Sequence requirements for integration of Moloney murine leukemia virus DNA in vitro.

Normal replication of Moloney murine leukemia virus (MoMLV) requires the integration of a DNA copy of the viral RNA genome into a chromosome of the host. In this work, we characterize the DNA sequences at the ends of the linear proviral precursor that are required for integration in the presence of MoMLV integration protein in vitro. We found that nine bases of MoMLV DNA at each end of a linear model substrate were sufficient for near-maximal levels of integration and that four bases of MoMLV DNA at each end were sufficient for low levels of correct integration. We also found that a 3'-terminal A residue was preferred for integration. We infer from the limited DNA sequence requirements for integration that factors in addition to DNA sequence direct integration protein to act at the ends of the viral DNA.     

The integration sites of endogenous and exogenous Moloney murine leukemia virus

Specific cDNA probes of Moloney and AKR murine leukemia viruses have been prepared to characterize the proviral integration sites of these viruses in the genomes of Balb/Mo and Balb/c mice. The genetically transmitted Moloney provirus of Balb/Mo mice was detected in a characteristic Eco RI DNA fragment of 16 x 10(6) daltons. No fragment of this size was detected in tissue DNAs from Balb/c mice infected as newborns with Moloney virus. We conclude that a viral integration site, occupied in preimplantation mouse embryos, is not necessarily occupied when virus infects cells in post-natal animals. Balb/Mo and Balb/c mice do carry the AkR structural gene in an Eco RI DNA fragment of 12 x 10(6) daltons. Further restriction analysis of this fragment indicated that both mouse lines carry one AKR-type provirus. Leukemogenesis in Balb/Mo and newborn infected Balb/c mice is accompanied by reintegration of Moloney viral sequences in new chromosomal sites of tumor tissues. Part of the reintegrated Moloney viral sequences are of subgenomic size. The AKR viral sequences, however, are not found in new sites. Further restriction analysis revealed that the development of Moloney virus-induced leukemia in Balb/Mo mice does not lead to detectable structural alteration of the genetically transmitted Moloney and AKR structural genes. Possible mechanisms of the reintegration process are also discussed.     

Infectious, linear, unintegrated DNA of Moloney murine leukemia virus.

A closed circular, double-stranded infectious DNA of Moloney leukemia virus has been described previously. The present report characterizes a second type of infectious, unintegrated viral DNA which is linear, largely double stranded, and of mass comparable to that of the closed circular viral DNA. The linear form is of nonpermuted sequence, and SalI endonuclease cleaves at one site 45% from one end.     

Assembly and catalysis of concerted two-end integration events by Moloney murine leukemia virus integrase

Retroviral integration results in the stable and coordinated insertion of the two termini of the linear viral DNA into the host genome. An in vitro concerted two-end integration reaction catalyzed by the Moloney murine leukemia virus (M-MuLV) integrase (IN) was used to investigate the binding and coordination of the two viral DNA ends. Comparison of the two-end integration and strand transfer assays indicates that zinc is required for efficient concerted integration utilizing plasmid DNA as target. Complementation assays using a pair of nonoverlapping integrase domains, consisting of the HHCC domain and the core/C-terminal region, yielded products containing the correct 4-base target site duplication. The efficiency of the coordinated two-end integration varied depending on the order of addition of the individual protein and DNA components in the complementation assay. Two-end integration was most efficient when the long terminal repeat (LTR) was premixed with either the target DNA or the HHCC domain. The preference for two-end integration through preincubation of the HHCC finger with the viral DNA supports the role of this domain in the recognition and/or positioning of the LTR.     

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.     

Inhibition of Moloney murine leukemia virus integration using polyamides targeting the long-terminal repeat sequences

The retroviral integrase (IN) carries out the integration of the viral DNA into the host genome. Both IN and the DNA sequences at the viral long-terminal repeat (LTR) are required for the integration function. In this report, a series of minor groove binding hairpin polyamides targeting sequences within terminal inverted repeats of the Moloney murine leukemia virus (M-MuLV) LTR were synthesized, and their effects on integration were analyzed. Using cell-free in vitro integration assays, polyamides targeting the conserved CA dinucleotide with cognate sites closest to the terminal base pairs were effective at blocking 3' processing but not strand transfer. Polyamides which efficiently inhibited 3' processing and strand transfer targeted the LTR sequences through position 9. Polyamides that inhibited integration were effective at nanomolar concentrations and showed subnanomolar affinity for their cognate LTR sites. These studies highlight the role of minor groove interactions within the LTR termini for retroviral integration.     

Mutants and pseudorevertants of Moloney murine leukemia virus with alterations at the integration site

Soon after infection, retroviruses synthesize a DNA copy of the genomic RNA and insert that DNA into the cellular genome by recombination at inverted repeat sequences at the termini of the viral genome. We have generated mutations that alter one terminus of the genome of Moloney murine leukemia virus (M-MuLV). Some mutations did not prevent integration of the viral DNA even though the very terminal bases were disrupted. Other mutations had dramatic effects on the efficiency of infection; in these cases the formation of preintegrative DNA was normal but the establishment of the productive provirus was prevented. One of these defective mutants gave rise to a pseudorevertant which differed from the wild type but displayed normal infectivity. An unusual number of bases of viral DNA were removed during the integration reaction carried out by this virus.     

Sequence-specific binding of DNA by the Moloney murine leukemia virus integrase protein.

Genetic studies have indicated that integration of retroviral DNA into the host genome depends on the presence of the inverted repeats at the free termini of the long terminal repeats on the unintegrated DNA and on the product of the 3' end of the pol gene (the integrase [IN] protein). While the precise function of the Moloney murine leukemia virus IN protein is uncertain, others have shown that it is a DNA-binding protein and functions in the processing of the inverted repeats prior to integration. By using site-directed mutagenesis, we cloned and expressed the IN protein in Escherichia coli. Crude extracts of total cellular protein were fractionated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, transferred to nitrocellulose filters, denatured in guanidine, renatured, and incubated with oligonucleotide probes. Single- and double-stranded oligonucleotides corresponding to the termini of unintegrated linear viral DNA were specifically bound by the IN protein in this assay. These data suggest that the role of the Moloney IN protein in the early steps of integration involves sequence-specific recognition of the DNA sequences found at the ends of the long terminal repeats.     

Mutational analysis of the carboxyl terminus of the Moloney murine leukemia virus integration protein.

The integration protein (IN) is required for retrovirus DNA integration into the host DNA. The function of the C terminus of the Moloney murine leukemia virus IN protein was examined. The terminal 28 amino acids were found to be nonessential. A linker insertion at position 6025, within a 36-amino-acid insertion not found in any other retrovirus, also produced viable virus.     

Gene product of Moloney murine leukemia virus required for proviral integration is a DNA-binding protein

The 3'-terminal portion of the retroviral pol gene encodes a function required for the formation of the integrated provirus soon after infection of sensitive cells. To permit the isolation of large quantities of the gene product, we expressed various portions of the pol gene of Moloney murine leukemia virus (M-MuLV) as trpE fusion proteins in Escherichia coli. The proteins were found to exhibit strong DNA-binding activity after extraction and renaturation by two different procedures. In the first method, proteins separated by polyacrylamide gel electrophoresis were blotted to nitrocellulose and assayed when bound to the support. The second procedure involved the isolation of proteins in an insoluble fraction, solubilization with guanidine, and renaturation. The characteristics of the binding activity are described and compared with those of authentic viral protein.     

Analysis of mutations in the integration function of Moloney murine leukemia virus: effects on DNA binding and cutting.

The 3' terminus of the pol gene of Moloney murine leukemia virus encodes the integration (IN) protein, required for the establishment of the integrated provirus. A series of six linker insertion mutations and two single-base substitutions were generated within the region encoding the IN protein. Mutations were initially generated within an Escherichia coli plasmid expressing the IN protein, and the resulting variants were assayed for DNA-binding activity. Mutations which altered conserved cysteine residues within a potential DNA finger-binding motif resulted in lower or variable DNA binding, which appeared to be the result of variable protein folding. Upon renaturation, these proteins were able to nonspecifically bind DNA in a manner similar to that of the other mutant IN proteins and the parent. When reconstructed back into full-length virus, seven of the eight mutations were lethal. All mutants produced a stable IN protein in virions and mediated normal conversion of the retroviral RNA to its three DNA forms. Fine-structure analysis of the linear double-stranded viral DNA indicated that all seven lethal alterations within the IN protein blocked the formation of the 3' recessed termini that normally precedes integration.     

Host cell cathepsins potentiate Moloney murine leukemia virus infection

The roles of cellular proteases in Moloney murine leukemia virus (MLV) infection were investigated using MLV particles pseudotyped with vesicular stomatitis virus (VSV) G glycoprotein as a control for effects on core MLV particles versus effects specific to Moloney MLV envelope protein (Env). The broad-spectrum inhibitors cathepsin inhibitor III and E-64d gave comparable dose-dependent inhibition of Moloney MLV Env and VSV G pseudotypes, suggesting that the decrease did not involve the envelope protein. Whereas, CA-074 Me gave a biphasic response that differentiated between Moloney MLV Env and VSV G at low concentrations, at which the drug is highly selective for cathepsin B, but was similar for both glycoproteins at higher concentrations, at which CA-074 Me inhibits other cathepsins. Moloney MLV infection was lower on cathepsin B knockout fibroblasts than wild-type cells, whereas VSV G infection was not reduced on the B-/- cells. Taken together, these results support the notion that cathepsin B acts at an envelope-dependent step while another cathepsin acts at an envelope-independent step, such as uncoating or viral-DNA synthesis. Virus binding was not affected by CA-074 Me, whereas syncytium induction was inhibited in a dose-dependent manner, consistent with cathepsin B involvement in membrane fusion. Western blot analysis revealed specific cathepsin B cleavage of SU in vitro, while TM and CA remained intact. Infection could be enhanced by preincubation of Moloney MLV with cathepsin B, consistent with SU cleavage potentiating infection. These data suggested that during infection of NIH 3T3 cells, endocytosis brings Moloney MLV to early lysosomes, where the virus encounters cellular proteases, including cathepsin B, that cleave SU.     

Structure of products of the Moloney murine leukemia virus endogenous DNA polymerase reaction.

We have investigated the process by which the single-stranded RNA genome of Moloney murine leukemia virus is copied into DNA in vitro. DNA synthesis if initiated near the 5' end of the genome, and the elongation of the growing chain occurs by a jumping mechanism whereby the DNA synthesized at the 5' end of the genome is elongated along the 3' end. Unique DNA fragments synthesized beyond the 5' end of the genome in vitro have, at their 5' and 3' ends, copies of unique sequences from the 5' and 3' ends of the genome. These flank a copy of the 49- to 60-nucleotide terminally redundant sequence. These results indicate that the terminal redundancy serves as a "bridge" to allow a DNA molecule synthesized at the 5' end of the genome to serve as a primer for synthesis from the 3' end.     

Isolation of recombinant DNA clones carrying complete integrated proviruses of Moloney murine leukemia virus

EcoRI DNA fragments from a Moloney murine leukemia virus (M-MuLV)-infected mouse fibroblast line (M-MuLV clone A9) were cloned in lambda phage Charon 4A cloning vector to derive clones containing integrated M-MuLV proviral DNA. A 10- to 16-megadalton class of EcoRI fragments was chosen for cloning, based on (i) its ability to induce XC-positive virus upon transfection of NIH/3T3 cells, and (ii) its content of a 0.8-megadalton viral KpnI fragment diagnostic for M-MuLV. Six recombinant DNA clones were isolated which contain a complete M-MuLV provirus, as judged by (i) restriction endonuclease mapping and (ii) the fact that all of the clones gave rise to XC-positive, NB-tropic virus upon DNA infection in NIH/3T3 cells. The sizes of the inserts were 12.0 (for three clones) or 12.5 megadaltons (for three clones). Restriction mapping indicated that these six clones represent five different M-MuLV proviral integrations into different cellular DNA sites.     

Moloney murine leukemia virus integration protein produced in yeast binds specifically to viral att sites.

The integration protein (IN) of Moloney murine leukemia virus (MuLV), purified after being produced in yeast cells, has been analyzed for its ability to bind its putative viral substrates, the att sites. An electrophoretic mobility shift assay revealed that the Moloney MuLV IN protein binds synthetic oligonucleotides containing att sequences, with specificity towards its cognate (MuLV) sequences. The terminal 13 base pairs, which are identical at both ends of viral DNA, are sufficient for binding if present at the ends of oligonucleotide duplexes in the same orientation as in linear viral DNA. However, only weak binding was observed when the same sequences were positioned within a substrate in a manner simulating att junctions in circular viral DNA with two long terminal repeats. Binding to att sites in oligonucleotides simulating linear viral DNA was dependent on the presence of the highly conserved CA residues preceding the site for 3' processing (an IN-dependent reaction that removes two nucleotides from the 3' ends of linear viral DNA); mutation of CA to TG abolished binding, and a CA to TA change reduced affinity by at least 20-fold. Removal of either the terminal two base pairs from both ends of the oligonucleotide duplex or the terminal two nucleotides from the 3' ends of each strand did not affect binding. The removal of three 3' terminal nucleotides, however, abolished binding, suggesting an essential role for the A residue immediately upstream of the 3' processing site in the binding reaction. These results help define the sequence requirements for att site recognition by IN, explain the conservation of the subterminal CA dinucleotide, and provide a simple assay for sequence-specific IN activity.     

Interaction of Moloney murine leukemia virus matrix protein with IQGAP

The matrix protein (MA) of the Moloney murine leukemia virus (M-MuLV) was found to interact with IQGAP1, a prominent regulator of the cytoskeleton. Mutational studies defined residues of MA critical for the interaction, and tests of viruses carrying MA mutations revealed a near-perfect correlation between binding and virus replication. The replication-defective mutants showed defects in both early and late stages of the life cycle. Four viable second-site revertant viruses were isolated from three different replication-defective parental mutants, and in all cases the interaction with IQGAP1 was restored by the suppressor mutations. The interaction of MA and IQGAP1 was readily detected in vitro and in vivo. Virus replication was potently inhibited by a C-terminal fragment of IQGAP1, and impaired by RNAi knockdown of IQGAP1 and 2. We suggest that the IQGAPs link the virus to the cytoskeleton for trafficking both into and out of the cell.