HIV introduction Human immunodeficiency virus (HIV) is a lentivirus [REF] (a member of the retrovirus family [REF]) that causes

acquired immunodeficiency syndrome (AIDS), [1,2] a condition in humans in which progressive failure of the immune system allows life-threatening opportunistic infections and cancers to thrive. Infection with HIV occurs by the transfer of blood, semen, vaginal fluid, pre-ejaculate, or breast milk. Within these bodily fluids, HIV is present as both free virus particles and virus within infected immune cells. The four major routes of transmission are unsafe sex, contaminated needles, breast milk, and transmission from an infected mother to her baby at birth (perinatal transmission) [REF]. Screening of blood products for HIV has largely eliminated transmission through blood transfusions or infected blood products in the developed world [REF]. HIV infection in humans is considered pandemic by the World Health Organization (WHO) [3]. Nevertheless, complacency about HIV may play a key role in HIV risk [4,5]. From its discovery in 1981 to 2006, AIDS killed more than 25 million people [6]. HIV infects about 0.6% of the world's population [6]. In 2009, AIDS claimed an estimated 1.8 million lives, down from a global peak of 2.1 million in 2004 [7]. Approximately 260,000 children died of AIDS in 2009 [7]. A disproportionate number of AIDS deaths occur in Sub-Saharan Africa, retarding economic growth and exacerbating the burden of poverty [8]. An estimated 22.5 million people (68% of the global total) live with HIV in sub-Saharan Africa, which is also home to 90% of the world's 16.6 million children orphaned by HIV [7]. Treatment with antiretroviral drugs reduces both the mortality and the morbidity of HIV infection [9]. Although antiretroviral medication is still not universally available, expansion of antiretroviral therapy programs since 2004 has helped to turn the tide of AIDS deaths and new infections in many parts of the world [7]. Intensified awareness and preventive measures, as well as the natural course of the epidemic, have also played a role. Nevertheless, an estimated 2.6 million people were newly infected in 2009 [7].

HIV infects vital cells in the human immune system such as helper T cells (specifically CD4+ T cells), macrophages, and dendritic cells [10]. HIV infection leads to low levels of CD4+ T cells through three main mechanisms: First, direct viral killing of infected cells; second, increased rates of apoptosis in infected cells; and third, killing of infected CD4+ T cells by CD8 cytotoxic lymphocytes that recognize infected cells. When CD4+ T cell numbers decline below a critical level, cell-mediated immunity is lost, and the body becomes progressively more susceptible to opportunistic infections [REF]. Most untreated people infected with HIV-1 eventually develop AIDS [11]. These individuals mostly die from opportunistic infections or malignancies associated with the progressive failure of the immune system [12]. HIV progresses to AIDS at a variable rate affected by viral, host, and environmental factors; most will progress to AIDS within 10 years of HIV infection: some will have progressed much sooner, and some will take much longer [13,14]. Treatment with anti-retrovirals increases the life expectancy of people infected with HIV. Even after HIV has progressed to diagnosable AIDS, the average survival time with antiretroviral therapy was estimated to be more than 5 years as of 2005 [15]. Without antiretroviral therapy, someone who has AIDS typically dies within a year [16].

Historical overview Discovery AIDS was first clinically observed between in 1981 in the United States. 17 Injection drug users and gay men with no known cause of impaired immunity showed symptoms of Pneumocystis carinii pneumonia (PCP), a rare opportunistic infection that was known to present itself in people with very compromised immune systems.18-20 Soon thereafter, additional gay men developed a previously rare skin cancer called Kaposi's sarcoma (KS).21-22 Many more cases of PCP and KS quickly emerged, alerting U.S. Centers for Disease Control and Prevention (CDC). A CDC task force was formed to monitor the outbreak. After recognizing a pattern of

anomalous symptoms presenting themselves in patients, the task force named the condition acquired immune deficiency syndrome (AIDS).23 In 1983, two separate research groups led by Robert Gallo and Luc Montagnier independently declared that a novel retrovirus may have been infecting AIDS patients, and published their findings in the same issue of the journal Science. 24, 25 Gallo claimed that a virus his group had isolated from an AIDS patient was strikingly similar in shape to other human T-lymphotropic viruses (HTLVs) his group had been the first to isolate. Gallo's group called their newly isolated virus HTLV-III. At the same time, Montagnier's group isolated a virus from a patient presenting lymphadenopathy (swelling of the lymph nodes) of the neck and physical weakness, two classic symptoms of AIDS. Contradicting the report from Gallo's group, Montagnier and his colleagues showed that core proteins of this virus were immunologically different from those of HTLV-I. Montagnier's group named their isolated virus lymphadenopathy-associated virus (LAV).23 HIV was chosen as a compromise between the two claims (LAV and HTLV-III). Whether Gallo or Montagnier deserve more credit for the discovery of the virus that causes AIDS has been a matter of considerable controversy. Together with his colleague Franoise Barr-Sinoussi, Montagnier was awarded one half of the 2008 Nobel Prize in Physiology or Medicine for his "discovery of human immunodeficiency virus".26 Harald zur Hausen also shared the Prize for his discovery that human papilloma virus leads to cervical cancer, but Gallo was left out.27 Gallo said that it was "a disappointment" that he was not named a corecipient.28 Montagnier said he was "surprised" Gallo was not recognized by the Nobel Committee: "It was important to prove that HIV was the cause of AIDS, and Gallo had a very important role in that. I'm very sorry for Robert Gallo." 27

Origins HIV is thought to have originated in non-human primates in sub-Saharan Africa and was transferred to humans late in the 19th or early in the 20th century. 29-31 The

first paper recognizing a pattern of opportunistic infections characteristic of AIDS was published in 1981.32 Both HIV-1 and HIV-2 are believed to have originated in West-Central Africa and to have jumped species (a process known as zoonosis) from non-human primates to humans. HIV-1 appears to have originated in southern Cameroon through the evolution of SIV(cpz), a simian immunodeficiency virus (SIV) that infects wild chimpanzees (HIV-1 descends from the SIV(cpz) endemic in the chimpanzee subspecies Pan troglodytes troglodytes).33,
34

The closest relative of HIV-2 is

SIV(smm), a virus of the sooty mangabey (Cercocebus atys atys), an Old World monkey living in litoral West Africa (from southern Senegal to western Ivory Coast).35 New World monkeys such as the owl monkey are resistant to HIV-1 infection, possibly because of a genomic fusion of two viral resistance genes. 36 HIV-1 is thought to have jumped the species barrier on at least three separate occasions, giving rise to the three groups of the virus, M, N, and O.37 There is evidence that humans who participate in bushmeat activities, either as hunters or as bushmeat vendors, commonly acquire SIV.38 However, SIV is a weak virus, it is typically suppressed by the human immune system within weeks of infection. It is thought that several transmissions of the virus from individual to individual in quick succession are necessary to allow it enough time to mutate into HIV.39 Furthermore, due to its relatively low person-to-person transmission rate, it can only spread throughout the population in the presence of one or more of highrisk transmission channels, which are thought to have been absent in Africa prior to the 20th century. Specific proposed high-risk transmission channels, allowing the virus to adapt to humans and spread throughout the society, depend on the proposed timing of the animal-to-human crossing. Genetic studies of the virus suggest that the most recent common ancestor of the HIV-1 M group dates back to approximately 1910.40 Proponents of this dating link the HIV epidemic with the emergence of colonialism and growth of large colonial African cities, leading to social changes, including a higher degree of sexual promiscuity, the spread of prostitution, and the

concomitant high frequency of genital ulcer diseases (such as syphilis) in nascent colonial cities.41 There is evidence that transmission rates of HIV during vaginal intercourse, while quite low under regular circumstances, may be increased tens, if not hundreds of times, if one of the partners suffers from an STD resulting in genital ulcers. Early 1900s colonial cities were notable due to their high prevalence of prostitution and genital ulcer STDs, to the degree that, as of 1928, as many as 45% of female residents of eastern Kinshasa were thought to have been prostitutes, and, as of 1933, around 15% of all residents of the same city were infected by one of the forms of syphilis.41 An alternative view holds that unsafe medical practices in Africa during years following World War II, such as unsterile reuse of single use syringes during mass vaccination, antibiotic and anti-malaria treatment campaigns, were the initial vector that allowed the virus to adapt to humans and spread.39, 42, 43 The earliest well documented case of HIV in a human dates back to 1959. 44 The virus may have been present in the United States as early as 1966, 45 but the vast majority of infections occurring outside sub-Saharan Africa (including the U.S.) can be traced back to a single unknown individual who got infected with HIV in Haiti and then brought the infection to the United States sometime around 1969.46 The epidemic then rapidly spread among high-risk groups (initially, sexually promiscuous gay men). By 1978, the prevalence of HIV-1 among gay male residents of New York and San Francisco was estimated at 5%, suggesting that several thousand individuals in the country had been infected by then. 46

Virology of HIV

HIV is a member of the genus Lentivirus,47 part of the family of Retroviridae.48 Lentiviruses have many morphologies and biological properties in common. Many species are infected by lentiviruses, which are characteristically responsible for

long-duration illnesses with a long incubation period.49 Lentiviruses are transmitted as single-stranded, positive-sense, enveloped RNA viruses. Upon entry into the target cell, the viral RNA genome is converted (reverse transcribed) into doublestranded DNA by a virally encoded reverse transcriptase that is transported along with the viral genome in the virus particle. The resulting viral DNA is then imported into the cell nucleus and integrated into the cellular DNA by a virally encoded integrase and host co-factors.50 Once integrated, the virus may become latent, allowing the virus and its host cell to avoid detection by the immune system. Alternatively, the virus may be transcribed, producing new RNA genomes and viral proteins that are packaged and released from the cell as new virus particles that begin the replication cycle anew. Two types of HIV have been characterized: HIV-1 and HIV-2. HIV-1 is the virus that was initially discovered and termed both LAV and HTLV-III. It is more virulent, more infective,
51

and is the cause of the majority of HIV infections globally. The

lower infectivity of HIV-2 compared to HIV-1 implies that fewer of those exposed to HIV-2 will be infected per exposure. Because of its relatively poor capacity for transmission, HIV-2 is largely confined to West Africa.35

Structure and genome HIV is different in structure from other retroviruses. It is roughly spherical
52

with a

diameter of about 120 nm, around 60 times smaller than a red blood cell, yet large for a virus.53 It is composed of two copies of positive single-stranded RNA that codes for the virus's nine genes enclosed by a conical capsid composed of 2,000 copies of the viral protein p24.54 The single-stranded RNA is tightly bound to nucleocapsid proteins, p7, and enzymes needed for the development of the virion such as reverse transcriptase, proteases, ribonuclease and integrase. A matrix composed of the viral protein p17 surrounds the capsid ensuring the integrity of the virion particle.54

This is, in turn, surrounded by the viral envelope that is composed of two layers of fatty molecules called phospholipids taken from the membrane of a human cell when a newly formed virus particle buds from the cell. Embedded in the viral envelope are proteins from the host cell and about 70 copies of a complex HIV protein that protrudes through the surface of the virus particle.54 This protein, known as Env, consists of a cap made of three molecules called glycoprotein (gp) 120, and a stem consisting of three gp41 molecules that anchor the structure into the viral envelope.55 This glycoprotein complex enables the virus to attach to and fuse with target cells to initiate the infectious cycle.55 Both these surface proteins, especially gp120, have been considered as targets of future treatments or vaccines against HIV.56 The RNA genome consists of at least seven structural landmarks (LTR, TAR, RRE, PE, SLIP, CRS, and INS), and nine genes (gag, pol, and env, tat, rev, nef, vif, vpr, vpu, and sometimes a tenth tev, which is a fusion of tat env and rev), encoding 19 proteins. Three of these genes, gag, pol, and env, contain information needed to make the structural proteins for new virus particles.54 For example, env codes for a protein called gp160 that is broken down by a cellular protease to form gp120 and gp41. The six remaining genes, tat, rev, nef, vif, vpr, and vpu (or vpx in the case of HIV-2), are regulatory genes for proteins that control the ability of HIV to infect cells, produce new copies of virus (replicate), or cause disease.54 The two Tat proteins (p16 and p14) are transcriptional transactivators for the LTR promoter acting by binding the TAR RNA element. The TAR may also be processed into microRNAs that regulate the apoptosis genes ERCC1 and IER3. 57,
58

The Rev protein (p19) is involved in shuttling RNAs from the nucleus and the

cytoplasm by binding to the RRE RNA element. The Vif protein (p23) prevents the action of APOBEC3G (a cell protein that deaminates DNA:RNA hybrids and/or interferes with the Pol protein). The Vpr protein (p14) arrests cell division at G2/M. The Nef protein (p27) down-regulates CD4 (the major viral receptor), as well as the MHC class I and class II molecules.59-61

Nef also interacts with SH3 domains. The Vpu protein (p16) influences the release of new virus particles from infected cells.54 The ends of each strand of HIV RNA contain an RNA sequence called the long terminal repeat (LTR). Regions in the LTR act as switches to control production of new viruses and can be triggered by proteins from either HIV or the host cell. The Psi element is involved in viral genome packaging and recognized by Gag and Rev proteins. The SLIP element (TTTTTT) is involved in the frame shift in the Gag-Pol reading frame required to make functional Pol.54

Tropism The term viral tropism refers to which cell types HIV infects. HIV can infect a variety of immune cells such as CD4+ T cells, macrophages, and microglial cells. HIV-1 entry to macrophages and CD4+T cells is mediated through interaction of the virion envelope glycoproteins (gp120) with the CD4 molecule on the target cells and also with chemokine coreceptors.55 Macrophage (M-tropic) strains of HIV-1, or non-syncitia-inducing strains (NSI) use the -chemokine receptor CCR5 for entry and are, thus, able to replicate in macrophages and CD4+ T cells.62 This CCR5 coreceptor is used by almost all primary HIV-1 isolates regardless of viral genetic subtype. Indeed, macrophages play a key role in several critical aspects of HIV infection. They appear to be the first cells infected by HIV and perhaps the source of HIV production when CD4+ cells become depleted in the patient. Macrophages and microglial cells are the cells infected by HIV in the central nervous system. In tonsils and adenoids of HIVinfected patients, macrophages fuse into multinucleated giant cells that produce huge amounts of virus. T-tropic isolates, or syncitia-inducing (SI) strains replicate in primary CD4+ T cells as well as in macrophages and use the -chemokine receptor, CXCR4, for entry.6264

Dual-tropic HIV-1 strains are thought to be transitional strains of HIV-1 and thus

are able to use both CCR5 and CXCR4 as co-receptors for viral entry.

The -chemokine SDF-1, a ligand for CXCR4, suppresses replication of T-tropic HIV-1 isolates. It does this by down-regulating the expression of CXCR4 on the surface of these cells. HIV that use only the CCR5 receptor are termed R5; those that use only CXCR4 are termed X4, and those that use both, X4R5. However, the use of coreceptor alone does not explain viral tropism, as not all R5 viruses are able to use CCR5 on macrophages for a productive infection 62 and HIV can also infect a subtype of myeloid dendritic cells,65 which probably constitute a reservoir that maintains infection when CD4+ T cell numbers have declined to extremely low levels. Some people are resistant to certain strains of HIV. 66 For example, people with the CCR5-32 mutation are resistant to infection with R5 virus, as the mutation stops HIV from binding to this coreceptor, reducing its ability to infect target cells. Sexual intercourse is the major mode of HIV transmission. Both X4 and R5 HIV are present in the seminal fluid, which is passed from a male to his sexual partner. The virions can then infect numerous cellular targets and disseminate into the whole organism. However, a selection process leads to a predominant transmission of the R5 virus through this pathway.67-69 How this selective process works is still under investigation, but one model is that spermatozoa may selectively carry R5 HIV as they possess both CCR3 and CCR5 but not CXCR4 on their surface 70 and that genital epithelial cells preferentially sequester X4 virus. 71 In patients infected with subtype B HIV-1, there is often a co-receptor switch in late-stage disease and T-tropic variants appear that can infect a variety of T cells through CXCR4. 72 These variants then replicate more aggressively with heightened virulence that causes rapid T cell depletion, immune system collapse, and opportunistic infections that mark the advent of AIDS.73 Thus, during the course of infection, viral adaptation to the use of CXCR4 instead of CCR5 may be a key step in the progression to AIDS. A number of studies with subtype B-infected individuals have determined that between 40 and 50% of AIDS patients can harbor viruses of the SI and, it is presumed, the X4 phenotypes.74-75

HIV-2 is much less pathogenic than HIV-1 and is restricted in its worldwide distribution. The adoption of "accessory genes" by HIV-2 and its more promiscuous pattern of coreceptor usage (including CD4-independence) may assist the virus in its adaptation to avoid innate restriction factors present in host cells. Adaptation to use normal cellular machinery to enable transmission and productive infection has also aided the establishment of HIV-2 replication in humans. A survival strategy for any infectious agent is not to kill its host but ultimately become a commensal organism. Having achieved a low pathogenicity, over time, variants more successful at transmission will be selected.76

Replication cycle Entry to the cell HIV enters macrophages and CD4+ T cells by the adsorption of glycoproteins on its surface to receptors on the target cell followed by fusion of the viral envelope with the cell membrane and the release of the HIV capsid into the cell. 77-78 Entry to the cell begins through interaction of the trimeric envelope complex (gp160 spike) and both CD4 and a chemokine receptor (generally either CCR5 or CXCR4, but others are known to interact) on the cell surface. 77-78 gp120 binds to integrin 47 activating LFA-1 the central integrin involved in the establishment of virological synapses, which facilitate efficient cell-to-cell spreading of HIV-1.79 The gp160 spike contains binding domains for both CD4 and chemokine receptors.77-78 The first step in fusion involves the high-affinity attachment of the CD4 binding domains of gp120 to CD4. Once gp120 is bound with the CD4 protein, the envelope complex undergoes a structural change, exposing the chemokine binding domains of gp120 and allowing them to interact with the target chemokine receptor.77-78 This allows for a more stable two-pronged attachment, which allows the N-terminal fusion peptide gp41 to penetrate the cell membrane. 77-78 Repeat sequences in gp41, HR1, and HR2 then interact, causing the collapse of the

extracellular portion of gp41 into a hairpin. This loop structure brings the virus and cell membranes close together, allowing fusion of the membranes and subsequent entry of the viral capsid.77-78 After HIV has bound to the target cell, the HIV RNA and various enzymes, including reverse transcriptase, integrase, ribonuclease, and protease are injected into the cell.77 During the microtubule-based transport to the nucleus, the viral single-strand RNA genome is transcribed into double-strand DNA, which is then integrated into a host chromosome. HIV can infect dendritic cells (DCs) by this CD4-CCR5 route, but another route using mannose-specific C-type lectin receptors such as DC-SIGN can also be used.80 DCs are one of the first cells encountered by the virus during sexual transmission. They are currently thought to play an important role by transmitting HIV to T-cells when the virus is captured in the mucosa by DCs. 80 The presence of FEZ-1, which occurs naturally in neurons, is believed to prevent the infection of cells by HIV.81

Replication and transcription Shortly after the viral capsid enters the cell, an enzyme called reverse transcriptase liberates the single-stranded (+)RNA genome from the attached viral proteins and copies it into a complementary DNA (cDNA) molecule.82 The process of reverse transcription is extremely error-prone, and the resulting mutations may cause drug resistance or allow the virus to evade the body's immune system. The reverse transcriptase also has ribonuclease activity that degrades the viral RNA during the synthesis of cDNA, as well as DNA-dependent DNA polymerase activity that creates a sense DNA from the antisense cDNA.83 Together, the cDNA and its complement form a double-stranded viral DNA that is then transported into the cell nucleus. The integration of the viral DNA into the host cell's genome is carried out by another viral enzyme called integrase.82

This integrated viral DNA may then lie dormant, in the latent stage of HIV infection.82 To actively produce the virus, certain cellular transcription factors need to be present, the most important of which is NF-B (NF kappa B), which is upregulated when T-cells become activated.84 This means that those cells most likely to be killed by HIV are those currently fighting infection. During viral replication, the integrated DNA provirus is transcribed into mRNA, which is then spliced into smaller pieces. These small pieces are exported from the nucleus into the cytoplasm, where they are translated into the regulatory proteins Tat (which encourages new virus production) and Rev. As the newly produced Rev protein accumulates in the nucleus, it binds to viral mRNAs and allows unspliced RNAs to leave the nucleus, where they are otherwise retained until spliced. 85 At this stage, the structural proteins Gag and Env are produced from the full-length mRNA. The full-length RNA is actually the virus genome; it binds to the Gag protein and is packaged into new virus particles. HIV-1 and HIV-2 appear to package their RNA differently; HIV-1 will bind to any appropriate RNA, whereas HIV-2 will preferentially bind to the mRNA that was used to create the Gag protein itself. This may mean that HIV-1 is better able to mutate (HIV-1 infection progresses to AIDS faster than HIV-2 infection and is responsible for the majority of global infections).

Assembly and release The final step of the viral cycle, assembly of new HIV-1 virons, begins at the plasma membrane of the host cell. The Env polyprotein (gp160) goes through the endoplasmic reticulum and is transported to the Golgi complex where it is cleaved by protease and processed into the two HIV envelope glycoproteins gp41 and gp120. These are transported to the plasma membrane of the host cell where gp41 anchors the gp120 to the membrane of the infected cell. The Gag (p55) and GagPol (p160) polyproteins also associate with the inner surface of the plasma membrane along with the HIV genomic RNA as the forming virion begins to bud

from the host cell. Maturation occurs either in the forming bud or in the immature virion after it buds from the host cell. During maturation, HIV proteases cleave the polyproteins into individual functional HIV proteins and enzymes. The various structural components then assemble to produce a mature HIV virion.86 This cleavage step can be inhibited by protease inhibitors. The mature virus is then able to infect another cell.

Genetic variability HIV differs from many viruses in that it has very high genetic variability. This diversity is a result of its fast replication cycle, with the generation of about 10 10 virions every day, coupled with a high mutation rate of approximately 3 x 10 5 per nucleotide base per cycle of replication and recombinogenic properties of reverse transcriptase.87-89 This complex scenario leads to the generation of many variants of HIV in a single infected patient in the course of one day.87 This variability is compounded when a single cell is simultaneously infected by two or more different strains of HIV. When simultaneous infection occurs, the genome of progeny virions may be composed of RNA strands from two different strains. This hybrid virion then infects a new cell where it undergoes replication. As this happens, the reverse transcriptase, by jumping back and forth between the two different RNA templates, will generate a newly synthesized retroviral DNA sequence that is a recombinant between the two parental genomes.87 This recombination is most obvious when it occurs between subtypes.87 The closely related simian immunodeficiency virus (SIV) has evolved into many strains, classified by the natural host species. SIV strains of the African green monkey (SIVagm) and sooty mangabey (SIVsmm) are thought to have a long evolutionary history with their hosts. These hosts have adapted to the presence of the virus,90 which is present at high levels in the host's blood but evokes only a mild immune response,91 does not cause the development of simian AIDS, 92 and

does not undergo the extensive mutation and recombination typical of HIV infection in humans.93 In contrast, when these strains infect species that have not adapted to SIV ("heterologous" hosts such as rhesus or cynomologus macaques), the animals develop AIDS and the virus generates genetic diversity similar to what is seen in human HIV infection.94 Chimpanzee SIV (SIVcpz), the closest genetic relative of HIV-1, is associated with increased mortality and AIDS-like symptoms in its natural host.95 SIVcpz appears to have been transmitted relatively recently to chimpanzee and human populations, so their hosts have not yet adapted to the virus. 90 This virus has also lost a function of the Nef gene that is present in most SIVs; without this function, T cell depletion is more likely, leading to immunodeficiency.95 Three groups of HIV-1 have been identified on the basis of differences in the envelope (env) region: M, N, and O.96 Group M is the most prevalent and is subdivided into eight subtypes (or clades), based on the whole genome, which are geographically distinct.97 The most prevalent are subtypes B (found mainly in North America and Europe), A and D (found mainly in Africa), and C (found mainly in Africa and Asia); these subtypes form branches in the phylogenetic tree representing the lineage of the M group of HIV-1. Coinfection with distinct subtypes gives rise to circulating recombinant forms (CRFs). In 2000, the last year in which an analysis of global subtype prevalence was made, 47.2% of infections worldwide were of subtype C, 26.7% were of subtype A/CRF02_AG, 12.3% were of subtype B, 5.3% were of subtype D, 3.2% were of CRF_AE, and the remaining 5.3% were composed of other subtypes and CRFs.98 Most HIV-1 research is focused on subtype B; few laboratories focus on the other subtypes. 99 The existence of a fourth group, "P", has been hypothesized based on a virus isolated in 2009. 100-102 The strain is apparently derived from gorilla SIV (SIVgor), first isolated from western lowland gorillas in 2006.100 The genetic sequence of HIV-2 is only partially homologous to HIV-1 and more closely resembles that of SIVsmm.

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