Point Mutations Associated with HIV-1 Drug Resistance, Evasion of the Immune Response and AIDS Pathogenesis

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Introduction
Point mutations within the human immunodeficiency virus-type 1 (HIV-1) genome confer resistance to antiretroviral drugs. The mutations also help the virus to evade the immune system response and influence transmission and progression of the disease, collectively called acquired immune deficiency syndrome (AIDS) pathogenesis. This chapter presents an up-to-date critical review of the literature and provides a synthesis of the current understanding of HIV-1 point mutations in relation to drug resistance, evasion of the immune system and AIDS pathogenesis. First, to prime the reader, the chapter briefly describes organization and function of the HIV-1 genes. It then pinpoints salient point mutations within specific HIV-1 genes associated with resistance to current antiretroviral drugs. In particular, it pays attention to the env gene and its product, the surface envelope (Env) glycoprotein because the Env protein mediates viral entry, tropism and disease progression. Thus the role and frequencies of point mutations within the env gene is related to HIV-1 transmission dynamics and progression to AIDS. The env gene and Env protein are further unpacked to provide a better understanding of the role point mutations within this gene play in helping the virus to evade antibody mediated immune response. Finally, drawing mainly from our recent research work, the chapter proposes an aptamer-based strategy for the design of desirable approaches that can help circumvent HIV-1 point mutations and delay or diminish drug resistance. www.intechopen.com (SU) gp120 (Figure 1). For a mature virion, on the inner leaflet of the envelope there are proteolytically processed gag gene products: a p17 matrix (MA) protein and a p24 capsid (CA) protein that form the core of the particle and an RNA-binding p7 nucleocapsid (NC) protein ( Figure 1 and Table 1). Other proteins present in the core of the virion particle are derived from the pol gene and perform enzymatic functions. HIV-1 gp120 and gp41 (env gene products) and pol gene products, reverse transcriptase (RT) and integrase (IN) are involved in early stages of the HIV life cycle and are primary targets of therapeutic drugs. Env, RT and IN are targets of antiretroviral drugs called entry inhibitors (EI), reverse transcriptase inhibitors (RTI) and integrase inhibitors (INI), respectively. Another pol gene product called protease (PR) is involved in later stages of the HIV life cycle and it is also a target of a class of antiretroviral drugs called protease inhibitors. Other HIV-1 genes encode auxiliary proteins (Figure 1).

Point mutations and HIV-1 drug resistance
Antiretroviral drugs are currently the cornerstone of controlling HIV-1 in infected individuals. To date, 24 antiretroviral drugs have been approved and licensed for the treatment of HIV-1 infection. Thirteen of these drugs are reverse transcriptase inhibitors (nine nucleoside and four non-nucleoside RT inhibitors); nine are protease inhibitors; a fusion inhibitor; a CCR5 inhibitor and an integrase inhibitor. However, despite the use of current antiretroviral drugs, HIV-1 can still persists in infected individuals by accumulating mutations, which make it resistant to one or more drugs. The antiretroviral drug resistant mutations can be identified by one or more of the following: (a) in vitro passage experiments or site directed mutagenesis studies (Clark et al., 2006); (b) mutagenically separated PCR (Frater et al., 2001); (c) drug susceptibility testing (Petropoulos et al., 2000;Svicher et al., 2011); (d) nucleotide sequencing of viruses from patients failing a specific drug (McNicholas et al., 2011;Shulman et al., 2004); (e) correlation studies between genotype at baseline and virologic response in patients exposed to a specific antiretroviral drug (Demeter et al., 2008). Currently, more than 200 mutations in the HIV-1 pol and env genes associated with resistance to current antiretroviral drugs, which include reverse transcriptase inhibitors (RTIs), protease inhibitors (PIs), integrase inhibitors (INI), fusion inhibitors and attachment inhibitors respectively, have been identified (V. A. Johnson et al., 2010;Shafer & Schapiro, 2008).

Point mutations associated with resistance to NRTIs
HIV-1 RT is a heterodimer made up of two subunits, p66 and p51 (di Marzo Veronese et al., 1986) and its crystal structure has been solved (Arnold et al., 1992;Kohlstaedt et al., 1992b). RT is important in the HIV-1 life cycle because it orchestrates synthesis of a linear doublestranded DNA copy of a viral single-stranded RNA genome. The knowledge that HIV-1 is a retrovirus immediately pinpointed RT synthesis of proviral DNA as a target for a class of antiretroviral drugs called RTIs. Indeed, RTIs including the prototype azidothymidine (AZT) also known as zidovudine (ZDV), were the first to be approved for clinical use (Fischl et al., 1987), and remains the mainstay of HIV-1 therapy. Nearly half of current antiretroviral drugs target the polymerase activity of RT. RTIs belong to one of the two broad categories: nucleoside RT inhibitors (NRTIs) and non-nucleoside RT inhibitors (NNRTIs).
NRTIs are structural analogues of natural substrate of DNA synthesis. They lack the 3hydroxyl group (3-OH) and hence they block HIV-1 replication by acting as chain terminators when incorporated into a viral DNA by RT. Two basic NRTI resistance mechanisms are exclusion and excision. The exclusion mechanism involves enhanced discrimination at the time the NRTI tri-phosphate is incorporated in the nascent DNA strand. The M184V/I point mutation (Figure 2 and Table 2), which selectively reduces incorporation of abacavir (ABC); emtricitabine (FTC) and lamivudine (3TC) NRTIs into a nascent DNA chain by steric hindrance (Gao et al., 2000;Sarafianos et al., 1999) is a common example of the exclusion mechanism. M184V point mutation causes a median 1.5-fold and 3.0-fold reduction in susceptibility to didanosine (ddI) and ABC, respectively in the PhenoSenseGT assay (Petropoulos et al., 2000;Rhee et al., 2004). However several clinical trials have shown that ABC and ddI retain clinical activity in the presence of M184V point mutation J. J. Eron, Jr. et al., 2007;Lanier et al., 2004a;Molina et al., 2005;M. A. Winters et al., 2003). The phenotypic and clinical significance of M184V is influenced by the presence or absence of other NRTI resistance mutations (Shafer & Schapiro, 2008). For instance, the presence of K65R or L74V (J. Eron, Jr. et al., 2006;Gallant et al., 2006;Moyle et al., 2005) point mutations ( Figure 2 and Table 2) in addition to the M184V is sufficient for high level resistance to ddI and ABC , particularly in patients with non-subtype B clades (Harrigan et al., 2000;Lanier et al., 2004a;Svarovskaia et al., 2007;M. A. Winters et al., 1997). K65R has emerged more rapidly during the in vitro passage of subtype C compared with subtype B isolates in the presence of increasing concentrations of TDF . The emergence of K65R mutation is suppressed maximally in regimens containing AZT compared with d4T (Roge et al., 2003). Thus, the K65R point mutation commonly causes intermediate resistance to tenofovir (TDF),  ABC, ddI, 3TC and FTC (Table 2), low level resistance to d4T, and increased susceptibility to AZT (Antinori et al., 2007;Lanier et al., 2004b). There is a body of evidence suggesting that K65R mutation occurs more commonly in low-income countries when patients with nonsubtype B HIV-1 strains are treated with d4T/ddI and d4T/3TC Hawkins et al., 2009) or TDF/3TC (Rey et al., 2009). In comparison to K65R, L74V point mutation causes intermediate resistance to ddI and ABC, and a slight increase in susceptibility to AZT and TDF (Rhee et al., 2006b). L74V and K65R mutations rarely occur in the same viruses (Shafer & Schapiro, 2008). However, several patients harbouring HIV-1 species containing the L74V point mutation while receiving a regimen containing ABC or ddI have been found to have minor variants containing K65R (Descamps et al., 2006;Svarovskaia et al., 2007). On the other hand, K65R and M184V mutations are primarily found in patients receiving the NRTI backbone TDF/3TC (Gallant et al., 2004;Margot et al., 2006) and less commonly ABC/3TC (Moyle et al., 2005) or TDF/FTC (Gallant et al., 2006;Smith et al., 2008). L74V and M184V mutations primarily occur in patients receiving ABC/3TC or ddI/3TC/FTC NRTI backbones (Descamps et al., 2006;Moyle et al., 2005;Sosa et al., 2005). In addition to the M184V, K65R and L74V mutations, which between them cause resistance to ABC, 3TC, FTC, TDF and ddI by the exclusion mechanism; there is a complex set of several mutations that involves Q151M causing multi-NRTI resistant ( Figure  2 and Table 2). All of the amino acids involved in the NRTIs resistance by the exclusion mechanism are in the fingers or the palm of RT ( Figure 2) and could potentially affect the binding of an incoming deoxy-nucleotide triphosphate (dNTP).  Table 2. Red show amino acid residues that confer multi-NRTI resistance (all the FDA approved drugs). Green show residues that affect all the FDA approved NRTIs except for TDF. Mutations of blue residues are associated with TAMs. Yellow shows residues that confer resistance to certain NRTIs (Table 2) by the exclusion mechanism. www.intechopen.com

NRTI TAMs (excision) Non-TAMs (exclusion) Multi-NRTI resistance mutations
*Denotes position of consensus amino acids that confer resistance to NRTIs when mutated. Dashes (-) indicate that the respective NNRTIs are not affected by the mutation(s). All amino acids are denoted by the single letter annotation, except for "Ins", which stands for one or more amino acid insertions. Mutations in bold are associated with high levels of phenotypic resistance or reduced virologic response. a K70R occur in HIV-1 isolates from patients receiving thymidine analogs. b V75I occurs in combination with Q151M. Modified from (Shafer & Schapiro, 2008). In contrast to the exclusion mechanism discussed above; the excision mechanism involves selective removal of the NRTI from the end of the viral DNA after it has been incorporated by RT (Arion et al., 1998;Boyer et al., 2001;Meyer et al., 1999;Meyer et al., 1998). This is ATPdependent excision and polymerization run in reverse, where an AZT resistant RT efficiently incorporates AZT triphosphate but has the enhanced ability to remove the incorporated AZT-monophosphate from the 5-end of the template strand (Arion et al., 1998;Meyer et al., 1998). The first and well studied example of excision mechanism involves a set of AZT resistant mutations such as M41L, D67N, K70R, L210W, T215F/Y and K219E ( Figure 1 and Table 1). These AZT resistant mutations are also called thymidine-analog mutations (TAMs) or excision-enhancing mutations (EEMs). In addition to AZT, the TAMs decrease susceptibility to d4T and to a lesser extent ABC, ddI and TDF (Whitcomb et al., 2003). TAMs are common in low income countries in which the fixed-dose combinations containing thymidine analogues are the mainstay of therapy (Ross et al., 2001). TAMs are also common in HIV-1 isolates from patients who began therapy in the pre-highly active antiretroviral therapy (HAART) era (De Luca et al., 2006;Rhee et al., 2005). The mutations accumulate in two distinct but overlapping patterns (De Luca et al., 2006;Gonzales et al., 2003;Marcelin et al., 2004b;Yahi et al., 1999). The type I pattern includes the M41L, L210W and T15Y mutations, respectively (De Luca et al., 2006;Miller et al., 2004). Type II patterns includes D67N, K70R, T215F and K219Q/E ( Figure 2). D67N point mutation also occurs commonly with type I TAMs (Cozzi-Lepri et al., 2005;Rhee et al., 2007). However, K70R and L210W rarely occur together (Yahi et al., 2000). Type I TAMs cause higher levels of phenotypic and clinical resistance to the thymidine analogs and cross resistance to ABC, ddI and TDF than do the type II TAMs (Cozzi-Lepri et al., 2005;De Luca et al., 2007;Lanier et al., 2004a;Miller et al., 2004). The clinical significance of the type II TAMs is not well understood.

Point mutations associated with resistance to NNRTIs
The NNRTIs block RT activity allosterically by binding to a hydrophobic pocket close to but not contiguous with the polymerase active site (Figure 3). The NNRTI-binding pocket (NNIBP), which exists only in bound RT (Kohlstaedt et al., 1992a;Ren et al., 1995a), consists of the following amino acid residues: L100; K101; K013; V106; T107; V108; V179, Y181; Y188; V189; G190; F227; W229; L234; Y318 and E138 ( Figure 3). Nearly all the of the NNRTI resistance mutations are within the NNIBP or adjacent to residues in the pocket Sarafianos et al., 2004). The NNRTI resistant mutations are broadly classified as: (a) primary mutations that cause high-level resistance to one or more NNRTIs and are the first to develop during the therapy; (b) secondary mutations that usually occur in combination with primary NNRTI resistance mutations (c) minor nonpolymorphic mutations that may occur alone or in combination with other NNRTI resistance mutations and (d) polymorphic accessory mutations that modulate the effects of other NNRTI resistance mutations (Shafer & Schapiro, 2008). There is low genetic barrier to NNRTI resistance. Only one or two mutations are required for high level resistance. Red shows residues where primary point mutations which cause resistance to nearly all the current NNRTIs occur. Green shows a residue where a major secondary mutation which causes resistance to EFV occurs. Blue shows unique residues which confers resistance to ETR when mutated. Yellow shows residues where miscellaneous mutations that cause resistance to at least one of the NNRTI may occur.
The most frequently observed primary point mutations in patients treated with approved NNRTIs are K103N and Y181C (Table 3). Each of the primary NNRTI resistance mutations, namely, K103N/S; V106A/M; Y181C/I/V, Y188L/C/H and G190A/S/E ( Figure 3 and Table 3) cause high level resistance to nevirapine (NVP) and variable resistance to efavirenz (EFV), ranging from about 2-fold for V106A and Y181C; 6-fold for G190A; 20-fold for K103N; and more than 50-fold for Y188L and G190S (Bacheler et al., 2001;Rhee et al., 2006b). Transient virologic responses to EFV-based salvage therapy regimen occur in some NNRTIexperienced patients but a sustained response is not common (Antinori et al., 2002;Delaugerre et al., 2001;Shulman et al., 2000;Walmsley et al., 2001). On the other hand, patients with any of the NNRTI resistance primary point mutations ( Figure 3 and Table 3) may benefit from etravirine (ETR) salvage therapy (Lazzarin et al., 2007;Madruga et al., 2007). However, point mutations at Y181 and to a lesser extent G190 compromise ETR response and may provide the foundation for the development of high-level ETR resistance (Lazzarin et al., 2007;Madruga et al., 2007). Major secondary and minor/miscellaneous mutations that usually occur in combination with one of the primary NNRTI resistance point mutations are depicted in Figure 3 and their respective effects on NNRTIs is summarized in Table 3.
Current understanding suggests that there are at least three classes of NNRTI-resistance mechanisms (Sarafianos et al., 2009). The first mechanism involves loss or change of key hydrophobic amino acid residues in the NNIBP such as Y181, Y188 and F227 ( Figure 3). Specific point mutations in one of these hydrophobic residues cause significant resistance through the loss of the aromatic ring interactions with NNRTIs (Das et al., 1996;Kohlstaedt et al., 1992a;Ren et al., 1995b;Ren et al., 2001;Ren et al., 2004). This causes high levels of resistance to the first generation NNRTIs, which are relatively rigid (Sarafianos et al., 2009). Second generation and more advanced NNRTI are designed with an inherent flexibility. This intrinsic flexibility called the wiggling and jiggling makes the second and subsequent generation NNRTIs to have compensatory interactions with RT that has mutations causing resistance to first generation NNRTIs (Das et al., 2008;Das et al., 2004). The second mechanism of NNRTI-resistance point mutations involves steric hindrance by amino acid residues in the central region of the NNIBP such as L100 and G190 ( Figure 3). The L100I point mutation confers high-level resistance to all the current NNRTIs (Table 3) by changing the shape of the pocket (Ren et al., 2004). In contrast G190A point mutation, which also confers high-level resistance to almost all the current NNRTIs (Table 3), causes a bulge (Sarafianos et al., 2004). The third and last mechanism of NNRTI-resistance point mutation involves pocket entrance mutations at the rim of the NNIBP such as K103N and K101E ( Figure 3). These point mutations cause resistance to first generation NNRTIs by interfering with the entry of NNRTIs into the NNIBP pocket (Hsiou et al., 2001;Ren et al., 2007). Second generation NNRTIs were designed to circumvent this problem. For instance, diarylpyrimidine NNRTIs such as ETR are able to inhibit HIV-1 isolates with K103N point mutation because they interact with the side chain of the mutated N103 residue Janssen et al., 2005).

NRTI Primary mutations Major secondary mutations
Minor and miscellaneous mutations *Denotes position of consensus amino acids that confer resistance to NNRTIs when mutated. Dashes (-) indicate that the respective NNRTIs are not affected by the mutation(s). All amino acids are denoted by the single letter annotation. Mutations in bold are associated with high levels of phenotypic resistance or reduced virologic response. Modified from (Shafer & Schapiro, 2008).

Point mutations associated with resistance to protease inhibitors
Resistance mutations in the protease genes are classified as major or minor (V. A. Johnson et al., 2010). Major mutations are defined as those selected first in the presence of the protease inhibitor (PI) antiretroviral drug or those substantially reducing drug susceptibility. These mutations tend to be the primary contact residues for drug binding (Figure 4). Major PI resistance mutations that are of the most clinical significance occur at 17 largely nonpolymorphic positions (Figure 4). Mutations at 13 of these 17 positions, including mutations at the substrate cleft positions L23, D30, V32, I47, G48, I50, V82 and I84; the flap positions M46 and I54; and the interior enzyme positions L76, N88 and L90 (Figure 4), reduce susceptibility to at least one PI (Table 4). Mutations at the other 4 positions (L24, L33, F53 and G73) are also important because they are common, non-polymorphic and cause resistance to several PI (Rhee et al., 2006b).

Point mutations associated with resistance to integrase inhibitors
The HIV-1 integrase catalyses the ligation of the viral 3-OH end to the 5-DNA of host chromosomal DNA through a process called the strand transfer (A. A. Johnson et al., 2006;Pommier et al., 2005 Figure 5). Some of the INI resistance mutations decrease susceptibility by themselves, whereas others compensate for the decrease fitness associated with other INI resistance mutations (Lataillade et al., 2007). There is a high level of cross-resistance between raltegravir and elvitegravir, as well as between these INI and the first generation of strand-transfer inhibitors, suggesting that the development of non cross-resistant INI will be challenging (Hombrouck et al., 2008;Shimura et al., 2008).
Common INI resistance point mutations are N155H and Q148H/R/K (Table 5), each of which can reduce raltegravir susceptibility by up to 25-fold (Cooper et al., 2008). Higher levels of raltegravir resistance occur with accumulation of additional mutations. E92Q mutation and the two polymorphic mutations L74M and G163R generally occur with N155H, while G140A/S generally occurs with Q148H/R/K (Cooper et al., 2008). Additional mutations (listed in Table  4) have been reported as being selected either in vitro or in vivo by raltegravir and include the non-polymorphic L74R, E138A/K, Y143R/C/H, N155S, H183P, Y226D/F/H, S230R, and D232N and the polymorphic mutations T97A and V151I (Delelis et al., 2011;Malet et al., 2008;Mouscadet et al., 2009). For both raltegravir and elvitegravir, virologic failure has generally been accompanied by 100-fold or greater decreases in susceptibility and the development of two or more INI resistance mutations. Mutations at amino acid residues E92, F121, G140, Q148 and N155 are associated with more than 10-fold decrease in susceptibility to both INI, whereas mutations at amino acid residues T66 and S147 are associated with marked decreases in susceptibility only to elvitegravir ( Figure 5 and Table 5).

Major mutations
*Denotes position of consensus amino acids that confer resistance to PI when mutated. Dashes (-) indicate that the respective PI are not affected by the mutation(s). All amino acids are denoted by the single letter annotation. Mutations in bold are associated with high levels of phenotypic resistance or reduced virologic response. Mutations in bold underline are relative contraindications to the use of specific PI. ATVr (atazanavir/r); DRVr (darunavir/r); FPVr (fosamprevanir); IDVr (indinavir/r); NFV (nelfinavir); SQVr (saquinavir/r); TPVr (tipranavir/r). Modified from (Shafer & Schapiro, 2008).

Point mutations associated with resistance to fusion inhibitors
The HIV-1 gp41 mediates the fusion between the virus and host cell membrane. Fusion inhibitors act by a dominant negative mechanism of action, preventing formation of the sixhelix bundle that is the driving force for the fusion process. Enfurvirtide, also known as T20 (Kilby et al., 1998;Wild et al., 1994), is the only FDA approved fusion inhibitor to date. Enfurvirtide has antiviral activity similar to most other classes of antiretroviral drugs discussed above; however resistance develop rapidly in patients receiving the drug for salvage therapy who do not receive a sufficient number of RTI, PI and INI drugs such as efavirenz, lopinanir/r and raltegravir, respectively. The emergence of enfurvirtide resistant strains followed by virologic rebound has been observed in some patients within two to four weeks (Cabrera et al., 2006;Lu et al., 2006).
Mutations in gp41 codons 36 to 45 (Figure 6), the region to which enfurvirtide binds, are primarily responsible for resistance to the drug (Marcelin et al., 2004c;Melby et al., 2006;Menzo et al., 2004;Mink et al., 2005;Sista et al., 2004;Su et al., 2006). The most common and potent T20-resistance mutations in this region are G36D/E, V38E/A, Q40H and N43D/K/S (Table 6). A single point mutation is generally associated with about 10-fold decreased susceptibility; whereas double point mutations can decrease susceptibility by more than 100-fold (Charpentier et al., 2011;Su et al., 2006;Xu et al., 2005). The replication fitness of recombinant viruses carrying the enfurvirtide-resistant mutations is relatively poor  and the mutant viruses rapidly revert to wild type phenotype in patients who discontinue the drug (Deeks et al., 2007). Some enfurvirtide-resistant mutations, particularly V38A/E mutations, are associated with significant CD4 count increases (Aquaro et al., 2006), presumably because mutations at residue V38 decrease HIV-1 replication or render the virus more susceptibility to a subset of neutralizing antibodies that target fusion intermediates (Reeves et al., 2005).

Fusion inhibitor Mutations
*Denotes position of consensus amino acids that confer resistance to enfuvirtide when mutated. All amino acids are denoted by the single letter annotation. Mutations in bold are associated with more 10fold phenotypic resistance or reduced virologic response in most clinical isolates. Modified from (Shafer & Schapiro, 2008). Table 6. Mutations in the gp41 env gene associated with resistance to fusion inhibitors

Point mutations associated with resistance to attachment inhibitors
Despite the important role gp120 plays in attachment of HIV-1 to the CD4 receptor and CCR5/CXCR4; maraviroc, which is a CCR5 antagonist, is the only FDA approved attachment inhibitor in clinical use to date. While gp120 binds to the N-terminus and the second extracellular loop region of CCR5 (Hartley et al., 2005), site-directed mutagenesis study suggests that small molecule attachment inhibitors such as maraviroc bind to a CCR5 pocket formed by the transmembrane helices (Dragic et al., 2000). Thus, although gp120 and maraviroc do not bind to the same site of the CCR5 coreceptor, maraviroc allosterically inhibit gp120 binding to the CCR5 coreceptor. Point mutations in gp120 that allow the virus to bind to the maraviroc-bound form of CCR5 in the drug insensitive manner have been described (Westby et al., 2007). Most of these mutations are found in the V3 loop of gp120 ( Figure 7A), which determines HIV-1 tropism (discussed in the next section). One study showed that reverse mutations at positions 316 and 323 in the V3 loop of maraviroc-resistant HIV-1 variants to their original sequence restored wild-type susceptibility to maraviroc, www.intechopen.com while reversion of either mutation resulted in a partially sensitive virus with reduced maximal inhibition (Westby et al., 2007). In another recent study maraviroc-resistant variants were isolated from the V3 loop library virus (HIV-1(V3lib)) containing the T199K and T275M plus 5 mutations in the V3 loop, namely I304V; F312W; T314A; E317D; I318V (Yuan et al., 2011). Furthermore, the same study showed that the profile of HIV-1(JR-FL) pseudotype containing I304V/F312W/T314A/E317D/I318V ( Figure 7A) in the V3 loop alone revealed a typical non-competitive resistance to maraviroc (Yuan et al., 2011). Generally, amino acids mutations in the V3 loop may include known polymorphism as well as novel substitutions, insertions, and deletions. Further complicating the genetic basis of maraviroc or CCR5 inhibitor resistance is the observation that the same inhibitor may select for different mutations in different HIV-1 isolates (Westby et al., 2007). The mutations observed in maraviroc-resistant strains isolated from patients are variable and differed for each HIV-1 isolate (Seclen et al., 2011;Tilton et al., 2011). Fig. 7. Illustration of amino acid residues in the V3 loop important for HIV-1 tropism and resistance to maraviroc. (A) The sequence of the V3 variable loop indicating the sites of unique X4 substitution sites when compared to R5 clones (underlined). The red residues indicate the positions where maraviroc resistance mutations occur. Together with T199K and T275M, these mutations confer complete resistance to maraviroc. (B) Ribbon diagram of gp120 V3 variable loop highlighting amino acid residues essential for co-receptor usage. X4 tropism is determined by positively charged residues at position 11, 24 and 25 (colour coded blue, green and magenta respectively). R5 tropism is determined by a negative or neutral residue at any of the three positions.

Point mutations associated with HIV-1 tropism, transmission dynamics and AIDS pathogenesis
The identification of CCR5 and CXCR4 chemokine receptors as having critical roles in the cellular entry of HIV-1 allowed development of a more precise system for defining the viral tropism. Prototype T-cell tropic strains use CXCR4, macrophage-tropic strains use CCR5, and dual-tropic strains use both CXCR4 and CCR5 chemokine receptors, culminating in the currently used nomenclature X4, R5 and R5X4 strains, respectively (Berger et al., 1998). The phenotype of HIV-1 that is transmitted in vivo is R5 even when the contaminating virus is a mixture of R5, R5X4 and X4. The reason for this selection of viral phenotype during transmission is not well understood. The viraemia present during primary infection consists of oligoclonal R5 virus, and this virus type generally persists during the asymptomatic phase of the infection until the onset of AIDS. Viral transmission and disease progression, collectively called AIDS pathogenesis, corresponds to evolution of coreceptor phenotype. In about half of all HIV-1 infections there is viral evolution towards dual (R5X4) tropism and CXCR4 usage that is prognostic for accelerated progression to AIDS (Collman et al., 1992;Doranz et al., 1996). Thus when the phenotype switch occurs, there is an initial evolution from CCR5 using viruses to those that can use both CCR5 and CXCR4 (Connor et al., 1997). While evolution towards usage of the CXCR4 coreceptor and emergence of X4 viruses coincides with a decline in CD4 + T cells and the onset of AIDS, the dominant theory is that X4 viruses are opportunistic infections that can only appear once the CD4 + T cells have dropped below a certain threshold level. Notwithstanding, in another 50% of individuals infected with HIV-1 there is no viral evolution towards CXCR4 usage or emergence of X4 viruses; R5 viruses persists from primary infection until one succumbs to AIDS. This suggests that R5 viruses are necessary and sufficient to cause AIDS.
The main determinants for coreceptor tropism resides in the V3 loop (Figure 7), although mutations outside of the V3 loop may also influence tropism, either in combination or independently of the V3 mutations (Hartley et al., 2005;W. Huang et al., 2008;Pastore et al., 2007;Pastore et al., 2006). Very few mutations in the V3 loop are sufficient for switching coreceptor use from CCR5 to CXCR4 in most HIV-1 isolates, but in some isolates additional mutations in V1/V2 (Pastore et al., 2006) and gp41 (W. Huang et al., 2008) are observed during coreceptor switching. The presence of positively charged amino acids at positions 11, 24 and 25 in the V3 loop, combined with other V3 sequence characteristics (Figure 7), have specificity of about 90% and sensitivity of 70-80% for predicting X4 tropism (Hartley et al., 2005;Jensen & van 't Wout, 2003;Sing et al., 2007). However, the number and type of mutations by which an R5 virus switches to X4 is complex and depends on the sequence of the baseline R5 virus Moncunill et al., 2008;Pastore et al., 2007;Pastore et al., 2006). The frequency and genetic basis for tropism switch is different for different HIV-1 subtypes (Hartley et al., 2005;. While differential use of CXCR4 or CCR5 by HIV-1 strains has been observed, the striking differences seen appear to be in how R5 and R5X4 viruses utilize the CCR5 coreceptor. Generally, R5-tropic viruses are particularly well adapted for CCR5 use Deng et al., 1996;Dragic et al., 1996), and can tolerate deletion or substitution of the CCR5 amino-terminal domain (Howard et al., 1999;Z. Wang et al., 1999). R5X4 viruses, however, are much more sensitive to mutations in CCR5, particularly in the amino terminal domain (Doranz et al., 1997). Thus, there is a cost associated with the benefit to use dual chemokine receptors as exhibited by R5X4 tropic strains that have more stringent requirements for CCR5 usage (Rana et al., 1997).
Host genetic factors can also influence HIV-1 tropism and transmission. Cohorts of HIV-1 infected individuals have been established based on specific clinical, virological and immunological criteria. One type of cohort contains individuals selected after frequent www.intechopen.com exposure to HIV-1 without seroconversion. A mutation in the CCR5 gene has been frequently found in this type of cohort (Zimmerman et al., 1997). This mutation is a deletion of 32 base pairs (Δ32), resulting in a defective CCR5 receptor. CD4 + T cells and macrophages carrying the Δ32 mutation are resistant to infection with R5 viruses. Homozygous individuals carrying the mutation on both alleles (1% of Caucasians) are almost completely refractory to infection with R5 HIV-1 strains Zimmerman et al., 1997). Very rare examples of infection of these individuals have been reported (O'Brien et al., 1997;Theodorou et al., 1997). In these cases the transmitted R5 viruses appear to use CXCR4. Individuals heterozygous for the Δ32 mutation are not protected from HIV-1 infection, but have slower rate of progression to AIDS. This is because the mutated form of the protein acts as a dominant negative mutant and prevents most of the wild-type CCR5 protein from reaching the cell surface. However, HIV-1 can evolve adaptive mutations on the V3 loop of gp120 that allows it to use defective or mutant CCR5 co-receptor. For instance, it has been shown that a N300Y adaptive point mutation on the V3 loop of gp120 enhances binding of HIV-1 to the mutant CCR5 coreceptor (Platt et al., 2001). A follow-up study from the same group has shown that in addition to the adaptive mutation on the V3 loop of gp120, an S193N point mutation on the V2 stem and loss of N-linked oligosaccharide from position N403 of gp120 help HIV-1 to efficiently use defective CCR5 co-receptor that has a badly damaged amino terminus (Platt et al., 2005). Taken together, these studies have important implications for understanding HIV-1 transmission dynamics and AIDS pathogenesis.
AIDS pathogenesis is multifactorial; it is a mixture of host and pathogen genetics combined with factors such as the immune response and viral adaptation. Viral adaptation include reverting and compensatory mutations such as the recently described T242N point mutation on the gag gene associated with progression to AIDS in South African patients (K. H. Huang et al., 2011). However in general, progression from acute infection is often accompanied by depletion of the CD4 + lymphocytes and this depletion is a major component of the eventual failure of the immune system and hence AIDS pathogenesis. Apoptosis is one mechanism that contributes to depletion of HIV-1 infected and uninfected cells, deterioration of the immune system and progression to AIDS, respectively (Cotton et al., 1997;Gougeon & Montagnier, 1993;Herbein et al., 1998;Joshi et al., 2011;C. J. Li et al., 1995;Zhu et al., 2011). HIV-induced apoptosis is triggered by a number of HIV-1 proteins, notably the envelope protein (Herbein et al., 1998;Ishikawa et al., 1998;Joshi et al., 2011;Micoli et al., 2006;Micoli et al., 2000). Four point mutations (A835W, A838W, A838I, and I842R) in the cytoplasm domain of the gp41 subunit of the envelope protein reduces HIV-induced apoptosis of target cells (Micoli et al., 2006). A recent study has shown that induction of bystander apoptosis in CCR5 expressing cells requires the fusogenic activity of gp41; and that V2E and V38A/E point mutation in the second amino acid and the heptad repeat 1 region of gp41 respectively, abrogated fusion activity and hence apoptosis induced by an R5 virus (Joshi et al., 2011). In an earlier study, the same group showed that a V513E point mutation in the fusion domain of gp41 resulted in a fusion-defective envelope protein that failed to induce apoptosis and that a G547D mutation in the gp41 N-terminal helix also reduced cell fusion capacity and apoptosis induced by an X4 virus (Garg et al., 2007).

Point mutations associated with evasion of antibody mediated immune response
Point mutations on HIV-1 envelope protein do not only influence HIV-1 tropism and disease progression, but they also help the virus to evade the antibody mediated immune response. The HIV-1 surface gp120 and the trans-membrane gp41 can elicit both virus-neutralizing and non-neutralizing antibodies during natural infection. Antibodies that lack neutralizing activity are often directed against the gp120 regions that are occluded on the assembled trimer, and which are exposed only upon shedding (Moore & Sodroski, 1996). Neutralizing antibodies, by contrast must access the functional envelope glycoprotein complex and typically recognize conserved or variable epitopes near the receptor binding sites (Posner et al., 1991;Sattentau & Moore, 1995;Trkola et al., 1996a;. Neutralising antibodies are also elicited by the membrane proximal external region (MPER) of gp41 (Frey et al., 2008;J. Wang et al., 2011;Zwick et al., 2001). The production and characterization of monoclonal antibodies of human and rodent origin have allowed a precise determination of the neutralization epitopes present on the HIV-1 gp120 (Moore & Ho, 1995;Poignard et al., 1996).
Using epitope maps in conjunction with the X-ray crystal structure of gp120 complexed with CD4 and a neutralizing monoclonal antibody (Kwong et al., 1998b), spatial organization of conserved neutralization epitopes on gp120 was determined (Wyatt et al., 1998). More broadly neutralizing antibodies recognize discontinuous, conserved epitopes in three regions of the gp120 (Figure 8). In HIV-1 infected individuals, the most abundant of these are directed against the CD4 binding site (CD4bs), and block gp120-CD4 interaction (Euler et al., 2011;Ho et al., 1991;Y. Li et al., 2011;Posner et al., 1991;Walker et al., 2009;Zhou et al., 2011). Less common are antibodies against epitopes induced or exposed upon CD4 binding (CD4i) (Thali et al., 1993). Both CD4i and V3 antibodies disrupt the binding of gp120-CD4 complexes to chemokine receptors (Trkola et al., 1996a;. Notwithstanding, none of the CD4i antibodies discovered to date such as 17b, 48d or X5 are broadly neutralizing. A third gp120 neutralization epitope is defined by a unique monoclonal antibody, 2G12 (Trkola et al., 1996b), which does not efficiently block receptor binding (Trkola et al., 1996a). Table 6 lists broadly neutralising monoclonal antibodies elicited by the HIV-1 gp120 and gp41 envelope proteins. Although high-titer antibodies are sustained in infected individuals throughout the course of infection, these antibodies have very poor neutralizing activity against autologous as well as representative primary HIV-1 isolates (Montefiori et al., 1996;Moog et al., 1997). This observation suggests that neutralizing antibodies that arise in response to HIV-1 infection may not be critical in limiting viral replication.
The escape of HIV-1 from effects of neutralizing antibodies includes mutations in gp120 and/or gp41. One of the seminal studies showed that A582T point mutation in the HIV-1 envelope gene made a laboratory adapted HIV-1 strain called HXB2 to be resistant to HIV-1 neutralizing human serum (Reitz et al., 1988). Another study published ten years later, showed that HIV-1 MN molecular clones which had 294N/K mutation before the V3 loop, 307N/I mutation proximal to the V3 loop, 327I/K mutation within V3 and 336N/I mutation after the V3 loop were resistant to human serum containing neutralizing antibodies directed at the immunodominant V3 loop of gp120 (Park et al., 1998). Point mutations in a highly conserved structural motif within the intracytoplasmic tail of gp41 rendered HIV-1 molecular clones resistant to broadly neutralizing polyclonal human serum antibodies (Kalia et al., 2005). Fig. 8. Ribbon diagram illustrating epitopes of the gp120 core, highlighting some amino acid residues that interact with broadly neutralizing antibodies. The CD4bs (red) and the CD4i co-receptor-binding surface (green) as well as residues implicated in 2G12 binding (blue) are depicted.
In a clinically relevant study, paediatric HIV-1 subtype C clinical isolates with K665S/R/N point mutation were found to be resistant to a 2F5 broadly neutralizing monoclonal antibody (Gray et al., 2006). This study confirmed earlier findings that the amino acid residue K665 is crucial for neutralization by 2F5 (Binley et al., 2004). In the same study, the Lynn Morris' group found out that all the paediatric HIV-1 subtype C clinical isolates analysed had a conserved 4E10 epitope (W672, F673, W680) consistent with their phenotypic sensitivity to the 4E10 monoclonal antibody (Gray et al., 2006). 4E10 recognizes an epitope containing the sequence NWF(D/N)IT (Zwick et al., 2001). Mutagenesis studies have shown that the amino acid residues W672, F673 and W680 are indispensable for recognition by 4E10 (Zwick et al., 2005). Indeed, a recently published study describing four subjects infected with viruses carrying rare MPER polymorphisms associated with resistance to 4E10 neutralization has shown that three subjects had W680 polymorphism, including a W680G point mutation (Nakamura et al., 2011). The study demonstrated that W680G point mutation was necessary to confer 4E10 resistant phenotype (Nakamura et al., 2011). In another subject a W680R point mutation caused variable resistant to 4E10 (Nakamura et al., 2011). A fourth subject possessed a F673L point mutation also associated with 4E10 resistance (Nakamura et al., 2011). Earlier, an independent study conducted in South Africa also showed that an HIV-1 subtype C virus isolated from a 7-year-old perinatally infected child with F673L point mutation was resistant to 4E10 (Gray et al., 2008). These case studies are examples of the challenges presented by point mutations in controlling HIV-1 infection by antibodies. Notwithstanding, recent discovery of additional and more potent broadly neutralizing antibodies such as VRC01 (X. Wu et al., 2010), PG9 and PG16 (Walker et al., 2009) is encouraging and boosts the impetus for HIV-1 vaccine design that may elicit broadly neutralizing antibodies (Burton & Weiss, 2010). Interfere with gp120-CD4 binding CD4bs antibodies compete with CD4 and with antibodies against CD4i epitope. PG9 and PG16 bind to trimer specific glycosylated epitope on the V1/V2 and V3 region. (Corti et al., 2010;Euler et al., 2011;Ho et al., 1991;Y. Li et al., 2011;Pancera et al., 2010;Posner et al., 1991;Walker et al., 2009; Glycans 2G12 Interfere with binding of gp120 to CCR5 chemokine receptor and DC-SIGN. Also prevent loss of CD4 + T cells. Antibody binding is dependent upon proper N-linked glycosylation. Recognize a mannose rich epitope on the "silent" face of gp120 (Binley et al., 2006;Calarese et al., 2003;Luo et al., 2011;Scanlan et al., 2003;Trkola et al., 1996b) MPER of gp41

Point mutations and anti-gp120 aptamers
Aptamers are synthetic oligonucleotide ligands that can be isolated in vitro against diverse targets including HIV-1 gp120 (Khati et al., 2003). Aptamers assume a defined threedimensional structure and generally bind functional sites on their respective targets. They possess the molecular recognition properties of monoclonal antibodies in terms of their high affinity and specificity (Khati, 2010). Unlike antibodies, aptamers can fold properly and retain activity in the intracellular environment. However, the majority of aptamers with potential therapeutic utility selected to date target extracellular proteins (Chen et al., 2003;Eyetech Study Group, 2002, 2003Green et al., 1995;Kim et al., 2003;Liu et al., 2009;Lupold et www.intechopen.com Fig. 9. A ribbon diagram of gp120 highlighting amino acid residues that interact with B40 aptamer. Residues in bold and single asterix are highly conserved among all HIV-1 isolates and significantly reduced binding of the aptamer by more than 10-fold when experimentally mutated to alanine, respectively (Joubert et al., 2010). Those residues with double or tripple asterix are moderately and significantly varaiable, respectively, in all HIV isolates and while they make contact with the aptamer they did not significantly affect binding of the aptamer when experimentally mutated to alanine, respectively. Mi et al., 2009;Nobile et al., 1998;Ruckman et al., 1998;Rusconi et al., 2004;Rusconi et al., 2002;Vinores, 2003;White et al., 2003).
Extracellular therapeutic targets such as HIV-1 gp120 have the advantage of ready access to aptamer intervention without the need for enabling access to cells. The aptamers against HIV-1 gp120 bound the protein with high affinity, high specificity and neutralized a broad range of R5 HIV-1 clinical isolates (Khati et al., 2003). In a recent review, these RNA aptamers were reported to have the most potent in vitro antiviral efficacy of all HIV-1 entry inhibitors described to date (Held et al., 2006), including antibodies. These aptamers prevented entry and suppressed viral replication in cultured human peripheral blood mononuclear cells (PBMC) by up to 10,000-fold (Khati et al., 2003). While the emergence of HIV-1 escape mutants and drug resistance variants appears to be inevitable, our goup and collaborators have recently shown that one extensively studied aptamer called B40 penetrate the highly variable exterior surfaces of gp120 and bind the conserved core at the heart of the CCR5-binding site (Cohen et al., 2008;Dey et al., 2005;Joubert et al., 2010), which the virus may be unable to mutate without compromising its fitness. Four amino acids (Q114, K117, K207, and I439) of gp120 ( Figure 9) that weakened binding of gp120 to B40 aptamer by at least 10-fold when experimentally mutated to alanine (Joubert et al., 2010) are naturally conserved among all HIV-1 isolates (Kwong et al., 1998a). This suggests that B40 makes direct contact with at least four conserved core residues on gp120 within the CCR5-binding site, which the virus cannot afford to mutate without losing selective advantage. These data bode well for the future development of aptamers such as B40 as an entry inhibitor drug, which will hopefully help circumvent HIV-1 point mutations and delay or diminish drug resistance. In order to maximally suppress or eradicate the virus; the aptamer or any other anti-HIV molecule in development should be used in combination with current antiretroviral drugs and the envisaged vaccines currently developed to elicit broadly neutralising antibodies.