splicing and RNA dimerization appear to be coupled. Indeed, when the RNAs underwent splicing, the FLSD' dimerization reached a frequency similar to co-transcriptional heterodimerization. Altogether, our results indicate that randomness of heterodimerization increases when RNAs are co-expressed during either transcription or splicing. Our results strongly support the notion that dimerization occurs in the nucleus, at or near the transcription and splicing sites, at areas of high viral RNA concentration

ZASC1 knockout mice exhibit an early bone marrow-specific defect in murine leukemia virus replication

Abstract Background ZASC1 is a zinc finger-containing transcription factor that was previously shown to bind to specific DNA binding sites in the Moloney murine leukemia virus (Mo-MuLV) promoter and is required for efficient viral mRNA transcription (J. Virol. 84:7473-7483, 2010). Methods To determine whether this cellular factor influences Mo-MuLV replication and viral disease pathogenesis in vivo, we generated a ZASC1 knockout mouse model and completed both early infection and long term disease pathogenesis studies. Results Mice lacking ZASC1 were born at the expected Mendelian ratio and showed no obvious physical or behavioral defects. Analysis of bone marrow samples revealed a specific increase in a common myeloid progenitor cell population in ZASC1-deficient mice, a result that is of considerable interest because osteoclasts derived from the myeloid lineage are among the first bone marrow cells infected by Mo-MuLV (J. Virol. 73: 1617-1623, 1999). Indeed, Mo-MuLV infection of neonatal mice revealed that ZASC1 is required for efficient early virus replication in the bone marrow, but not in the thymus or spleen. However, the absence of ZASC1 did not influence the timing of subsequent tumor progression or the types of tumors resulting from virus infection. Conclusions These studies have revealed that ZASC1 is important for myeloid cell differentiation in the bone marrow compartment and that this cellular factor is required for efficient Mo-MuLV replication in this tissue at an early time point post-infection

Differential multimerization of Moloney murine leukemia virus integrase purified under nondenaturing conditions

Retroviral integrases (IN) catalyze the integration of the reverse-transcribed viral DNA into the host genome, an essential process leading to virus replication. For Moloney murine leukemia virus (M-MuLV) IN, the limited solubility of the recombinant protein has restricted the development of biophysical and structural analyses. Herein, recombinant M-MuLV IN proteins, either full length or two nonoverlapping domain constructs, were purified under non-denaturing conditions from solubilized bacterial extracts by Ni2+-NTA resins. Additionally, WT IN was further purified by heparin chromatography. All of the purified proteins were shown to be active and stable. WT M-MuLV IN chromatographed with a peak corresponding with a dimer by gel filtration chromatography. In contrast, the single point mutant C209A IN migrated predominantly as a tetramer. For both proteins, fractions in equilibrium between dimers and tetramers were competent to assemble concerted two-end integrations and yielded a unique strand-transfer profile in the presence of a 28-mer U5 oligonucleotide substrate, indicative of a distinct conformation within the synaptic complex. This specific target-site selection was not observed with a shorter 20-mer U5 substrate. These studies provide the foundation for biophysical and structural analysis on M-MuLV IN and the mechanism of retroviral integration

MoMuLV-ts-1: A Unique Mouse Model of Retrovirus-Induced Lymphoma Transmitted by Breast Milk

Our laboratory has developed a murine model of lymphoma via breast milk transmission of MoMuLV-ts-1 (Moloney murine leukemia virus-temperature sensitive mutant-1). Uninfected offspring suckled from infected surrogate mothers become infected and develop lymphoma. Multiple gene integration sites of ts-1 into the infected mouse genome including tacc3, aurka, ndel1, tpx2, p53, and rhamm were identified, and mRNA expressions were quantitated. These genes produce centrosomal proteins, which may be involved in abnormal chromosomal segregation leading to aneuploidy or multiploidy, thus causing lymphoma. Since there is no report to date on this retroviral model leading to centrosomal abnormality, and causing lymphoma development, this is a valuable and unique model to study the centrosomal involvement in lymphomagenesis