Sequential activation of the three protomers in the Moloney murine leukemia virus Env.

Viral membrane fusion proteins of class I are trimers in which the protomeric unit is a complex of a surface subunit (SU) and a fusion active transmembrane subunit (TM). Here we have studied how the protomeric units of Moloney murine leukemia virus envelope protein (Env) are activated in relation to each other, sequentially or simultaneously. We followed the isomerization of the SU-TM disulfide and subsequent SU release from Env with biochemical methods and found that this early activation step occurred sequentially in the three protomers, generating two asymmetric oligomer intermediates according to the scheme (SU-TM)3 → (SU-TM)2TM → (SU-TM)TM2 → TM3 This was the case both when activation was triggered in vitro by depleting stabilizing Ca2+ from solubilized Env and when viral Env was receptor triggered on rat XC cells. In the latter case, the activation reaction was too fast for direct observation of the intermediates, but they could be caught by alkylation of the isomerization active thiol.

X-ray crystal structure of the N-terminal region of Moloney murine leukemia virus integrase and its implications for viral DNA recognition.

The retroviral integrase (IN) carries out the integration of a dsDNA copy of the viral genome into the host DNA, an essential step for viral replication. All IN proteins have three general domains, the N-terminal domain (NTD), the catalytic core domain, and the C-terminal domain. The NTD includes an HHCC zinc finger-like motif, which is conserved in all retroviral IN proteins. Two crystal structures of Moloney murine leukemia virus (M-MuLV) IN N-terminal region (NTR) constructs that both include an N-terminal extension domain (NED, residues 1-44) and an HHCC zinc-finger NTD (residues 45-105), in two crystal forms are reported. The structures of IN NTR constructs encoding residues 1-105 (NTR1-105 ) and 8-105 (NTR8-105 ) were determined at 2.7 and 2.15 Å resolution, respectively and belong to different space groups. While both crystal forms have similar protomer structures, NTR1-105 packs as a dimer and NTR8-105 packs as a tetramer in the asymmetric unit. The structure of the NED consists of three anti-parallel ß-strands and an α-helix, similar to the NED of prototype foamy virus (PFV) IN. These three ß-strands form an extended ß-sheet with another ß-strand in the HHCC Zn2+ binding domain, which is a unique structural feature for the M-MuLV IN. The HHCC Zn2+ binding domain structure is similar to that in HIV and PFV INs, with variations within the loop regions. Differences between the PFV and MLV IN NEDs localize at regions identified to interact with the PFV LTR and are compared with established biochemical and virological data for M-MuLV. Proteins 2017; 85:647-656. © 2016 Wiley Periodicals, Inc.

On the conformational stability of the smallest RNA kissing complexes maintained through two G·C base pairs.

Two identical 5'GACG3' tetra-loop motifs with different stem sequences (called H2 and H3) are found in the 5' end region of Moloney Murine Leukemia Virus (MMLV) genomic RNA. They play important roles in RNA dimerization and encapsidation through two identical tetra-loops (5'GACG3') forming a loop-to-loop kissing complex, the smallest RNA kissing complex ever found in nature. We examined the effects of a loop-closing base pair as well as a stem sequence on the conformational stability of the kissing complex. UV melting analysis and gel electrophoresis were performed on eight RNA sequences mimicking the H2 and H3 hairpin tetra-loops with variation in loop-closing base pairs. Our results show that changing the loop-closing base pair from the wildtype (5'A·U3' for H3, 5'U·A3' for H2) to 5'G·C3'/5'C·G3' has significant effect on the stability of the kissing complexes: the substitution to 5'C·G3' significantly decreases both thermal and mechanical stability, while switching to the 5'G·C3' significantly increases the mechanical stability only. The kissing complexes with the wildtype loop-closing base pairs (5'A·U3' for H3 and 5'U·A3' for H2) show different stability when attached to a different stem sequence (H2 stem vs. H3 stem). This suggests that not only the loop-closing base pair itself, but also the stem sequence, affects the conformational stability of the RNA kissing complex.

Long time-course monitoring of ZFP809-mediated gene silencing in transgene expression driven by promoters containing MLV-derived PBS.

Expression of Moloney murine leukemia virus (MoMLV)-typed retroviral vectors is strictly suppressed in immature cells such as embryonic stem cells. The phenomenon known as gene silencing is primed by the sequence-specific binding of the zinc finger protein 809 (ZFP809) to the primer-binding site of the vectors. However, it has yet to be determined whether the ZFP809-mediated gene silencing is maintained over a long period. In this study, we established an experimental system that can monitor gene silencing during a long-term cell culture using flow cytometry technology combined with fluorescent reporters for the expression of ZFP809 and the transgene expression driven by the promoters of interest. Time-course analysis using our system revealed that ZFP809 maintains gene silencing effect even at a longtime period. Furthermore, our system was useful for the monitoring of ZFP809-mediated gene silencing regardless of the types of vectors and cell lines.

The third to fifth zinc fingers play an essential role in the binding of ZFP809 to the MLV-derived PBS.

Members of the kruppel-associated box-containing zinc finger protein (KRAB-ZFP) family mediate a number of cellular processes through binding to target DNA sequences via zinc fingers. Generally, zinc fingers recognize three-nucleotide sequences; however, this rule is not universally applicable. Zinc finger protein 809 (ZFP809) belongs to the KRAB-ZFP family and functions in repressing the expression of Moloney murine leukemia virus (MoMLV) via sequence-specific binding to the primer-binding site (PBS) located downstream of the MoMLV-long terminal repeat (LTR) and the induction of epigenetic modifications at LTR, such as repressive histone modifications and de novo DNA methylation. Previously, we demonstrated the role of the first to fifth zinc fingers of ZFP809 in binding to MLV PBS, indicating these zinc fingers do not recognize MLV PBS as a three-nucleotide sequence. Therefore, in the present study, we constructed truncated and mutated zinc fingers and examined their ability to bind to MLV PBS. The third to fifth zinc fingers of ZFP809 were found to be essential for binding to MLV PBS. Furthermore, the results of the present study indicate that other zinc fingers, which were not directly involved in binding to MLV PBS, may function in potentiating binding and stable protein expression. Further characterization of the amino acid sequences of zinc fingers will help further elucidate the functions and features of KRAB-ZFP and other zinc finger proteins.

Amino acid substitutions away from the RNase H catalytic site increase the thermal stability of Moloney murine leukemia virus reverse transcriptase through RNase H inactivation.

We have previously used site-directed mutagenesis to introduce basic residues (i.e., Arg; Lys) in the nucleic acid binding cleft of the Moloney murine leukemia virus reverse transcriptase (MMLV RT) in order to increase its template-primer (T/P) binding affinity. Three stabilizing mutations (i.e., E286R, E302K, and L435R) were identified (Yasukawa et al., 2010). Now, we studied the mechanism by which those mutations increase the thermal stability of the RT. The three single-mutants (E286R, E302K, and L435R), an RNase H-deficient MMLV RT (carrying the RNase H-inactivating mutation D524A), a quadruple mutant (E286R/E302K/L435R/D524A, designated as MM4) and the wild-type enzyme (WT) were produced in Escherichia coli. All RTs exhibited similar dissociation constants (Kd) for heteropolymeric DNA/DNA (2.9-6.5 nM) and RNA/DNA complexes (1.2-2.9 nM). Unlike the WT, mutant enzymes (E286R, E302K, L435R, D524A, and MM4) were devoid of RNase H activity, and were not able to degrade RNA in RNA/DNA complexes. These results suggest that the mutations, E286R, E302K, and L435R increase the thermostability of MMLV RT not by increasing its affinity for T/P but by abolishing its RNase H activity.

A structure-based mechanism for tRNA and retroviral RNA remodelling during primer annealing.

To prime reverse transcription, retroviruses require annealing of a transfer RNA molecule to the U5 primer binding site (U5-PBS) region of the viral genome. The residues essential for primer annealing are initially locked in intramolecular interactions; hence, annealing requires the chaperone activity of the retroviral nucleocapsid (NC) protein to facilitate structural rearrangements. Here we show that, unlike classical chaperones, the Moloney murine leukaemia virus NC uses a unique mechanism for remodelling: it specifically targets multiple structured regions in both the U5-PBS and tRNA(Pro) primer that otherwise sequester residues necessary for annealing. This high-specificity and high-affinity binding by NC consequently liberates these sequestered residues--which are exactly complementary--for intermolecular interactions. Furthermore, NC utilizes a step-wise, entropy-driven mechanism to trigger both residue-specific destabilization and residue-specific release. Our structures of NC bound to U5-PBS and tRNA(Pro) reveal the structure-based mechanism for retroviral primer annealing and provide insights as to how ATP-independent chaperones can target specific RNAs amidst the cellular milieu of non-target RNAs.

The Nef-like effect of murine leukemia virus glycosylated gag on HIV-1 infectivity is mediated by its cytoplasmic domain and depends on the AP-2 adaptor complex.

UNLABELLED: Human immunodeficiency virus type 1 (HIV-1) Nef enhances the infectivity of progeny virions. However, Nef is dispensable for the production of HIV-1 virions of optimal infectivity if the producer cells are superinfected with certain gammaretroviruses. In the case of the ecotropic Moloney murine leukemia virus (M-MLV), the Nef-like effect is mediated by the glycosylated Gag (glycoGag) protein. We now show that the N-terminal intracellular domain of the type II transmembrane protein glycoGag is responsible for its effect on HIV-1 infectivity. In the context of a fully active minimal M-MLV glycoGag construct, truncations of the cytoplasmic domain led to a near total loss of activity. Furthermore, the cytoplasmic domain of M-MLV glycoGag was fully sufficient to transfer the activity to an unrelated type II transmembrane protein. Although the intracellular region of glycoGag is relatively poorly conserved even among ecotropic and xenotropic MLVs, it was also fully sufficient for the rescue of nef-deficient HIV-1 when derived from a xenotropic virus. A mutagenic analysis showed that only a core region of the intracellular domain that exhibits at least some conservation between murine and feline leukemia viruses is crucial for activity. In particular, a conserved YXXL motif in the center of this core region was critical. In addition, expression of the µ2 subunit of the AP-2 adaptor complex in virus producer cells was essential for activity. We conclude that the ability to enhance HIV-1 infectivity is a conserved property of the MLV glycoGag cytoplasmic domain and involves AP-2-mediated endocytosis. IMPORTANCE: The Nef protein of HIV-1 and the entirely unrelated glycosylated Gag (glycoGag) protein of a murine leukemia virus (MLV) similarly enhance the infectiousness of HIV-1 particles by an unknown mechanism. MLV glycoGag is an alternative version of the structural viral Gag protein with an extra upstream region that provides a cytosolic domain and a plasma membrane anchor. We now show for the first time that the cytosolic domain of MLV glycoGag contains all the information needed to enhance HIV-1 infectivity and that this function of the cytosolic domain is conserved despite limited sequence conservation. Within the cytosolic domain, a motif that resembles a cellular sorting signal is critical for activity. Furthermore, the enhancement of HIV-1 infectivity depends on an endocytic cellular protein that is known to interact with such sorting signals. Together, our findings implicate the endocytic machinery in the enhancement of HIV-1 infectivity by MLV glycoGag.

Deamination intensity profiling of human APOBEC3 protein activity along the near full-length genomes of HIV-1 and MoMLV by HyperHRM analysis.

Enzymatic deamination of cytidines in DNA is an intrinsic component of antibody maturation and retroviral resistance, but can also be a source of HIV drug resistance and cancer-causing mutations. Here, we developed a high-throughput method based on high resolution melt (HRM) analysis called HyperHRM that can screen genomic DNA for rare hypermutated proviral sequences and accurately quantify the number of C-to-T or G-to-A mutations in each sequence. We demonstrate the effectiveness of the approach by profiling in parallel the intensity of the DNA mutator activity of all seven human APOBEC3 proteins on the near full-length sequence of HIV-1 and the Moloney murine leukemia virus. Additionally, HRM was successfully used to identify hypermutated proviral sequences in peripheral blood mononuclear cells from an HIV-1 patient. These results exemplify the effectiveness of HRM-based approaches for hypermutation quantification and for the detection of hypermutated DNA sequences potentially associated with disease or retroviral drug resistance.

EBP1, a novel host factor involved in primer binding site-dependent restriction of moloney murine leukemia virus in embryonic cells.

Mouse embryonic cells are unable to support the replication of Moloney murine leukemia virus (MLV). The integrated viral DNA is transcriptionally silenced, largely due to binding of host transcriptional repressors to the primer binding site (PBS) of the provirus. We have previously shown that a PBS DNA-binding repressor complex contains ZFP809 and TRIM28. Here, we identified ErbB3-binding protein 1 (EBP1) to be a novel component of the ZFP809-TRIM28 silencing complex and show that EBP1 depletion reduces PBS-mediated retroviral silencing.

Asymmetric polymerase chain reaction provides alternatives for preparation of (GT)5-tailed duplex DNA promoter for promoter trapping.

Synthesis of (GT)5-tailed duplex DNA promoter is an important first step for purifying transcription complexes by promoter trapping purification. In our previous publication, we showed that the purification of the c-jun promoter using lambda exonuclease digestion of polymerase chain reaction (PCR) produced DNA with single-stranded tails. Asymmetric PCR can also produce tailed single strands that can be annealed to yield the desired promoter. An effective method uses asymmetric PCR and double digestion. After PCR, first a restriction enzyme, in this case SacII, cuts duplex strands remaining after asymmetric PCR, leaving 5' phosphoryl ends susceptible to a second digestion with lambda exonuclease to effectively degrade any duplex. The resulting single strands are then annealed to produce a duplex DNA with a single-stranded (GT)5 tail at the 3' end of each strand of the duplex. Unlike the previously described method, this novel procedure produces the desired tailed promoter devoid of any untailed duplex.

Targeted entry via somatostatin receptors using a novel modified retrovirus glycoprotein that delivers genes at levels comparable to those of wild-type viral glycoproteins.

Here we report a novel viral glycoprotein created by replacing a natural receptor-binding sequence of the ecotropic Moloney murine leukemia virus envelope glycoprotein with the peptide ligand somatostatin. This new chimeric glycoprotein, which has been named the Sst receptor binding site (Sst-RBS), gives targeted transduction based on three criteria: (i) a gain of the use of a new entry receptor not used by any known virus; (ii) targeted entry at levels comparable to gene delivery by wild-type ecotropic Moloney murine leukemia virus and vesicular stomatitis virus (VSV) G glycoproteins; and (iii) a loss of the use of the natural ecotropic virus receptor. Retroviral vectors coated with Sst-RBS gained the ability to bind and transduce human 293 cells expressing somatostatin receptors. Their infection was specific to target somatostatin receptors, since a synthetic somatostatin peptide inhibited infection in a dose-dependent manner and the ability to transduce mouse cells bearing the natural ecotropic receptor was effectively lost. Importantly, vectors coated with the Sst-RBS glycoprotein gave targeted entry of up to 1 × 10(6) transducing U/ml, a level comparable to that seen with infection of vectors coated with the parental wild-type ecotropic Moloney murine leukemia virus glycoprotein through the ecotropic receptor and approaching that of infection of VSV G-coated vectors through the VSV receptor. To our knowledge, this is the first example of a glycoprotein that gives targeted entry of retroviral vectors at levels comparable to the natural capacity of viral envelope glycoproteins.

Selective inactivation of M-MuLV RT RNase H activity by site-directed PEGylation: an improved ability to synthesize long cDNA molecules.

Moloney murine leukemia virus reverse transcriptase (M-MuLV RT) is a domain structured enzyme that has the N-terminally located DNA polymerization activity and C-terminally located RNase H activity, which interferes with the efficient synthesis of long cDNA molecules. Here we present the PEGylation as a tool for engineering the M-MuLV RT derivative deficient in RNase H activity. We demonstrate that site-directed chemical modification (SDCM) of the RNase H domain by selectively PEGylating C635, one of the eight cysteine residues present in the reverse transcriptase (RT), specifically inactivated its ribonucleolytic activity. As a consequence, the efficiency of long cDNA molecules synthesis by modified enzyme was greatly increased.

Structure of a conserved retroviral RNA packaging element by NMR spectroscopy and cryo-electron tomography.

The 5'-untranslated regions of all gammaretroviruses contain a conserved "double-hairpin motif" (Ψ(CD)) that is required for genome packaging. Both hairpins (SL-C and SL-D) contain GACG tetraloops that, in isolated RNAs, are capable of forming "kissing" interactions stabilized by two intermolecular G-C base pairs. We have determined the three-dimensional structure of the double hairpin from the Moloney murine leukemia virus ([Ψ(CD)](2), 132 nt, 42.8 kDa) using a (2)H-edited NMR-spectroscopy-based approach. This approach enabled the detection of (1)H-(1)H dipolar interactions that were not observed in previous studies of isolated SL-C and SL-D hairpin RNAs using traditional (1)H-(1)H correlated and (1)H-(13)C-edited NMR methods. The hairpins participate in intermolecular cross-kissing interactions (SL-C to SL-D' and SLC' to SL-D) and stack in an end-to-end manner (SL-C to SL-D and SL-C' to SL-D') that gives rise to an elongated overall shape (ca 95 Å×45 Å×25 Å). The global structure was confirmed by cryo-electron tomography (cryo-ET), making [Ψ(CD)](2) simultaneously the smallest RNA to be structurally characterized to date by cryo-ET and among the largest to be determined by NMR. Our findings suggest that, in addition to promoting dimerization, [Ψ(CD)](2) functions as a scaffold that helps initiate virus assembly by exposing a cluster of conserved UCUG elements for binding to the cognate nucleocapsid domains of assembling viral Gag proteins.

Secondary structure of the mature ex virio Moloney murine leukemia virus genomic RNA dimerization domain.

Retroviral genomes are dimeric, comprised of two sense-strand RNAs linked at their 5' ends by noncovalent base pairing and tertiary interactions. Viral maturation involves large-scale morphological changes in viral proteins and in genomic RNA dimer structures to yield infectious virions. Structural studies have largely focused on simplified in vitro models of genomic RNA dimers even though the relationship between these models and authentic viral RNA is unknown. We evaluate the secondary structure of the minimal dimerization domain in genomes isolated from Moloney murine leukemia virions using a quantitative and single nucleotide resolution RNA structure analysis technology (selective 2'-hydroxyl acylation analyzed by primer extension, or SHAPE). Results are consistent with an architecture in which the RNA dimer is stabilized by four primary interactions involving two sets of intermolecular base pairs and two loop-loop interactions. The dimerization domain can independently direct its own folding since heating and refolding reproduce the same structure as visualized in genomic RNA isolated from virions. Authentic ex virio RNA has a SHAPE reactivity profile similar to that of a simplified transcript dimer generated in vitro, with the important exception of a region that appears to form a compact stem-loop only in the virion-isolated RNA. Finally, we analyze the conformational changes that accompany folding of monomers into dimers in vitro. These experiments support well-defined structural models for an authentic dimerization domain and also emphasize that many features of mature genomic RNA dimers can be reproduced in vitro using properly designed, simplified RNAs.

Late domain-independent rescue of a release-deficient Moloney murine leukemia virus by the ubiquitin ligase itch.

Moloney murine leukemia virus (MoMLV) Gag utilizes its late (L) domain motif PPPY to bind members of the Nedd4-like ubiquitin ligase family. These interactions recruit components of the cell's budding machinery that are critical for virus release. MoMLV Gag contains two additional L domains, PSAP and LYPAL, that are believed to drive residual MoMLV release via interactions with cellular proteins Tsg101 and Alix, respectively. We found that overexpression of Tsg101 or Alix failed to rescue the release of PPPY-deficient MoMLV via these other L domains. However, low-level expression of the ubiquitin ligase Itch potently rescued the release and infectivity of MoMLV lacking PPPY function. In contrast, other ubiquitin ligases such as WWP1, Nedd4.1, Nedd4.2, and Nedd4.2s did not rescue this release-deficient virus. Efficient rescue required the ubiquitin ligase activity of Itch and an intact C2 domain but not presence of the endophilin-binding site. Additionally, we found Itch to immunoprecipitate with MoMLV Gag lacking the PPPY motif and to be incorporated into rescued MoMLV particles. The PSAP and LYPAL motifs were dispensable for Itch-mediated virus rescue, and their absence did not affect the incorporation of Itch into the rescued particles. Itch-mediated rescue of release-defective MoMLV was sensitive to inhibition by dominant-negative versions of ESCRT-III components and the VPS4 AAA ATPase, indicating that Itch-mediated correction of MoMLV release defects requires the integrity of the host vacuolar sorting protein pathway. RNA interference knockdown of Itch suppressed the residual release of the MoMLV lacking the PPPY motif. Interestingly, Itch stimulation of the PPPY-deficient MoMLV release was accompanied by the enhancement of Gag ubiquitination and the appearance of new ubiquitinated Gag proteins in virions. Together, these results suggest that Itch can facilitate MoMLV release in an L domain-independent manner via a mechanism that requires the host budding machinery and involves Gag ubiquitination.

Expression artifact with retroviral vectors based on pBMN.

While characterizing various splice forms of p120 catenin, we observed what appeared to be a novel posttranslational modification of p120, resulting in a higher molecular weight form that was dependent on the splicing pattern. Further investigation revealed the higher molecular weight form to be a fusion protein between sequences encoded by the retroviral vector and p120. We found that the publicly available sequence of the vector we used does not agree with the experimental sequence. We caution other investigators to be aware of this potential artifact.

Mutations affecting the MA portion of the v-Abl protein reveal a conserved role of Gag in Abelson murine leukemia virus (MLV) and Moloney MLV.

Abelson murine leukemia virus (Ab-MLV) arose from a recombination between gag sequences in Moloney MLV (Mo-MLV) and the c-abl proto-oncogene. The v-Abl oncoprotein encoded by Ab-MLV contains MA, p12, and a portion of CA sequences derived from the gag gene of Mo-MLV. Previous studies indicated that alteration of MA sequences affects the biology of Mo-MLV and Ab-MLV. To understand the role of these sequences in Ab-MLV transformation more fully, alanine substitution mutants that affect Mo-MLV replication were examined in the context of Ab-MLV. Mutations affecting Mo-MLV replication decreased transformation, while alanine mutations in residues dispensable for Mo-MLV replication did not. The altered v-Abl proteins displayed aberrant subcellular localization that correlated to transformation defects. Immunofluorescent analyses suggested that aberrant trafficking of the altered proteins and improper interaction with components of the cytoskeleton were involved in the phenotype. Similar defects in localization were observed when the Gag moiety containing these mutations was expressed in the absence of abl-derived sequences. These results indicate that MA sequences within the Gag moiety of the v-Abl protein contribute to proper localization by playing a dominant role in trafficking of the v-Abl molecule.

RNase H activity: structure, specificity, and function in reverse transcription.

This review compares the well-studied RNase H activities of human immunodeficiency virus, type 1 (HIV-1) and Moloney murine leukemia virus (MoMLV) reverse transcriptases. The RNase H domains of HIV-1 and MoMLV are structurally very similar, with functions assigned to conserved subregions like the RNase H primer grip and the connection subdomain, as well as to distinct features like the C-helix and loop in MoMLV RNase H. Like cellular RNases H, catalysis by the retroviral enzymes appears to involve a two-metal ion mechanism. Unlike cellular RNases H, the retroviral RNases H display three different modes of cleavage: internal, DNA 3' end-directed, and RNA 5' end-directed. All three modes of cleavage appear to have roles in reverse transcription. Nucleotide sequence is an important determinant of cleavage specificity with both enzymes exhibiting a preference for specific nucleotides at discrete positions flanking an internal cleavage site as well as during tRNA primer removal and plus-strand primer generation. RNA 5' end-directed and DNA 3' end-directed cleavages show similar sequence preferences at the positions closest to a cleavage site. A model for how RNase H selects cleavage sites is presented that incorporates both sequence preferences and the concept of a defined window for allowable cleavage from a recessed end. Finally, the RNase H activity of HIV-1 is considered as a target for anti-virals as well as a participant in drug resistance.