Moloney Murine Leukemia Virus Replication Research Articles

Effect of betulinic acid and its ionic derivatives on M-MuLV replication

Murine leukemia virus (MuLV) is a retrovirus known causing leukemia and neurological disorders in mice, and its viral life cycle and pathogenesis have been investigated extensively over the past decades. As a natural antiviral agent, betulinic acid is a pentacyclic triterpenoid that can be found in the bark of several species of plants (particularly the white birch). One of the hurdles for betulinic acid to release its antiviral potency is its poor water solubility. In this study, we synthesized more water-soluble ionic derivatives of betulinic acid, and examined their activities against Moloney MuLV (M-MuLV). The mouse fibroblast cells stably infected with M-MuLV, 43D cells, were treated with various doses of betulinic acids and its derivatives, and the viral structural protein Gag in cells and media were detected by western blots. Two ionic derivatives containing the benzalkonium cation were found to inhibit the virus production into media and decreased Gag in cells. However, a cell proliferation assay showed that the benzalkonium cation inhibited the growth of 43D cells, suggesting that our ionic derivatives limited virus production through the inhibition of metabolism in 43D cells. Interestingly, all of these betulinic acid compounds exhibited a minimum impact on the processing and release of Gag from 43D cells, which outlines the differences of viral maturation between MuLV and human immunodeficiency virus.

Modulation of stop codon read-through efficiency and its effect on the replication of murine leukemia virus.

ABSTRACTTranslational readthrough—suppression of termination at a stop codon—is exploited in the replication cycles of several viruses and represents a potential target for antiviral intervention. In the gammaretroviruses, typified by Moloney murine leukemia virus (MuLV), gag and pol are in the same reading frame, separated by a UAG stop codon, and termination codon readthrough is required for expression of the viral Gag-Pol fusion protein. Here, we investigated the effect on MuLV replication of modulating readthrough efficiency. We began by manipulating the readthrough signal in the context of an infectious viral clone to generate a series of MuLV variants in which readthrough was stimulated or reduced. In carefully controlled infectivity assays, it was found that reducing the MuLV readthrough efficiency only 4-fold led to a marked defect and that a 10-fold reduction essentially abolished replication. However, up to an ∼8.5-fold stimulation of readthrough (up to 60% readthrough) was well tolerated by the virus. These high levels of readthrough were achieved using a two-plasmid system, with Gag and Gag-Pol expressed from separate infectious clones. We also modulated readthrough by silencing expression of eukaryotic release factors 1 and 3 (eRF1 and eRF3) or by introducing aminoglycosides into the cells. The data obtained indicate that gammaretroviruses tolerate a substantial excess of viral Gag-Pol synthesis but are very sensitive to a reduction in levels of this polyprotein. Thus, as is also the case for ribosomal frameshifting, antiviral therapies targeting readthrough with inhibitory agents are likely to be the most beneficial.IMPORTANCE Many pathogenic RNA viruses and retroviruses use ribosomal frameshifting or stop codon readthrough to regulate expression of their replicase enzymes. These translational “recoding” processes are potential targets for antiviral intervention, but we have only a limited understanding of the consequences to virus replication of modulating the efficiency of recoding, particularly for those viruses employing readthrough. In this paper, we describe the first systematic analysis of the effect of increasing or decreasing readthrough efficiency on virus replication using the gammaretrovirus MuLV as a model system. We find unexpectedly that MuLV replication is only slightly inhibited by substantial increases in readthrough frequency, but as with other viruses that use recoding strategies, replication is quite sensitive to even modest reductions. These studies provide insights into both the readthrough process and MuLV replication and have implications for the selection of antivirals against gammaretroviruses.

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

BackgroundZASC1 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).MethodsTo 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.ResultsMice 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.ConclusionsThese 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.

Tetherin is a key effector of the antiretroviral activity of type I interferon in vitro and in vivo

Tetherin (Bst-2 CD317) is a cell-surface protein whose expression is induced by IFNα. Although tetherin expression causes the retention of retrovirus particles on the surface of infected cells, it is not known whether tetherin inhibits retroviral replication or pathogenesis in vivo. Mutation of tetherin antagonists often has little effect on retroviral replication in vitro, and, although tetherin can reduce the yield of extracellular viral particles, some studies suggest that tetherin actually enhances direct cell-to-cell viral transmission. We generated tetherin-deficient mice to determine the effect of this protein on murine retrovirus replication and pathogenesis. We find that tetherin markedly inhibits the replication of Moloney murine leukemia virus (Mo-MLV) and is required for the antiretroviral activity of IFNα to be fully manifested in vitro. Surprisingly, Mo-MLV replication and disease progression was not significantly different in WT and tetherin-deficient mice, but this finding was explained by the fact that Mo-MLV infection did not induce detectable tetherin expression on candidate target cells in vivo. Indeed, IFNα induction was required to reveal the anti-Mo-MLV activity of tetherin in vivo. Moreover, LP-BM5, an MLV strain that has been demonstrated to induce immune activation and IFNα expression, achieved higher levels of viremia and induced exaggerated pathology in tetherin-deficient mice. These data indicate that tetherin is a bona fide antiviral protein and can reduce retroviral replication and disease in vivo.

Moloney Leukemia Virus 10 (MOV10) Protein Inhibits Retrovirus Replication

Moloney leukemia virus 10 (MOV10) protein is a superfamily-1 RNA helicase, and it is also a component of the RNA-induced silencing complex. Recent studies have shown that MOV10 plays an active role in the RNA interference pathway. Here, we report that MOV10 inhibits retrovirus replication. When it was overexpressed in viral producer cells, MOV10 was able to reduce the infectivity of human immunodeficiency virus type 1 (HIV-1), simian immunodeficiency virus, and murine leukemia virus. Conversely, when MOV10 expression was reduced by small interfering RNAs, HIV-1 infectivity was increased. Consistently, silencing of MOV10 expression in a human T cell line enhanced HIV-1 replication. Furthermore, we found that MOV10 interacts with HIV-1 nucleocapsid protein in an RNA-dependent manner and is packaged into virions. It blocks HIV-1 replication at a postentry step. In addition, we also found that HIV-1 could suppress MOV10 protein expression to counteract this cellular resistance. All of these results indicate that MOV10 has a broad antiretroviral activity that can target a wide range of retroviruses, and it could be actively involved in host defense against retroviral infection.

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.

Mismatch Extension During Strong Stop Strand Transfer and Minimal Homology Requirements for Replicative Template Switching During Moloney Murine Leukemia Virus Replication

Reverse transcription requires two replicative template switches, called minus and plus strand strong stop transfer, and can include additional, recombinogenic switches. Donor and acceptor template homology facilitates both replicative and recombinogenic transfers, but homology-independent determinants may also contribute. Here, improved murine leukemia virus-based assays were established and the effects of varying extents of mismatches and complementarity between primer and acceptor template regions were assessed. Template switch accuracy was addressed by examining provirus structures, and efficiency was measured using a competitive titer assay. The results demonstrated that limited mismatch extension occurred readily during both minus and plus strand transfer. A strong bias for correct targeting to the U3/R junction and against use of alternate regions of homology was observed during minus strand transfer. Transfer to the U3/R junction was as accurate with five bases of complementarity as it was with an intact R, and as few as 3 nt targeted transfer to a limited extent. In contrast, 12 base recombinogenic acceptors were utilized poorly and no accurate switch was observed when recombination acceptors retained only five bases of complementarity. These findings confirm that murine leukemia virus replicative and recombinogenic template switches differ in homology requirements, and support the notion that factors other than primer–template complementarity may contribute to strong stop acceptor template recognition.

Characterization of producer cell-dependent restriction of murine leukemia virus replication.

We previously reported that the human bronchocarcinoma cell line A549 produces poorly infectious gibbon ape leukemia virus-pseudotyped Moloney murine leukemia virus (MLV). In contrast, similar amounts of virions recovered from human fibrosarcoma HT1080 cells result in 10-fold-higher transduction rates (G. Duisit, A. Salvetti, P. Moullier, and F. Cosset, Hum. Gene Ther. 10:189-200, 1999). We have now extended this initial observation to other type-C envelope (Env) pseudotypes and analyzed the mechanism involved. Structural and morphological analysis showed that viral particles recovered from A549 (A549-MLV) and HT1080 (HT1080-MLV) cells were normal and indistinguishable from each other. They expressed equivalent levels of mature Env proteins and bound similarly to the target cells. Furthermore, incoming particles reached the cytosol and directed the synthesis of linear viral DNA equally efficiently. However, almost no detectable circular DNAs could be detected in A549-MLV-infected cells, indicating that the block of infection resulted from defective nuclear translocation of the preintegration complex. Interestingly, pseudotyping of A549-MLV with vesicular stomatitis virus glycoprotein G restored the amount of circular DNA forms as well as the transduction rates to HT1080-MLV levels, suggesting that the postentry blockage could be overcome by endocytic delivery of the core particles downstream of the restriction point. Thus, in contrast to the previously described target cell-dependent Fv-1 (or Fv1-like) restriction in mammalian cells (P. Pryciak and H. E. Varmus, J. Virol. 66:5959-5966, 1992; G. Towers, M. Bock, S. Martin, Y. Takeuchi, J. P. Stoye, and O. Danos, Proc. Natl. Acad. Sci. USA 97:12295-12299, 2000), we report here a new restriction of MLV replication that relies only on the producer cell type.

RNase H Activity Is Required for High-Frequency Repeat Deletion during Moloney Murine Leukemia Virus Replication

It has been postulated that retroviral recombination, like strong stop template switching, requires the RNase H activity of reverse transcriptase. To address this hypothesis, Moloney murine leukemia virus-based vectors, which were designed to test the recombination-related property of direct repeat deletion, were encapsidated in virions engineered to contain phenotypic mixtures of wild-type and RNase H catalytic site point mutant reverse transcriptase. Integrated provirus titers per milliliter were determined for these phenotypically mixed virions, and vector proviruses were screened to determine what percentage contained repeat deletions. The results revealed a steady decline in direct repeat deletion frequency that correlated with decreases in functional RNase H, with greater than fourfold decreases in repeat deletion frequency observed when 95% of virion reverse transcriptase was RNase H defective. Parallel experiments were performed to address effects of molar excesses of RNase H relative to functional DNA polymerase. These experiments demonstrated that increasing the stoichiometry of RNase H relative to the amount of functional DNA polymerase had minimal effects on direct repeat deletion frequency. DNA synthesis was error prone when directed principally by RNase H mutant reverse transcriptase, suggesting a role for RNase H catalytic integrity in the fidelity of intracellular reverse transcription.

Effects of limiting homology at the site of intermolecular recombinogenic template switching during Moloney murine leukemia virus replication.

A Moloney murine leukemia virus-based single-replication-cycle assay was developed to study the effects of limiting the extent of template and primer strand complementarity on recombinogenic template switching. This system mimicked forced copy choice recombination in which nascent DNA transfers from the end of a donor template to an acceptor position on the other copackaged RNA. When acceptor target regions with different extents of complementarity to the transferring DNA were tested, efficient recombination occurred with as few as 14 complementary nucleotides. The frequencies of correct targeting, transfer-associated errors, mismatch extension, and transfer before reaching the end of the donor template were determined. All four molecular events occurred, with their proportions varying depending on the nature of acceptor/transferring DNA complementarity. When complementarity was severely limited, recombination was inefficient and most products resulted from aberrant second-strand transfer rather than from forced template switching between RNAs. Other classes of reverse transcription products, including some that resulted from template switching between virus and host sequences, were also observed when homology between the acceptor and donor was limited.

Inhibition of Murine AIDS (MAIDS) Development in C57BL/6J Mice by Tyrphostin AG-1387

We previously showed that certain tyrphostin derivatives, known as protein tyrosine kinase inhibitors, also act as topoisomerase I-specific antagonists and inhibit Moloney murine leukemia virus replication in vitro in acutely and chronically infected cells. However, an accurate portrayal of retroviral-induced disease cannot rely exclusively on extrapolations from in vitro data. Therefore, experiments with animal models are essential for evaluating the efficacy of a specific drug in vivo. In this study, we examined the effect of tyrphostin AG-1387 on murine AIDS (MAIDS) development in C57BL/6J mice injected with the LP-BM5 virus mixture. A single dose of tyrphostin, administered together with or 24 h post virus inoculation, decreased the development of MAIDS symptoms as measured by spleen and lymph node weight, the T-cell response to concanavalin A (con A), and spleen architecture. Furthermore, weekly treatment with tyrphostins totally abolished MAIDS symptoms and prevented the viral infection of the spleen cells as measured by the absence of viral RNA and the restoration of T-cell function in these spleens. These results implicate that prolonged treatment with tyrphostins is needed for the prevention of MAIDS development in infected mice and suggest that it may be applied as a legitimate remedy for the treatment of retroviral-induced diseases.

Selection of optimal polypurine tract region sequences during Moloney murine leukemia virus replication.

Retrovirus plus-strand synthesis is primed by a cleavage remnant of the polypurine tract (PPT) region of viral RNA. In this study, we tested replication properties for Moloney murine leukemia viruses with targeted mutations in the PPT and in conserved sequences upstream, as well as for pools of mutants with randomized sequences in these regions. The importance of maintaining some purine residues within the PPT was indicated both by examining the evolution of random PPT pools and from the replication properties of targeted mutants. Although many different PPT sequences could support efficient replication and one mutant that contained two differences in the core PPT was found to replicate as well as the wild type, some sequences in the core PPT clearly conferred advantages over others. Contributions of sequences upstream of the core PPT were examined with deletion mutants. A conserved T-stretch within the upstream sequence was examined in detail and found to be unimportant to helper functions. Evolution of virus pools containing randomized T-stretch sequences demonstrated marked preference for the wild-type sequence in six of its eight positions. These findings demonstrate that maintenance of the T-rich element is more important to viral replication than is maintenance of the core PPT.

Effects of 3' untranslated region mutations on plus-strand priming during moloney murine leukemia virus replication.

A conserved purine-rich motif located near the 3' end of retroviral genomes is involved in the initiation of plus-strand DNA synthesis. We mutated sequences both within and flanking the Moloney murine leukemia virus polypurine tract (PPT) and determined the effects of these alterations on viral DNA synthesis and replication. Our results demonstrated that both changes in highly conserved PPT positions and a mutation that left only the cleavage-proximal half of the PPT intact led to delayed replication and reduced the colony-forming titer of replication defective retroviral vectors. A mutation that altered the cleavage proximal half of the PPT and certain 3' untranslated region mutations upstream of the PPT were incompatible with or severely impaired viral replication. To distinguish defects in plus-strand priming from other replication defects and to assess the relative use of mutant and wild-type PPTs, we examined plus-strand priming from an ectopic, secondary PPT inserted in U3. The results demonstrated that the analyzed mutations within the PPT primarily affected plus-strand priming whereas mutations upstream of the PPT appeared to affect both plus-strand priming and other stages of viral replication.

Tyrphostin AG-555 inhibits early and late stages of Moloney murine leukemia virus replication cycle

We have previously shown that certain tyrphostin derivatives, known as protein tyrosine blockers, inhibited Moloney murine leukemia virus (Mo-MuLV) replication in acutely and chronically infected NIH/3T3 cells, without affecting cell viability or growth. In our present work, we examined the stages in the viral life cycle that are affected by tyrphostin AG-555. We found that this drug inhibited the integration of the viral DNA into the host genome in acutely infected cells. This compound also reduced the level of viral RNA and specifically inhibited viral protein synthesis in NIH/3T3/Mo-MuLV chronically infected cells while no effect on the cellular beta-actin was observed. Since tyrphostin AG-555 inhibited both the early stages (integration process) and the late stages (viral protein synthesis) in the virus life cycle, it offers a potential advantage over other compounds which affect only one stage in the viral life cycle. Therefore, tyrphostin AG-555 may be considered as a potent antiretroviral drug.

The p15 matrix protein of moloney murine leukemia virus is a phosphotyrosine protein

Retroviruses have been etiologically implicated with leukemia in humans and animals. Understanding the virus life cycle, the proteins and enzymes involved in its replication, is essential for developing potent anti-viral drugs. Phosphorylation of retroviral proteins may alter their shape in such a way as to increase or inhibit their biological activities and thus influence the replication and pathogenic potential of the retroviruses. In our previous work, we demonstrated that non-cytotoxic doses of tyrphostins (protein tyrosine kinase blockers) inhibit moloney murine leukemia virus (Mo-MuLV) replication in acutely and chronically infected cells. In an attempt to understand their mode of action as anti-MoMuLV drugs, we examined the possibility that a viral protein is phosphorylated in tyrosine. Indeed, in our present work, we show that the p15 matrix protein (MA) of MoMuLV is a phosphotyrosine protein and is the only viral protein which is phosphorylated on tyrosine. Moreover, treatment of Mo-MuLV/NIH/ 3T3 chronically infected cells with tyrphostin AG-555 specifically inhibits the synthesis of p15 and other viral proteins but does not affect the synthesis of cellular proteins. Our results suggest that tyrosine phosphorylation of p15 MA protein may play a pivotal role in Mo-MuLV replication.

Recombinant mink cell focus-inducing virus and long terminal repeat alterations accompany the increased leukemogenicity of the Mo+PyF101 variant of Moloney murine leukemia virus after intraperitoneal inoculation

We recently showed that different routes of inoculation affect the leukemogenicity of the Mo+PyF101 variant of Moloney murine leukemia virus (M-MuLV). Intraperitoneal (i.p.) inoculation of neonatal mice with Mo+PyF101 M-MuLV greatly enhanced its leukemogenicity compared with subcutaneous (s.c.) inoculation. We previously also suggested that the leukemogenicity defect of Mo+PyF101 M-MuLV when inoculated s.c. may result from the inability of this virus to form env gene recombinant (mink cell focus-inducing [MCF]) virus. In this study, virus present in end-stage tumors and in preleukemic animals inoculated i.p. by Mo+PyF101 M-MuLV was characterized. In contrast to s.c. inoculation, all tumors from i.p.-inoculated mice contained high levels of recombinant MCF virus. Furthermore, Southern blot analyses demonstrated that the majority of the tumors contained altered Mo+PyF101 M-MuLV long terminal repeats. The U3 regions from several tumors with altered long terminal repeats were cloned by PCR amplification. Sequence analyses indicated that the M-MuLV 75-bp tandem repeat in the enhancer region was triplicated. This amplification was also previously observed in mice infected s.c. with a pseudotypic mixture of Mo+PyF101 M-MuLV and Mo+PyF101 MCF virus. The enhancer triplication was an early event, and it occurred within 2 weeks postinfection. Recombinant MCF viruses were not detected by Southern blot analyses until 4 weeks postinfection. Thus, the M-MuLV enhancer triplication event was initially important for efficient propagation of ecotropic Mo+PyF101 M-MuLV. The increased leukemogenicity following i.p. inoculation could be explained if the triplication enhances Mo+PyF101 M-MuLV replication in the bone marrow and bone marrow infection is required for recombinant MCF virus formation.

The leukemogenic potential of an enhancer variant of Moloney murine leukemia virus varies with the route of inoculation

We previously showed that the Mo+PyF101 variant of Moloney murine leukemia virus (M-MuLV) is poorly leukemogenic when inoculated subcutaneously (s.c.) into neonatal mice. We recently found that intraperitoneal (i.p.) inoculation of neonatal mice with the same virus significantly enhanced its leukemogenicity. In this study, infections of neonatal mice by the two different routes of inoculation were compared. We studied replication of the virus in vivo to identify critical preleukemic events. These would be observed in mice inoculated i.p. by Mo+PyF101 M-MuLV but not when inoculation was s.c. Infectious center assays indicated that regardless of the route of inoculation, Mo+PyF101 M-MuLV showed delayed infection of the thymus compared with wild-type M-MuLV. On the other hand, i.p.-inoculated mice showed more rapid appearance of infectious centers in the bone marrow than did s.c.-inoculated animals. Thus, the enhanced leukemogenicity of i.p. inoculation correlated with efficient early infection of the bone marrow and not with early infection of the thymus. These results suggest a role for bone marrow infection for efficient leukemogenesis in Mo+PyF101 M-MuLV-infected mice. Consistent with this notion, if bone marrow infection was decreased by injecting 10- to 12-day-old animals i.p., leukemogenicity resembled that of s.c. inoculation. Thus, two cell types that are critical for the induction of efficient leukemia were implicated. One cell delivers virus from the site of s.c. inoculation (the skin) to the bone marrow and is apparently restricted for Mo+PyF101 M-MuLV replication. The second cell is in the bone marrow, and its early infection is required for efficient leukemogenesis.

Exogenous Nucleosides Promote the Completion of MoMLV DNA Synthesis in G0-Arrested Balb c/3T3 Fibroblasts

We studied Moloney murine leukemia virus replication in newly infected Balb c/3T3 cells brought to the G0 phase by serum depletion. Using the polymerase chain reaction method, we showed that Moloney murine leukemia virus can be efficiently internalized in nonproliferating fibroblasts, although reverse transcription of the viral RNA in these cells remains uncompleted. It seems likely that a lower availability of deoxyribonucleotides in G0-arrested cells is responsible for this premature termination of the reverse transcription step. Accordingly, the addition of high concentrations of nucleosides to the culture medium of nondividing cells simultaneously with infection enables them to complete the reverse transcription process, without reinitiating the cell cycle. Inhibition of reverse transcription by hydroxyurea confirms the dependence of this retroviral step on the intracellular nucleotide pool rather than on the precise arrest point of the host cell cycle. Furthermore, the pyrimidine nucleotide pool, and more particularly the cytidine pool, appears to play a central regulatory role in this step.

Inhibition of Moloney murine leukemia virus replication by tyrphostins, tyrosine kinase inhibitors

We have previously shown that topoisomerase I (topo I) antagonist inhibited retrovirus replication. Since tyrphostins, synthetic compounds and protein tyrosine kinases (PTKs) blockers, inhibited topo I activity (manuscript in preparation) we examined their ability to inhibit Moloney murine leukemia virus (Mo-MuLV) replication. We found that non-cytotoxic doses of tyrphostin derivatives (AG-555, AG-18) blocked or substantially reduced Mo-MuLV replication in acute or chronically infected NIH/3T3 cells. Our experiments suggest that the antiviral effect of these tyrphostin derivatives was not the result of antiproliferative activity. However, the tyrphostin derivatives used in our present investigation differ in their ability to inhibit Mo-MuLV replication. Furthermore, as expected from stereospecific competitive inhibitors, the antiviral effect is not a general characteristic of all tyrphostin derivatives, since AG-213 does not affect Mo-MuLV replication. Our results indicate that these tyrphostin derivatives may represent a novel class of antiretroviral drugs.

Defects in Moloney murine leukemia virus replication caused by a reverse transcriptase mutation modeled on the structure of Escherichia coli RNase H.

We have studied a mutant Moloney murine leukemia virus with a deletion in reverse transcriptase (RT) which is predicted to make its RNase H domain resemble structurally that of human immunodeficiency virus RT. This deletion was based on improved RNase H homology alignments made possible by the recently solved three-dimensional structure for Escherichia coli RNase H. This mutant Moloney murine leukemia virus RT was fully active in the oligo(dT)-poly(rA) DNA polymerase assay and retained nearly all of wild-type RT's RNase H activity in an in situ RNase H gel assay. However, proviruses reconstructed to include this deletion were noninfectious. Minus-strand strong-stop DNA was made by the deletion mutant, but the amount of minus-strand translocation was intermediate to the very low level measured with RNase H-null virions and the high level seen with wild-type RT. The average length of translocated minus-strand DNA was shorter for the deletion mutant than for wild type, suggesting that mutations in the RNase H domain of RT also affect DNA polymerase activity.