MHC II proteins are heterodimeric glycoproteins that form complexes with antigenic peptides in order to elicit a CD4+ adaptive immune response. Even though there have been numerous MHC II-peptide crystal structures solved, there is little insight into the dynamic process of peptide loading. Through biochemical and biophysical studies, it has been shown that MHC II adopt multiple conformations throughout the peptide loading process. At least one of these conformations is stabilized by the MHC II-like homologue, HLA-DM. The main focus of this thesis is to elucidate alternate conformers of MHC II in an effort to better understand the structural features that enable HLA-DM catalyzed peptide loading. In this thesis, two altered conformations of HLA-DR were investigated, one modeled in the absence of peptide using molecular dynamics, and one stabilized by the mutation αF54C. The model for the peptide-free form of HLA-DR1 was derived from a molecular dynamics simulation. In this model, part of the alpha-subunit extended-strand region proximal to the peptide binding groove is folded into the peptide-binding groove such that the architecture of the critical peptide binding pocket, P1, as well as the invariant hydrogen bonding network were maintained. Biochemical studies aimed at validating the predicted structural changes were consistent with the model generated from the simulations. Next, structural studies were carried out on an MHC II mutant, αF54C, which was shown to have unique peptide binding characteristics as well as enhanced susceptibility to HLA-DM. Although this mutation did not affect the affinity for peptide, there was a striking increase in the rate of intrinsic peptide release. Both αF54C and αF54A were over 100-fold more susceptible to HLADM catalyzed peptide release than wild type as well as other mutants introduced along the peptide binding groove. In addition, mutation of the αF54 position results in a higher affinity for HLA-DM, which, unlike wild type, is detectable by surface plasmon resonance. Crystallographic studies resulted in a 2.3 Å resolution structure for the αF54C-Clip complex. There were two molecules in the asymmetric unit, one of which had no obvious deviations from other MHC II-pep complexes and one which had a conformational change as a result of a crystal contact on the αF51 residue, a residue which has been shown to be involved in the HLA-DM/HLA-DR binding interface. The crystal structure of wild type HLA-DR1- Clip was also solved, but did not have the altered conformation even though there was a similar crystal contact at the αF51. These data suggest the altered conformation seen in the mutant structure, results from increased lability in the extended stand region due to the αF54C mutation. As a result of this work, we have developed a new mechanistic model for how structural features of MHC II influence DM mediated peptide release.

Systems biology approaches have allowed for comprehensive understanding of complicated biological processes. Here, we’ve developed a global phenotypic profiling method by improving upon transposon mutagenesis methods for identifying genes required for bacterial growth in various conditions. By using the massively parallel power of Illumina sequencing, we precisely redefined the genes required for the growth of Mycobacterium Tuberculosis (Mtb) in vitro. This adapted technique provided more informative data with both increased dynamic range and resolution. As a result, we quantitatively assessed the fitness of individual mutants, as well as identified sub-genic essentiality. Mtb is well adapted to its nutrient-limiting intracellular niche. One important and novel adaptation is its ability to consume cholesterol for both energy and carbon. A combination of this genome-wide phenotypic analysis and global metabolite profiling was used to define the dedicated cholesterol catabolic pathway, as well as important transcriptional and metabolic adaptations required for the consumption of this carbon source. We identified the methylcitrate cycle (MCC) and an unexpected gluconeogenic route as essential pathways. Furthermore, we found that the cholesterol-dependent transcriptional induction of these metabolic enzymes was also essential for growth on this substrate, a function mediated by the Rv1129c regulatory protein. Using a combination of genetic and chemical methods to inhibit these pathways, we show that cholesterol represents a significant source of carbon during intracellular growth in macrophages. Finally, we have begun to define the mechanism by which lipids, such as cholesterol, are imported into the cell by investigating the assembly of the ABC-like lipid transporter, Mce1. The subunits of this system are localized to the cell wall and data is provided to support a novel mechanism for Mce-dependent import of lipids, such as cholesterol. In sum, this global analysis of host cholesterol utilization during infection provides insight into each step of this complicated process; import into the bacterial cell, the degradation of the molecule into primary metabolites, and the transformation of these metabolites into carbon and energy.

In response to telomere shortening, oxidative stress, DNA damage or aberrant activation of oncogenes, normal somatic cells exit the cell cycle and enter an irreversible growth arrest termed senescence. The limited proliferative capacity imposed by senescence on cells impedes the accumulation of mutations necessary for tumorigenesis and prevents proliferation of cells at risk of neoplastic transformation. Opposite to the tumor suppressor function, accumulation of senescent cells in adult organisms is thought to contribute to aging by depleting the renewal capacity of tissues and stem/progenitor cells, and by interfering with tissue homeostasis and functions. The Antagonistic Pleiotropy Theory of senescence proposes that senescence is beneficial early in life by acting as a tumor suppressor, but harmful late in life by contributing to aging. Recent studies have provided evidence strongly supporting the tumor suppressor function of senescence, however, direct evidence supporting the role of senescence in aging remains largely elusive. In this thesis, I describe studies to test the Antagonistic Pleiotropy Theory of senescence in tumorigenesis and aging. The approach that I have taken is to alter the senescence response in vivo by changing the expression of a senescence regulator in mice. The consequence of altered senescence response on tumorigenesis and stem cell self-renewal was investigated. The senescence regulator I studied is Smurf2, which has been shown previously to activate senescence in culture. I hypothesized that the senescence response will be impaired by Smurf2 deficiency in vivo. Consequently, Smurf2-deficient mice will develop tumors at an increased frequency, but also gain enhanced self-renewal capacity of stem/progenitor cells with age. I generated a Smurf2-deficient mouse model, and found that Smurf2 deficiency attenuated p16 expression and impaired the senescence response in primary cells and tissues. Smurf2-deficient mice exhibited an increased susceptibility to spontaneous tumorigenesis, indicating that Smurf2 is a tumor suppressor. At the premalignant stage of tumorigenesis, a defective senescence response was documented in the Smurf2-deficient mice, providing a mechanistic link between impaired senescence response and increased tumorigenesis. The majority of tumors developed in Smurf2-deficent mice were B-cell lymphomas with an origin in germinal centers of the spleen and a phenotype resembling human diffuse large B-cell lymphoma (DLBCL). I discovered that Smurf2 mediated ubiquitination of YY1, a master regulator of germinal centers. Stabilization of YY1 in the absence of Smurf2 was responsible for increased cell proliferation and drove lymphomagenesis in Smurf2-deficient mice. Consistently, a significant decrease of Smurf2 expression was observed in human primary DLBCL samples, and more importantly, a low level of Smurf2 expression in DLBCL correlated with poor survival prognosis. Moreover, I found that hematopoietic stem cells (HSCs) in Smurf2-deficient mice had enhanced function compared to wild-type controls. This enhanced stem cell function was associated with increased cell proliferation and decreased p16 expression, suggesting that defective senescence response in Smurf2-deficient mice leads to increased self-renewal capacity of HSCs. My study, for the first time, offers direct genetic evidence of an important tumor suppressor function for Smurf2 as well as its function in contributing to stem cell aging. Collectively, these findings provide strong evidence supporting the Antagonistic Pleiotropy Theory of senescence in tumorigenesis and aging.

Repair of DNA double-strand breaks via homologous recombination is an essential pathway for vertebrate cell development and maintenance of genome integrity throughout the organism’s lifetime. The Rad51 enzyme provides the central catalytic function of homologous recombination while many other proteins are involved in regulation and assistance of Rad51 activity, including a group of five proteins referred to as Rad51 paralogs (Rad51B, Rad51C, Rad51D, Xrcc2, Xrcc3). At the start of my work, cellular studies of human Rad51 (HsRad51) had shown only that it forms distinct nuclear foci in response to DNA damage. Additionally, no information regarding the cellular localization, potential DNA damage-induced redistribution or cellular functions for any of the Rad51 paralog proteins was available. Therefore, the goals of this work were to (1) present a more complete description of the cellular localization and DNA damage-induced redistribution of Rad51 and the two paralog proteins known to specifically associate with Rad51, Rad51C and Xrcc3, and (2) to define specific functional roles for Rad51C and Xrcc3 in mediating Rad51 activity. I focused on the use of cellular, RNAi and immunofluorescence methods to study endogenous Rad51, Rad51C and Xrcc3 in human cells. In my initial studies we showed for the first time that Xrcc3 forms distinct foci in both the nucleus and cytoplasm independent of DNA damage, that the distribution of these foci did not change significantly throughout the time course of DNA damage and repair, and that Xrcc3 focus formation is independent of Rad51. Additionally, and unlike most previously published images of nuclear Rad51, we found that the majority of DNA damage-induced nuclear Rad51 foci do not colocalize with gamma H2AX, a histone marker used to indicate the occurrence of DNA double strand breaks. As a consequence of these initial outcomes, a significant amount of effort was devoted to developing and optimizing immunofluorescence methods. Importantly, we developed a purification method for commercially available monoclonal antibodies against Rad51C and Xrcc3 that significantly improved their reactivity and specificity. My next study concentrated on Rad51C. Similar to Xrcc3, we found for the first time that Rad51C forms distinct nuclear and cytoplasmic foci independent of DNA damage and Rad51. An additional and surprising outcome was our discovery that Rad51C plays an important role in regulating the ubiquitination and proteasome-mediated degradation of Rad51. While biochemical functions for Rad51 paralog proteins had been suggested in the literature, this was the first demonstration of a specific biochemical function for Rad51C that contributes directly to the Rad51 activity in the homologous recombination pathway. Our improved immunofluorescence methods allowed us to see the accumulation of Rad51, Rad51C and Xrcc3 at the nuclear periphery early in response to DNA damage, suggesting the existence of a DNA damage-dependent trafficking mechanism that promoted movement of these proteins from the cytoplasm to the nucleus. This led to further studies in which we show distinct co-localization of cytoplasmic Rad51 with actin as well as alpha and beta tubulin. Using both immunofluorescence and sub-cellular fractionation methods our recent results strongly suggest that trafficking of Rad51 to the nucleus in response to DNA damage is regulated at least in part by its association with cytoskeletal proteins, and involves movement of both existing pools of Rad51 and newly synthesized protein. In a particularly exciting development, in collaboration with Leica Microsystems and Dr. Joerg Bewersdorf at The Jackson Laboratory, Bar Harbor, ME., I have been able to exploit a new technology, 4Pi microscopy, to provide the first images of endogenous nuclear proteins using this method. Results presented in this thesis have added significantly to a more complete understanding of cellular localization Rad51, Rad51C and Xrcc3, and have provided important insights into possible mechanisms of cellular trafficking of Rad51 in response to response to DNA damage. Additionally, we have defined a specific function for Rad51C in its regulation of Rad51 ubiquitination. These findings open several new avenues of investigation for furthering our understanding of the cellular and molecular functions of proteins with critical roles in the maintenance of genome integrity in human cells.

KCNQ1 is a homotetrameric voltage-gated potassium channel expressed in cardiomyocytes and epithelial tissues. However, currents arising from KCNQ1 have never been physiologically observed. KCNQ1 is able to provide the diverse potassium conductances required by these distinct cell types through coassembly with and modulation by type I transmembrane β-subunits of the KCNE gene family. KCNQ1-KCNE K+ channels play important physiological roles. In cardiac tissues the association of KCNQ1 with KCNE1 gives rise to IKs, the slow delayed outwardly rectifying potassium current. IKs is in part responsible for repolarizing heart muscle, and is therefore crucial in maintaining normal heart rhymicity. IKschannels help terminate each action potential and provide cardiac repolarization reserve. As such, mutations in either subunit can lead to Romano-Ward Syndrome or Jervell and Lange-Nielsen Syndrome, two forms of Q-T prolongation. In epithelial cells, KCNQ1-KCNE1, KCNQ1-KCNE2 and KCNQ1-KCNE3 give rise to potassium currents required for potassium recycling and secretion. These functions arise because the biophysical properties of KCNQ1 are always dramatically altered by KCNE co-expression. We wanted to understand how KCNE peptides are able to modulate KCNQ1. In Chapter II, we produce partial truncations of KCNE3 and demonstrate the transmembrane domain is necessary and sufficient for both assembly with and modulation of KCNQ1. Comparing these results with published results obtained from chimeric KCNE peptides and partial deletion mutants of KCNE1, we propose a bipartite modulation residing in KCNE peptides. Transmembrane modulation is either active (KCNE3) or permissive (KCNE1). Active transmembrane KCNE modulation masks juxtamembranous C-terminal modulation of KCNQ1, while permissive modulation allows C-terminal modulation of KCNQ1 to express. We test our hypothesis, and demonstrate C-terminal Long QT point mutants in KCNE1 can be masked by active trasnsmembrane modulation. Having confirmed the importance the C-terminus of KCNE1, we continue with two projects designed to elucidate KCNE1 C-terminal structure. In Chapter III we conduct an alanine-perturbation scan within the C-terminus. C-terminal KCNE1 alanine point mutations result in changes in the free energy for the KCNQ1-KCNE1 channel complex. High-impact point mutants cluster in an arrangement consistent with an alphahelical secondary structure, "kinked" by a single proline residue. In Chapter IV, we use oxidant-mediated disulfide bond formation between non-native cysteine residues to demonstrate amino acid side chains residing within the C-terminal domain of KCNE1 are close and juxtaposed to amino acid side chains on the cytoplasmic face of the KCNQ1 pore domain. Many of the amino acids identified as high impact through alanine perturbation correspond with residues identified as able to form disulfide bonds with KCNQ1. Taken together, we demonstrate that the interaction between the C-terminus of KCNE1 and the pore domain of KCNQ1 is required for the proper modulation of KCNQ1 by KCNE1, and by extension, normal IKs function and heart rhymicity.

Drug resistance is the most important factor that influences the successful treatment of individuals infected with the human immunodeficiency virus type 1 (HIV-1), the causative organism of the acquired immunodeficiency syndrome (AIDS). Tremendous advances in our understanding of HIV and AIDS have led to the development of Highly Active Antiretroviral Therapy (HAART), a combination of drugs that includes HIV-1 reverse transcriptase, protease, and more recently, integrase and entry inhibitors, to combat the virus. Though HAART has been successful in reducing AIDS-related morbidity and mortality, HIV rapidly evolves resistance leading to therapy failure. Thus, a better understanding of the mechanisms of resistance will lead to improved drugs and treatment regimens. Protease inhibitors (PIs) play an important role in anti-retroviral therapy. The development of resistance mutations within the active site of the protease greatly reduces its affinity for the protease inhibitors. Frequently, these mutations reduce catalytic efficiency of the protease leading to an overall reduction in viral fitness. In order to overcome this loss in fitness the virus evolves compensatory mutations within the protease cleavage sites that allow the protease to continue to recognize and cleave its substrates while lowering affinity for the PIs. Improved knowledge of this substrate co-evolution would help better understand how HIV-1 evolves resistance and thus, lead to improved therapeutic strategies. Sequence analyses and structural studies were performed to investigate co-evolution of HIV-1 protease and its cleavage sites. Though a few studies reported the co-evolution within Gag, including the protease cleavage sites, a more extensive study was lacking, especially as drug resistance was becoming increasingly severe. In Chapter II, a small set of viral sequences from infected individuals were analyzed for mutations within the Gag cleavage sites that co-occurred with primary drug resistance mutations within the protease. These studies revealed that mutations within the p1p6 cleavage site coevolved with the nelfinavir-resistant protease mutations. As a result of increasing number of infected individuals being treated with PIs leading to the accumulation of PI resistant protease mutations, and with increasing efforts at genotypic and phenotypic resistance testing, access to a larger database of resistance information has been made possible. Thus in Chapter III, over 39,000 sequences were analyzed for mutations within NC-p1, p1-6, Autoproteolysis, and PR-RT cleavage sites and several instances of substrate co-evolution were identified. Mutations in both the NC-p1 and the p1-p6 cleavage sites were associated with at least one, if not more, primary resistance mutations in the protease. Previous studies have demonstrated that mutations within the Gag cleavage sites enhance viral fitness and/or resistance when they occur in combination with primary drug resistance mutations within the protease. In Chapter III viral fitness in the presence and absence of cleavage site mutations in combination with primary drug resistant protease mutations was analyzed to investigate the impact of the observed co-evolution. These studies showed no significant changes in viral fitness. Additionally in Chapter III, the impact of these correlating mutations on phenotypic susceptibilities to various PIs was also analyzed. Phenotypic susceptibilities to various PIs were altered significantly when cleavage site mutations occurred in combination with primary protease mutations. In order to probe the underlying mechanisms for substrate co-evolution, in Chapter IV, X-ray crystallographic studies were performed to investigate structural changes in complexes of WT and D30N/N88D protease variants and the p1p6 peptide variants. Peptide variants corresponding to p1p6 cleavage site were designed, and included mutations observed in combination with the D30N/N88D protease mutation. Structural analyses of these complexes revealed several correlating changes in van der Waals contacts and hydrogen bonding as a result of the mutations. These changes in interactions suggest a mechanism for improving viral fitness as a result of co-evolution. This thesis research successfully identified several instance of co-evolution between primary drug resistant mutations in the protease and mutations within NC-p1 and p1p6 cleavage sites. Additionally, phenotypic susceptibilities to various PIs were significantly altered as a result of these correlated mutations. The structural studies also provided insights into the mechanism underlying substrate co-evolution. These data advance our understanding of substrate co-evolution and drug resistance, and will facilitate future studies to improve therapeutic strategies.

Runx1 is a master regulator of hematopoiesis, required for the initiation of definitive hematopoiesis in the embryo and essential for appropriate differentiation of many hematopoietic lineages in the adult. The roles of Runx1 in normal hematopoiesis are juxtaposed with the high frequency of Runx1 mutations and translocations in leukemia. Leukemia associated Runx1 mutations that retain DNA-binding ability have truncations or frame shifts that lose C-terminal domains. These domains are important for subnuclear localization of Runx1 and protein interactions with co-factors. The majority of leukemia associated Runx1 translocations also replace the C-terminus of Runx1 with chimeric fusion proteins. The common loss of Runx1 C-terminal domains in hematopoietic diseases suggests a possible common mechanism. We developed a panel of mutations to test the functions of these domains in vitro, and then developed mouse models to examine the consequences of losing Runx1 C-terminal domains on hematopoietic development and leukemogenesis in vivo. We previously observed that overexpression of a subnuclear targeting defective mutant of Runx1 in a myeloid progenitor cell line blocks differentiation. Gene expression analysis before differentiation was initiated revealed that the mutant Runx1 was already deregulating genes important for maturation. Furthermore, promoters of the suppressed genes were enriched for binding sites of known Runx1 co-factors, indicating a non-DNA-binding role for the mutant Runx1. To investigate the in vivo function of Runx1 C-terminal domains, we generated two knock-in mouse models; a C-terminal truncation, Runx1Q307X, and a point mutant in the subnuclear targeting domain, Runx1 HTY350-352AAA . Embryos homozygous for Runx1 Q307X phenocopy a complete Runx1 null and die in utero from central nervous system hemorrhage and lack of definitive hematopoiesis. Embryos homozygous for the point mutation Runx1HTY350-352AAA bypass embryonic lethality, but have hypomorphic Runx1 function. Runx1HTY350-352AAA results in defective growth control of hematopoietic progenitors, deregulation of B-lymphoid and myeloid lineages, as well as maturation delays in megakaryocytic and erythroid development. Runx1 localizes to subnuclear domains to scaffold regulatory machinery for control of gene expression. This work supports the role of transcription factors interacting with nuclear architecture for greater biological control, and shows how even subtle alterations in that ability could have profound effects on normal biological function and gene regulation.

In the Drosophila brain, memories are processed and stored in two mirrorsymmetrical structures composed of approximately 5,000 neurons called Mushroom Bodies (MB). Depending on their axonal extensions, neurons in the MB can be further classified into three different subgroups: αβ, α’β’ and γ. In addition to the morphological differences between these groups of neurons, there is evidence of functional differences too. For example, it has been previously shown that while neurotransmission from α’β’ neurons is required for consolidation of olfactory memory, output from αβ neurons is required for its later retrieval. To gain insight into the functional properties of these discrete neurons we analyzed whether they were different at the level of gene expression. We generated an intersectional genetic approach to exclusively label each population of neurons and permit their purification. Comparing expression profiles, revealed a large number of potentially interesting molecular differences between the populations. We focused on the finding that the MB αβ neurons, which are the presumed storage site for transcription-dependent long-term memory, express high levels of mRNA for transposable elements and histones suggesting that these neurons likely possess unique genomic characteristics. For decades, transposable elements (TE) were considered to be merely “selfish” DNA elements inserted at random in the genome and that they their sole function was to self-replicate. However, new studies have started to arise that indicate TE contribute more than just “junk” DNA to the genome. Although it is widely believed that mobilization of TE destabilize the genome by insertional mutagenesis, deletions and rearrangements of genes, some rearrangements might be advantageous for the organism. TE mobilization has recently been documented to occur in some somatic cells, including in neuronal precursor cells (NPCs). Moreover, mobilization in NPCs seems to favor insertions within neuronal expressed genes and in one case the insertion elevated the expression. During the last decade, the discovery of the small RNA pathways that suppress the expression and mobilization of TE throughout the animal have helped to uncover new functions that TE play. In this work, we demonstrate that proteins of the PIWI-associated RNA pathway that control TE expression in the germline are also required to suppress TE expression in the adult fly brain. Moreover, we find that they are differentially expressed in subsets of MB neurons, being under represented in the αβ neurons. This finding suggests that the αβ neurons tolerate TE mobilization. Lastly, we demonstrate by sequencing αβ neuron DNA that TE are mobile and we identify >200 de novo insertions into neurally expressed genes. We conclude that this TE generated mosaicism, likely contributes a new level of neuronal diversity making, in theory, each αβ neuron genetically different. In principle the stochastic nature of this process could also render every fly an individual.

DNA damage induces mitotic exit delays through a process that requires the spindle assembly checkpoint (SAC), which blocks the metaphase to anaphase transition in the presence of unaligned chromosomes. Using time-lapse confocal microscopy in syncytial Drosophila embryos, we show that DNA damage leads to arrest during prometaphase and anaphase. In addition, functional GFP fusions to the SAC components MAD2 and Mps1, and the SAC target Cdc20 relocalize to kinetochore through anaphase arrest, and a null mad2mutation blocks damage induced prometaphase and anaphase arrest. We also show that the DNA damage signaling kinase Chk2 is required for damage induced metaphase and anaphase arrest, and that a functional GFP-Chk2 fusion localizes to kinetochores and centrosomes through mitosis. In addition, in the absence of Chk2, we find that DNA damage sufficient to fragment centromere DNA does not delay mitotic exit. We conclude that DNA damage signaling through Chk2 triggers Mad2-dependent delays in mitotic progression, both before or after the metaphase-anaphase transition.

Primate lentiviruses encode four “accessory proteins” including Vif, Vpu, Nef, and Vpr/ Vpx. Vif and Vpu counteract the antiviral effects of cellular restrictions to early and late steps in the viral replication cycle. The functions of Vpx/ Vpr are not well understood. This study presents evidence that the Vpx proteins of HIV-2/ SIVSMpromote HIV-1 infection by antagonizing an antiviral restriction in myeloid cells. Fusion of macrophages in which Vpx was essential for virus infection, with COS cells in which Vpx was dispensable for virus infection, generated heterokaryons that supported infection by wild-type SIV but not Vpx-deleted SIV. The restriction potently antagonized infection of macrophages by HIV-1, and expression of Vpx in macrophages in transovercame the restriction to HIV-1 and SIV infection. Similarly, the cellular restriction is the obstacle to transduction of macrophages by MLV. Neutralization of the restriction by Vpx rendered macrophages permissive to MLV infection. Vpx was ubiquitylated and both ubiquitylation and the proteasome regulated the activity of Vpx. The ability of Vpx to counteract the restriction to HIV-1 and SIV infection was dependent upon the HIV-1 Vpr interacting protein, damaged DNA binding protein 1 (DDB1), and DDB1 partially substituted for Vpx when fused to Vpr. This study further demonstrates that this restriction prevents transduction of quiescent monocytes by HIV-1. Although terminally differentiated macrophages are partially permissive to HIV-1, quiescent monocytes, which are macrophage precursors, are highly refractory to lentiviral infection. Monocyte-HeLa heterokaryons were resistant to HIV-1 infection, while heterokaryons formed between monocytes and HeLa cells expressing Vpx were permissive to HIV-1 infection, suggesting the resistance of quiescent monocytes to HIV-1 transduction is governed by a restriction factor. Encapsidation of Vpx within HIV-1 virions conferred the ability to infect quiescent monocytes. Introduction of Vpx into monocytes by pre-infection also rendered quiescent monocytes permissive to HIV-1 infection. Infection of monocytes by HIV-1 either with or without Vpx did not have an effect on temporal expression of CD71. In addition, Vpx increased permissivity of CD71– and CD71+cells to HIV-1 infection with no apparent bias. These results confirm that Vpx directly renders undifferentiated monocytes permissive to HIV-1 transduction without inducing their differentiation. The introduction of Vpx did not significantly alter APOBEC3G complex distribution, suggesting a restriction other than APOBEC3G was responsible for the resistance of monocytes to HIV-1. Collectively our results indicate that macrophages and monocytes harbor a potent antiviral restriction that is counteracted by the Vpx protein. The relative ability of primate lentiviruses and gammaretroviruses to transduce non-dividing myeloid-cells is dependent upon their ability to neutralize this restriction.