High visual sensitivity is a common but important characteristic of animal eyes. It is especially critical for night vision. In animal eyes, photoreceptors are the first to receive the incoming rays of light and they convert the light signals to electrical signals before passing the information to interneurons in the eye and finally to the brain. To function in dim light conditions, photoreceptors have developed high sensitivities to light. It is reported that both mammalian rod photoreceptors and Drosophilaphotoreceptors can detect single photons. The high sensitivities of photoreceptors largely depend on a high content of rhodopsin, a light-stimulated G protein-coupled receptor (GPCR), in light sensory organelles, outer segments in mammals and rhabdomeres in Drosophila. Two shared characteristics, the tightly packed photoreceptive membrane and the high concentration of rhodopsin in the membrane, work together to enable the photoreceptors to achieve the high content of rhodopsin in photosensory organelles in both mammals and Drosophila. In this thesis, I have used the Drosophilaeye as a model system to study the molecular mechanisms required for the maintenance of these two characteristics. In the second chapter, I present a new molecular mechanism of preventing Gq-mediated rhabdomeral degeneration. A new gene named tadr (for torn and diminished rhabdomeres), when mutated, leads to visual sensitivity reduction and photoreceptor degeneration. Degeneration in the tadr mutant is characterized by shrunken and disrupted rhabdomeres. The TADR protein interacts in vitro with the major light receptor Rh1 rhodopsin, and genetic reduction of the Rh1 level suppresses the tadr-induced degeneration, suggesting the degeneration is Rh1-dependent. Nonetheless, removal of phospholipase C (PLC), a key enzyme in phototransduction, and that of Arr2 fail to inhibit rhabdomeral degeneration in the tadr mutant background. Biochemical analyses reveal that, in the tadr mutant, the Gq protein of Rh1 is defective in dissociation from the membrane during light stimulation. Importantly, reduction of Gq level by introducing a hypomorphic allele of Gαq gene greatly inhibits the tadr degeneration phenotype. These results may suggest that loss of a potential TADR-Rh1 interaction leads to an abnormality in the Gqsignaling, which in turn triggers rhabdomeral degeneration independent of the PLC phototransduction cascade. We propose that TADR-like proteins may also protect photoreceptors from degeneration in mammals including humans. In the third chapter, I present a Drosophila CUB- and LDLa-domain transmembrane protein CULD that counteracts the visual arrestin Arr1-mediated endocytosis to retain rhodopsin in rhabdomeral membrane. CULD is mostly localized in rhabdomeres, but is also detected in scarce rhodopsin endocytic vesicles that contain Arr1. An intracellular region of CULD interacts with Arr1 in vitro. In both culdmutant and knockdown flies, a large amount of rhodopsin is mislocalized in the cell body of photoreceptors through lightdependent, Arr1-mediated endocytosis, leading to reduction of photoreceptor sensitivity. Expressing a wild-type CULD protein in photoreceptors, but not a mutant variant lacking the Arr1-interacting site, rescues both the rhodopsin mislocalization and the low sensitivity phenotypes. Once rhodopsin has been internalized in adult mutant flies, it is reversed only by expression of CULD but not by blocking endocytosis, suggesting that CULD promotes recycling of endocytosed rhodopsin to the rhabdomere. Our results demonstrate an important role of CULD in the maintenance of membrane rhodopsin density and photoreceptor sensitivity. We propose that a common cellular function of CUB- and LDLa-domain proteins, in both mammals and invertebrates, is to concentrate receptors including GPCRs in particular regions of cell membrane. In summary, the work addressed in this thesis has identified new molecular mechavii nisms underlying the maintenance of visual sensitivity in Drosophila, either through preventing Gq-mediated rhabdomeral degeneration or through antagonizing arrestin-mediated rhodopsin endocytosis. This work has advanced our understanding of visual biology and the general regulatory mechanisms of GPCR signaling, and may provide valuable clues to pathologic studies of human retinal degeneration disorders.

Innate immunity is the first line of defense against invading pathogens. It functions to eliminate pathogens and also to control infections. The innate immune response is also important for the development of pathogen-specific adaptive immune responses. As a result, the study of innate immune signaling pathways is crucial for understanding the interactions between host and pathogen. Unlike mammals, insects lack a classical adaptive immune response and rely mostly on innate immune responses. Innate immune mechanisms have been widely studied in the fruit fly, Drosophila melanogaster. The genetic and molecular tools available in the Drosophila system make it an excellent model system for studying immunity. Furthermore, the innate immune signaling pathways used by Drosophila show strong homology to those of vertebrates making them ideal for studying these pathways. Drosophila immunity relies on cellular and humoral innate immune responses to fight pathogens. The hallmark of the Drosophilahumoral immune response is the rapid induction of antimicrobial peptide genes in the fat body. The production of these antimicrobial peptides is regulated by two immune signaling pathways-Toll and Immune Deficency (IMD) pathways. The Toll pathway responds to many Gram-positive bacterial and fungal infections , while the IMD pathway is potently activated by DAP-type peptidoglycan (PGN) from Gram-negative bacteria and certain Gram-positive bacteria. Two receptors, PGRP-LC and PGRP-LE, are able to recognize DAP-type PGN at the cell surface or in the cytosol, respectively, and trigger the IMD pathway. Upon binding DAP-type PGN, both PGRP-LC and PGRP-LE dimerize/ multimerize and signal to the downstream components of IMD pathway. It is unclear how the receptor activates its downstream components. My work has focused on understanding the molecular events that take place at the receptors following there activation. In these studies I have identified a common motif in the N-terminal domains of both the receptors, known as the RHIM-like domain. The RHIM-like domain is critical for signaling by either receptor, but the mechanism(s) involved remain unclear. IMD, a downstream component of the pathway, associates with both PGRP-LC and -LE but the interaction of PGRP-LC with IMD is not mediated through its RHIM-like domain. Also, mutations affecting the PGRP-LC RHIM-like motif are defective in all known downstream signaling events. However, the RHIM-like mutant receptors are capable of serving as a platform for the assembly of all known components of a receptor proximal signaling complex. These results suggest that another, unidentified component of the IMD signaling pathway may function to mediate interaction with the RHIM-like motif. I performed a yeast two-hybrid screen to identify proteins that might interact with the receptor PGRP-LC through its RHIM- like domain. With this approach, two new components of the IMD pathway were identified. The first component I characterized is called Rudra and it is a critical feedback inhibitor of peptidoglycan receptor signaling. The other factor is known as RYBP, it includes a highly conserved ubiquitin binding motif (NZF), and RNAi studies suggest it is a critical component of the IMD pathway. The identification and characterization of these two new components of the IMD pathway has provided a new insight into the molecular events that take place proximal to the receptor.

N. meningitidis is a common commensal of the human upper respiratory tract and a leading cause of bacterial meningitis and septicemia worldwide. The classical pathway of complement (C) is essential for both naturally acquired and vaccine induced immunity against N. meningitidis. Qualitative and/or quantitative differences in anti-meningococcal antibodies (Abs) in serum is one reason for variations in C-dependent bactericidal Ab activity among individuals. I showed that IgG isolated from select individuals could block killing of group B meningococci by Abs that were otherwise bactericidal. Ligand overlay immunoblots revealed that these blocking IgG Abs were directed against a meningococcal antigen called H.8, Killing of meningococci in reactions containing bactericidal mAbs and human blocking Abs was restored when blocking Ab binding to meningococci was inhibited (or competed for) using either synthetic peptides corresponding to H.8 or a non-blocking mAb against H.8. Further, genetic deletion of H.8 from target organisms abrogated blocking. The Fc region of the blocking IgG was required for blocking because F(ab)2 fragments alone generated by pepsin treatment were ineffective. Blocking required IgG glycosylation; deglycosylation of blocking IgG with peptide:N-glycanase (PNGase) eliminated blocking. C4 deposition mediated by a bactericidal mAb directed against a meningococcal vaccine candidate, called factor H-binding protein (fHbp), was reduced by blocking Ab. Anti-fHbp-mediated C4 deposition was unaffected, however, by deglycosylated blocking IgG. Although preliminary, our data suggests blocking of serum bactericidal activity by human anti-H.8 blocking antibody may require mannan-binding lectin (MBL), which itself is a complement activator. Also, whether MBL recruits a complement inhibitor(s) that facilitates blocking remains to be determined. In conclusion, we have identified H.8 as a meningococcal target for novel blocking antibodies that are commonly found in human serum. Blocking Ab may reduce the efficacy of meningococcal vaccines. We propose that outer membrane vesicle-containing meningococcal vaccines may be more efficacious if purged of subversive immunogens such as H.8.

Among heat shock proteins, Hsp90 is unusual because it is not required for the proper folding of most cellular proteins but rather is disproportionally linked to the activation of signal transduction proteins including over forty kinases and many steroid hormone receptors. Mutated forms of many Hsp90 clients are causative agents in cancer, making Hsp90 a promising pharmacological target. Many small molecular inhibitors have been identified that competitively bind to the ATP binding site of Hsp90, some of which are in clinical trials as anticancer agents. Although the activation of kinase and hormone receptor clients by Hsp90 and its co-chaperones has been extensively studied, the molecular mechanism of client protein activation is poorly understood. Hsp90 is a dimeric chaperone containing three domains: the N-terminal (N) and middle (M) domains contribute directly to ATP binding and hydrolysis and the C-terminal (C) domain mediates dimerization. At physiological concentration, Hsp90 predominantly forms dimers, but the possibility that full-length monomers might also function in cells has not been tested. In Chapter 3, we used a single-chain strategy to design a full-length Hsp90 monomer (NMCC). The resulting construct was predominantly monomeric at physiological concentration and did not function to support yeast viability as the sole Hsp90. NMCC Hsp90 was also defective at ATP hydrolysis and the activation of kinase and steroid hormone receptor clients in yeast cells. The ability to support yeast growth was rescued by the addition of a coiled-coil dimerization domain, indicating that the parental single-chain construct is functionally defective because it is monomeric. After finding that a full-length Hsp90 monomer containing only one ATPase site was unable to support yeast viability or activate Hsp90 clients, we set out to further explore the role of ATPase activity in client protein activation. Approximately 10 % of the yeast proteome binds to Hsp90 making it important to study Hsp90 function in the cellular environment where all binding partners are present. In Chapter 4, we observed that co-expression of different Hsp90 subunits in Saccharomyces cerevisiae caused unpredictable synthetic growth defects due to cross-dimerization. We engineered super-stabilized Hsp90 dimers that resisted cross-dimerization with endogenous Hsp90 and alleviated the synthetic growth defect. We utilized these super-stabilized dimers to analyze the ability of ATPase mutant homodimers to activate known Hsp90 client proteins in yeast cells. We found that ATP binding and hydrolysis by Hsp90 are both required for the efficient maturation of the glucocorticoid hormone receptor (GR) and v-src confirming the critical role of ATP hydrolysis in the maturation of steroid hormone receptors and kinases in vivo. In addition to its role in the activation of signal transduction client proteins, Hsp90 has been shown to suppress the in vitro aggregation of numerous hard-to-fold proteins. In Chapter 5, we examine the role of charge in Hsp90 anti-aggregation activity. The charge on Hsp90 is largely concentrated in two highly acidic regions. We found that deletion of both charge-rich regions dramatically impaired Hsp90 anti-aggregation activity. Addition of an acid-rich region with a distinct amino acid sequence to our double-deleted Hsp90 construct rescued the anti-aggregation activity of Hsp90 indicating that the net charge contributes to its anti-aggregation activity. The in vitro anti-aggregation activity of Hsp90 studied in Chapter 5 occurs in the absence of ATP. However, all of the biologically important functions of Hsp90 in cells identified to date, including the maturation of kinases and nuclear steroid hormone receptors, clearly require ATP hydrolysis. Why does Hsp90 robustly hinder the aggregation of hard-to-fold proteins without ATP in vitro, but in vivo uses ATP hydrolysis for all of its essential functions? By utilizing separation of function Hsp90 variants (that specifically lack in vitro anti-aggregation activity) we have begun to address this question. We find that anti-aggregation deficient Hsp90 is unable to support yeast growth under stressful conditions, potentially due to reduced cellular expression. Interestingly, the ATP-independent anti-aggregation activity of Hsp90 has no measureable impact on cellular function. Thus, hindering the aggregation of most hard-to- fold proteins by Hsp90 (independent of ATP hydrolysis) does not appear to be important for cell function. These results suggest a cellular model where the Hsp40/60/70 machinery is responsible for hindering the aggregation of most hard-to-fold proteins while Hsp90 assists in the maturation of a select set of clients in an ATP-dependent fashion, potentially aided by its inherent anti-aggregation properties.

The innate immune system is the first line of defense against invading pathogens. Recognition of microbial ligands by the innate immune system relies on germ-line encoded, evolutionarily conserved receptors called pattern recognition receptors (PRRs). Toll-like receptors (TLRs) are one such family of PRRs and are involved in innate defenses to a variety of microbes. At the core of TLR signaling pathways are Toll interleukin-1 receptor (TIR) domain containing adapter proteins. Much of the specificity of TLR pathways arise from the differential use of these adapter proteins. The TLR signaling cascade that ensues upon ligand recognition is marked by finely orchestrated molecular interactions between the receptor and the TIR domain containing adapter proteins, as well as various downstream kinases and effector molecules. Conserving the structural integrity of the TLR components is thus essential for maintaining a robust host defense system. Sometimes, changes in a protein can be brought about by single nucleotide polymorphisms (SNPs). Studies carried out in this thesis focus on polymorphisms in MyD88 adapter-like (Mal) and myeloid differentiation protein 88 (MyD88), two TIR domain-containing adapter proteins, which incidentally are also highly polymorphic. Mal is a 235 amino acid protein that is involved in TLR2 and TLR4 signaling. The known polymorphisms in the coding region of Mal were screened with an aim to identify SNPs with altered signaling potential. A TIR domain polymorphism, D96N, was found to be completely defective in TLR2 and TLR4 signaling. Immortalized macrophage-like cell lines expressing D96N have impaired cytokine production as well as NF-κB activation. The reason for this loss-of-function phenotype is the inability of Mal D96N to bind the downstream adapter MyD88, an event necessary for signaling to occur. Genotyping studies reveal a very low frequency of this polymorphism in the population. Similar SNP analysis was carried out in myeloid differentiation protein 88 (MyD88). MyD88 is a key signaling adapter in TLR signaling; critical for all TLR pathways except TLR3. In reporter assays, a death domain variant, S34Y, was found to be inactive. Importantly, in reconstituted macrophage-like cell lines derived from knockout mice, MyD88 S34Y was severely compromised in its ability to respond to all MyD88-dependent TLR ligands. S34Y mutant has a dramatically different localization pattern as compared to wild type MyD88. Unlike wild type MyD88, S34Y is unable to form distinct foci in the cells but is present diffused in the cytoplasm. IRAK4, a downstream kinase, colocalizes with MyD88 in these aggregates or “Myddosomes”. S34Y MyD88, however, is unable to assemble into Myddosomes, thus demonstrating that proper cellular localization of MyD88 is a feature required for MyD88 function. This thesis thus describes two loss‐of‐function polymorphisms in TLR adapter proteins Mal and MyD88. It sheds light not only on the structural aspects of signaling by these two proteins, but also has implications for the development of novel pharmaceutical agents.

HCV afflicts many millions of people globally, and antiviral therapies are often ineffective and intolerable. The Food and Drug Administration approved the HCV protease inhibitors telaprevir and boceprevir in May 2011, marking an important milestone in anti-HCV research over the past two decades. Nevertheless, severe drug side effects of combination therapy – flu-like symptoms, depression and anemia – limit patient adherence to treatment regimens. The acquisition of resistance challenges the long-term efficacy of antiviral therapies, including protease inhibitors, as suboptimal dosing allows for the selection of drug resistant viral variants. A better understanding of the molecular basis of drug resistance is therefore central to developing future generation protease inhibitors that retain potency against a broader spectrum of HCV strains. To this end, my research characterizes the molecular basis of drug resistance against HCV protease inhibitors. Chapter II defines the mode of substrate recognition by the common volume shared by NS3/4A substrate products – the substrate envelope. Chapter III then correlates patterns of drug resistance to regions where drugs protrude from the substrate envelope. Lastly, Chapter IV elucidates the molecular underpinnings of resistance against four leading protease inhibitors – telaprevir, danoprevir, vaniprevir and MK-5172 – and provides practical approaches to designing novel drugs that are less susceptible to resistance. I ultimately hope my work appeals to the broader biomedical community of virologists, medicinal chemists and clinicians, who struggle to understand HCV and other human pathogens in the face of rapid disease evolution.

Spinal muscular atrophy (SMA) is an autosomal recessive neurodegenerative disorder that is the leading genetic cause of infantile death. SMA is caused by homozygous deletion or mutation of the survival of motor neuron 1 gene (SMN1). The SMN2 gene is nearly identical to SMN1, however is alternatively spliced. The close relationship to SMN1 results in SMN2 being a very power genetic modifier of SMA disease severity and a target for therapies. In this study we attempt to characterize novel chemical compounds identified as potential activators of the SMN2 gene. Additionally, we sought to determine the regulatory role individual HDAC proteins use to control expression of full length protein from the SMN2 gene. We used quantitative PCR to determine the effects of novel compounds and shRNA silencing of individual HDACs on the steady state levels of a SMN2-luciferase reporter transcripts. We determined that the compounds identified in multiple reporter high throughput screens increased SMN protein levels via transcriptional activation of the SMN2 gene. Other compounds identified in the same screen functioned post-transcriptionally, possibly stabilizing the SMN protein itself by decreasing degradation. Furthermore, we determined that reduction of individual HDAC proteins was sufficient to increase SMN protein levels in a transgenic reporter system. Knockdown of class I HDAC proteins preferentially activated the reporter by increased promoter transcription. Silencing of class II HDAC proteins maintained transcriptional activity; however silencing of HDAC 5 and 6 also appeared to enhance inclusion of an alternatively spliced exon. This collective work defines a quantitative RNA based protocol to determine mechanism of SMN reporter increase in response to any chosen treatment method. Additionally, this work highlights HDAC proteins 2 and 6 as excellent investigative targets. These data are important to the basic understanding of SMN expression regulation and the refinements of current therapeutic compounds as well as the development of novel SMA therapeutics.

Human papillomavirus infection is the cause of more than 99% of cervical cancer cases. The current vaccine is ineffective therapeutically; highlighting the need for continued papillomavirus research. One avenue that could be explored in this regard is the function of the papillomavirus E2 regulatory proteins. HPV E2 represses expression of the viral E6 and E7 oncoproteins. Reintroduction of E2 into cervical carcinoma cells results in growth arrest and cellular senescence. Understanding the mechanism of how E2 regulates the early promoter may be key to developing new therapeutic and prophylactic vaccines. Here, we describe regulation of E2 through acetylation and possibly through direct interaction with a novel cellular interacting protein, RINT1. Histone acetyltransferase (HAT) proteins have been demonstrated to interact with Bovine Papillomavirus (BPV) and Human Papillomavirus (HPV) E2 proteins as well as enhance E2 dependant transcription luciferase reporter plasmid containing E2 binding sites. We demonstrate that HATs p300, CBP, and pCAF are limiting for E2 dependant transcriptional activation and that each protein functions independently. We have also identified that BPV-1 E2 is a substrate for acetylation by p300. Mutants of E2 that cannot be acetylated on lysines 111 or 112, display abnormal transcriptional phenotypes. Cells deficient in p300 display similar transcriptional defects that are intensified by CBP depletion. We propose that acetylation of BPV-1 E2 is necessary for transcriptional activation. Acetylation generates a binding site through which a co-factor may interact via a bromodomain. Regulation of E2 dependent transcriptional activation through a post-transcriptional modification represents a novel method through which BPV-1 controls gene expression. We also present evidence for a direct interaction between BPV-1 E2 and the cellular factor RINT1. This interaction does not appear to be critical for transcriptional regulation; however, several other functional pathways are indicated by the cellular complexes in which RINT1 functions. Some of these, such as ER/Golgi vesicular transport and hTERT independent telomere maintenance, are pathways in which E2 has no known role. Further investigation into regulation and consequences of E2 acetylation and the biological significance of the interaction between E2 and RINT1 could prove important in understanding the complex role of E2 in papillomavirus infection.

Vesicle budding, membrane trafficking, and lipid metabolism depend on the switching of Arf and Rab GTPases from the inactive GDP bound state to the active GTP bound state. However, Arf and Rab GTPases have intrinsic rates of GDP to GTP exchange that are much slower (hours to days) than the time scale of the relevant trafficking processes (seconds or less). In cells, the activation of Arf and Rab GTPases is tightly regulated by guanine nucleotide exchange factors (GEFs) with Sec7 or Vps9 domains, respectively. Full length Cytohesins, which have a domain architecture consisting of heptad repeats, a Sec7 domain, a pleckstrin homology (PH) domain, and a polybasic motif, have 100-fold lower exchange activity than the isolated Sec7 domain. Insights into the low exchange activity were obtained by structural, biochemical and kinetic analyses. It was found that the Sec7-PH domain linker and a C-terminal amphipathic helix physically block the docking sites for the switch regions of Arf GTPases. Mutations within either element result in partial or complete relief of autoinhibition. Autohibition is also strongly relieved by phosphorylation of protein kinase C (PKC) sites in the polybasic motif of Cytohesin-1 or by phosphoinositide head group-dependent binding of active Arf6. Despite unrelated folds, Sec7 and Vps9 domains engage cognate GTPases in a strikingly similar manner and supply a critical acidic residue that interacts with an invariant lysine residues from phosphate binding (P) loop of the GTPase in the nucleotide free complex. The key acidic residues have also been proposed to disrupt the Mg2+ binding site; however, it is not known whether disruption of Mg2+ binding contributes to the rate limiting step for nucleotide release. To investigate the kinetic mechanism for catalysis of nucleotide exchange in the absence of autoinhibitory interactions, a detailed stopped flow kinetic analysis of the intrinsic and GEF mediated exchange reactions was conducted for the isolated catalytic cores. Using three different fluorescence methods to monitor Mg2+ dissociation, formation of the nucleotide free intermediate, and subsequent nucleotide binding, the catalytic cores of Cytohesin-1 and Rabex-5 were found to robustly accelerate nucleotide exchange on Arf1 and Rab5, respectively, by at least 105- fold at physiological concentrations of Mg2+. The acceleration of nucleotide exchange was reduced by roughly an order of magnitude at sub-micromolar concentrations of Mg2+. In addition, the Cytohesin-1 and Rabex-5 catalytic cores have similarly high catalytic efficiencies (kcat/KM) as well as high lower limits on both the rate (kcat) and steady state (KM) constants for GDP release at physiological as well as low Mg2+ concentration. The limits on kcat and KM are comparable to the highest values reported for other well characterized GEFs and likely reflect dual requirements of membrane targeting and autoregulatory mechanisms for tight control of catalytic output. These results provide a solid structural and mechanistic foundation for future experiments to investigate the spatial-temporal dynamics of Cytohesin and Rabex-5 activation in cellular contexts.

Around thirty years ago HIV-1 was identified, and from that point the known epidemic has grown to over 30 million infected individuals. Early on in the course of HIV-1 research, viruses were classified as either syncytia inducing, CXCR4-using, T-cell tropic or non-syncytia inducing, CCR5-using, macrophage tropic. Since that time, several groups have shown that this is an oversimplification. There is a great deal of diversity amongst CCR5-using HIV-1 variants. There remains a great deal to be discovered regarding HIV-1 CCR5-tropism and how this affects other aspects of HIV-1 infection. The CD4 binding site (CD4bs) on the HIV-1 envelope plays a major role in determining the capacity of R5 viruses to infect primary macrophages. Thus, envelope determinants within or proximal to the CD4bs have been shown to control the use of low CD4 levels on macrophages for infection. These residues affect the affinity for CD4 either directly or indirectly by altering the exposure of CD4 contact residues. In this thesis, a single amino acid determinant is described in the V1 loop that also modulates macrophage tropism. I identified an E153G substitution that conferred high levels of macrophage infectivity for several heterologous R5 envelopes, while the reciprocal G153E substitution abrogated infection. Shifts in macrophage tropism were associated with dramatic shifts in sensitivity to the V3 loop monoclonal antibody (MAb), 447-52D and soluble CD4, as well as more modest changes in sensitivity to the CD4bs MAb, b12. These observations are consistent with an altered conformation or exposure of the V3 loop that enables the envelope to use low CD4 levels for infection. The modest shifts in b12 sensitivity suggest that residue 153 impacts on the exposure of the CD4bs. However, the more intense shifts in sCD4 sensitivity suggest additional mechanisms that likely include an increased ability of the envelope to undergo conformational changes following binding to suboptimal levels of cell surface CD4. In summary, a conserved determinant in the V1 loop modulates the V3 loop to prime low CD4 use and macrophage infection. In addition to determinants, this thesis seeks to evaluate the roles of macrophage tropic and non-macrophage tropic envelopes during the course of infection. Non-macrophage tropic virus predominates in immune tissue throughout infection, even in individuals suffering from HIV-associated dementia (HAD) who are known to carry many macrophage tropic viruses. There must be some advantage for these non-macrophage tropic viruses allowing them to persist in immune tissue throughout the disease. This thesis demonstrates that there is no advantage for these viruses to directly infect CD4+ T-cells, nor is there an advantage for them to be preferentially transmitted by dendritic cells to CD4+ T-cells. Given that transmitted/founder (T/F) viruses may preferentially interact with α4β7, and T/F viruses are non-macrophage tropic, I tested whether non-mac viruses could utilize α4β7 to their advantage. These experiments show that macrophage tropism does not play a role in gp120 interactions with α4β7. I evaluated whether there was a distinct disadvantage to macrophage tropic Envs, given their ability to infect dendritic cells and possibly stimulate the innate immune response. Using infected monocyte-derived dendritic cells (MDDCs), it was shown that mac-tropic Envs do not generate a significant immune response. These experiments demonstrate that there does not appear to be any advantage to non-macrophage tropic Envs, and that macrophage tropic Envs are able to infect CD4+ T-cells more efficiently, as well as DCs.