HIV virology

Stuart Turville : Kirby Institute, Department of Medicine, University of New South Wales, Sydney NSW
Robert Oelrichs : Global HV/AIDS Program, The World Bank, Washington, DC USA

The acquired immune deficiency syndrome (AIDS) is caused by the human immunodeficiency virus (HIV). HIV-1 was initially identified by Luc Montagnier at the Institute Pasteur, Paris, in 19831 and was then more fully characterised in 1984 by Robert Gallo in Washington 2 and Jay Levy in San Francisco. 3, 4 A second virus, HIV-2, was isolated from West African patients in 1986. 5 Viruses similar to HIV-1 and HIV-2 have been isolated from chimpanzees and wild African monkeys. 6 It is most likely that HIV-1 and HIV-2 crossed species from primates to humans in Africa several times over the last hundred years with the earliest known samples of HIV-1 isolated from preserved tissues samples from 1959 and 1960. 6-8 The genetic diversity of these two HIV-1 isolates at that time estimates the most recent common ancestor for HIV-1 to be around 1908. 6-8

HIV structure and organisation 

HIV is classified in the family Retroviridae, subfamily Lentivirinae, and genus Lentivirus. 9, 10 The structure of HIV follows the typical pattern of a complex retrovirus family, comprising a single- stranded, positive-sense ribonucleic acid (RNA) genome of about 9.7 kilobases that encodes from structural, enzymatic and accessory proteins. An HIV virion is largely spherical in shape, with a diameter of 100 to 120 nm, and encloses its genome within a fullerene (cone-shaped) capsid core. The virus is enveloped with a lipid bilayer derived from the host cell, thus incorporating proteins found in the host membrane, such as the major histocompatibility (MHC) molecules, 11, 12 CD44 and lymphocyte function-associated antigen 1 (LFA-1). 13 Embedded within this membrane are the viral glycoproteins; the gp120 surface glycoprotein and the gp41 transmembrane glycoprotein. Each envelope subunit displays on the virion surface as a trimer of the outer envelope gp120, non-covalently liked to a trimer of gp41, the transmembrane protein that anchors this glycoprotein complex to the surface of the virion. The envelope protein is the most variable component of HIV, although gp120 itself is structurally divided into highly variable (V) and more constant (C) regions. The constant regions of the gp120 envelope are often associated with the ‘non-neutralising face’, as they are buried within the trimer. 14, 15 While the variable domains are associated with the ‘neutralising face’ of the envelope glycoprotein that is exposed to the humoral immune system and thus variability in this context is derived from immune escape. The variable domains also give the capacity of the virus to change receptor usage. In particular the V3 loop of HIV gp120 can change the virus’s ability to use the HIV coreceptors CXCR4 and CCR5 (receptors for HIV will be covered later in this chapter). Of note, the envelope glycoprotein gp120 has over half its molecular weight attributed to carbohydrate structures, in the form of complex and high mannose N-linked glycans. These glycans decorate the majority of the exposed outer gp120 surface and are known as the ‘silent face’, given their ability to protect this face of the envelope from the humoral immune response.14, 15

The viral structural proteins are derived from the Gag polyprotein, which is present in approximately 2400 copies in an immature virion, according to current estimates. 16 Viral maturation leads to the cleavage of the Gag polyprotein, giving rise to matrix (MA; p17) and capsid (CA; p24) proteins, as well as the smaller peptides nucleocapsid (NC; p7), p6, p2 and p1.17 Matrix associates with the inner membrane of the mature virion, while capsid forms the viral core, enclosing two single strands of viral RNA. The core structure is a cone shape comprised of approximately 1500 capsid proteins assembled into hexameric and pentameric rings. 18, 19 Viral proteins found within the capsid core include the NC, which is found associated with the viral RNA, as well as protease (PR), integrase (IN) and reverse transcriptase (RT) proteins. 20

The genomic organisation of HIV is extremely efficient. Use of all three reading frames (the triplet codes) of the genetic sequence permits overlapping of gene-coding regions. There are nine genes of HIV (Figure HIV genomic structure). These encode proteins that may be broadly classified into structural, catalytic, regulatory, and accessory classes (Table 1).

1.1
 
1.1Figure: HIV genomic structure 

Notes: (a) HIV virion structure highlighting envelope (gp120, gp41) and structural (gag p17, gag p24) proteins. RT = reverse transcriptase; ssRNA = single-stranded RNA. (b) The single-stranded RNA genome of HIV efficiently encodes nine major structural and catalytic proteins by using overlapping parts of the genome. Additionally, the nucleic acid secondary and tertiary structure performs functions independent of translation. LTR = long-terminal repeat

In addition to the functions performed by these proteins in the viral lifecycle, the nucleic acid sequence of the virus possesses intrinsic functions. For example, the Rev responsive element, within the coding region for gp41, interacts with the Rev protein to assist export of spliced RNA transcripts from the nucleus of the cell. 21 The long-terminal repeat (LTR) region has a transcription-promoter function in the integrated deoxyribonucleic acid (DNA) provirus, 22 and contains regions essential for reverse transcription, integration into the host-cell genome and genomic RNA dimerisation. 23

HIV life cycle

The life cycle of a retrovirus is that of an obligatory intracellular parasite, and thus HIV cannot replicate outside human cells (Figure HIV life cycle). 

Table 1 Summary of HIV-1 gene products and their functions
Viral gene Viral protein Designation and size (kDa) Function References
Structural Proteins

 Gag

   
Matrix  MA, p17 Targets virion assembly through binding to PI(4,5)P2 domains at the plasma membrane; binds gp41 to incorporate Env in nascent virions 24-27
Capsid  CA, p24 Forms the capsid-rich core via hexameric and pentameric CA structures; mediates Gag oligomerisation and assembly 18, 19, 28
Nucleocapsid  NC, p7 Binds viral RNA and packages the genome in the core; mediates Gag oligomerisation and assembly 29-31 
p6  p6 Promotes membrane fission of nascent virions via PTAP-Tsg101 recruitment of the ESCRT complex; binds Vpr protein and packages it in the core 32-34 
Env  Surface envelope glycoprotein  SU, gp120 Envelope surface protein; binds to CD4 receptor and chemokine receptors on target cell 35-38 
Transmembrane envelope glycoprotein  TM, gp41 Envelope transmembrane protein; mediates fusion via fusion peptide 39, 40 
Pol   Reverse Transcriptase  RT, p66, p51 RNA-dependent-DNA-polymerase; converts viral ssRNA to linear DNA 41, 42 
Integrase  IN, p32 Facilitates integration of HIV DNA into host genome; involved in dynein-mediated transport of the RTC. 43-46 
Protease  PR, p10 Cleavage of polyprotein components of Gag-Pol  47, 48 
Viral gene Viral protein Designation and size (kDa) Function References
Regulatory Proteins
Tat Trans-activator of Transcription  Tat, p14 Activator of transcription of HIV RNA; essential for virus replication 49-52
Rev Regulator of virion protein expression  Rev, p19 Post-transcriptional regulation of viral genes, including export of viral RNA from the nucleus
 
21, 53
Accessory Proteins
Nef Negative regulatory factor Nef, p27  Infectivity enhancement; CD4 and MHC class I down-regulation; cellular activation and signalling 54-60
Vpr Viral protein R  Vpr, p15   Helps in nuclear transport of HIV post entry; induces G2 cell cycle arrest in infected cells 61-66
Vpu Viral protein U
 
Vpu, p16  Enhances the release of virus particles by counter-acting the interferon induced restriction factor tetherin; promotes CD4 degradation 67-69
Vif Virion infectivity factor  Vif, p23  Counteracts the anti-viral activity of APOBEC3 protein group. The latter protein group are HIV restriction factors that hypermutate HIV genomes and render them non-functional.  70-72
figure 1.2The lifecycle of HIV-1

Source: Furtado MR, Callaway DS, Phair JP, Kunstman KJ, Stanton JL, Macken CA, et al. Persistence of HIV-1 transcription in peripheral-blood mononuclear cells in patients receiving potent antiretroviral therapy. N Engl J Med 1999;340:1614-22. Used with permission

HIV binding and entry

Infection of the host cell commences when HIV binds to specific receptors on the cell membrane. In general, the interaction requires the recognition of two host-cell surface-receptor proteins by the viral gp120 envelope protein. The presence or absence of these cellular proteins restricts the range of host-cell types that are susceptible to infection by a strain of HIV.

The first HIV receptor described was the CD4 protein 35 which is present predominantly on cells of the T lymphocyte and myeloid lineages. The distribution of CD4 receptors has been thought to restrict HIV susceptibility to cells of the lymphocyte, monocyte/macrophage and other CD4-expressing lineages, although there is precedent for HIV-1, HIV-2 and the related virus simian immunodeficiency virus (SIV) to be able to fuse with cells not expressing CD4 23. 73, 74

Subsequently, the requirement of a second coreceptor for viral entry was recognised. 75-80 This function may be performed by a range of proteins within the class of seven-transmembrane receptors, although the most important are CCR5 (CC chemokine receptor type 5) and CXCR4 (CXC chemokine receptor type 4). The seven- transmembrane class of receptors is very large, containing over 100 related proteins. Several of these proteins have been shown to facilitate the binding of HIV in vitro.81 The in vivo significance of entry via these minor coreceptors, however, remains unclear.

After HIV gp120 binds to CD4 receptor and the coreceptor, a conformational change in gp41 causes insertion of the N-terminal hydrophobic fusion-peptide region into the target-cell membrane.82 This insertion results in membrane fusion and the entry of the viral particle contents into the cytoplasm, a process critically dependent upon interactions between the N- and C-terminal regions of the gp41 ectodomain. This intra-protein interaction led to the discovery of a novel class of antivirals called fusion inhibitors, e.g. T-20 (enfuvirtide) which is a short peptide that mimics the structure of the conserved C-terminal region of gp41. 83

Although all HIV strains will recognise and bind to CD4, affinity for either CCR5 or CXCR4 varies. These differences account for the observed distinct tropisms between HIV strains. Binding ability and tropism of the virus are dependent on the protein structure of gp120. Particular patterns of sequence of the V3 and V4 variable regions, and other regions of gp120, relate to CD4 binding and differential coreceptor affinity. 81, 84, 85

In general, viral strains that bind to CCR5 (R5 strains) infect macrophages, activated CD4 T lymphocyte cells and subsets of resting CD4 T cells where CCR5 is expressed. The majority of strains present in vivo are R5 strains and variants of R5 strains known as founder viral strains (see below) are selected during the process of sexual HIV transmission. Other HIV strains that recognise CXCR4 (X4 strains) can exist, but by contrast, infect only T cells and T cell lines and are only present in approximately 40 to 50% of clinical cases during the latter stages of HIV disease. 81 The reason for the lack of CXCR4 variants in early stages of HIV disease, is presently hypothesised to be related to their susceptibility to the adaptive immune response in addition to other factors that limit their growth. 86 The adaptive immune hypothesis does fit well with the time that these isolates emerge, as it is typically at the latter stages of HIV disease when the immune system is being steadily degraded. In theory CXCR4 isolates can emerge via two mechanisms. Firstly, by CXCR4 viruses being transmitted alongside CCR5 using strains or secondly by progressive genetic evolution of a CCR5-using strain to a CXCR4-using strain. To date the majority of scientific observations support the latter hypothesis. 87 Indeed, some investigators argue that all HIV-positive patients would generate a X4-usingHIV strain, if given enough time (i.e. patients can succumb to the effects of R5 HIV before the emergence of X4 strains). The emergence of X4 strains is often clinically associated with subsequent accelerated loss in CD4 T cells and as a consequence accelerated disease progression due to fundamental lack of total CD4 T cells. This latter phenomena is a consequence of increased HIV target cells that can be accessed by X4 HIV strains. For instance, while CCR5 expression is restricted to a small memory/activated CD4 T cell subset, CXCR4 is present on upwards of 90% of all CD4 T cells. 88-90 Historically the growth of X4 strains in vitro has been characterised by the presence of syncytial cells, which are formed by the fusion of multiple infected cells and can be observed by light microscopy. This classification, however, is largely based on the relative expression of CXCR4 on T cell lines used in these assays. The expression of CCR5 expression to similar levels as CXCR4 can readily result in the formation of syncytia when using R5 strains that replicate at similar levels. Indeed the mechanistic basis of syncytial formation is the level of HIV envelope expressed on the infected donor cells and the HIV receptor expression (CD4 and CCR5 or CXCR4) on the neighbouring recipient cell. This scenario is largely based on the replication of the virus within the donor cell and indicative of overall viral production. Indeed investigators have demonstrated the spontaneous formation of syncytia in cell lines that are exposed to high titer virus. 91

Importantly the knowledge of the role of CD4 and associated chemokine receptors led to a new area in HIV therapeutics. A number of small molecules that block binding of HIV to either CCR5 and CXCR4 has been described. Two randomised, placebo-controlled clinical trials (known as MOTIVATE 1 and 2)using the CCR5 inhibitor maraviroc have demonstrated, in conjunction with present optimised therapies, greater virological and immunological efficacy profiles compared to placebo only control. However this was in the context of participants having only detectable R5 HIV. 92, 93 X4-specific chemokine receptor inhibitors also exist, the first generation AMD3100 and the second generation AMD11070. Curiously AMD3100 was discovered in anti-HIV drug screens prior to the knowledge that chemokine receptors work in concert with CD4 to allow HIV entry. 94, 95 Initial trials in humans using the bioavailable AMD11070 CXCR4 inhibitor observed positive virological outcomes, but further development of this drug class has been delayed due to observations of liver damage in animal models. 96

The pre-integration complex

The genetic information of HIV is contained within an RNA genome. Following infection of a new host cell, the RNA genome is first reverse transcribed into single-stranded DNA that is then further transcribed to double-stranded DNA. These two polymerase steps are performed by viral reverse transcriptase, which is co-packaged in the viral particle. Self-priming of the single-stranded RNA and DNA and removal of the transcribed RNA strand occur by a complex series of steps dependent upon interactions between the viral LTR and host-cell enzymes. The double-stranded DNA genome forms a complex with host-cell and viral proteins (including matrix, integrase and Vpr) that is actively transported to the nucleus. 97, 98

During the early steps of the HIV-1 replication cycle, the virus counteracts specific host proteins that have evolved to limit retroviral replication. Two key host cell factors that influence early viral events are i) a protein called human tripartite motif 5 alpha (TRIM5alpha) and ii) sterile alpha motif (SAM) domain and Histidine- Aspartic (HD) domain-containing protein 1 (SAMHD1). TRIM5alpha acts in the early steps of HIV-1 replication cycle, soon after the entry process and before reverse transcription. 99 TRIM5alpha restricts retroviral infection by specifically recognising HIV-1 capsid and promoting its rapid, premature disassembly. 100, 101 TRIM5alpha from rhesus macaques and African green monkeys inhibit HIV-1 replication, whereas the human homologue is inactive against SIV and HIV-1, leading to the susceptibility of human cells to both viruses. For SAMHD1, the primary mode of action was initially hypothesised to be by cleaving nucleotides needed for efficient HIV reverse transcription and subsequent integration. This action is particularly apparent in resting cells like macrophages, dendritic cells and resting CD4 T cells, where there is already a limited nucleotide pool. However recent studies have observed SAMHD1 to also act on HIV RNA and restrict HIV infection through RNase activity 102 and it is presently unclear whether it is the RNase activity combined with dNTPase activity that combines to restrict HIV infection. Observations by Ryoo and colleagues 102 currently support RNase activity only, but require further independent confirmation.

Integration and transcription

The double-stranded HIV genome is then either integrated into the host-cell genome by means of DNA splicing, performed by the viral integrase, or forms stable DNA circles.103 In the context of chromosomal integration, this process is not random, as recent studies have observed preferential HIV DNA integration in active transcriptional sites within infected cells.104 The integrated form of HIV is known as the provirus and takes the form shown in Figure HIV life cycle, with identical LTR copies flanking the coding regions. Proviral DNA is replicated as part of the normal cell genome and may persist in this form for long periods and through many rounds of mitotic cell division. Furthermore integration into long-lived resting CD4 T cells provides challenges in HIV cure attempts, as the virus in these cell types in typically latent.

The 5’ end of the LTR now functions as a promoter, regulating the production of RNA transcripts dependent on the presence of host-cell transcription factors (such as promoter-specific transcription factor, SP1, and nuclear factor - kappa beta) and the viral protein Tat. 105 The transcribed HIV RNA molecules may either be spliced in preparation for translation of viral proteins, or exported from the nucleus in an unspliced form for packaging into newly produced virions. Nuclear export of spliced RNA is assisted by the viral protein Rev.

HIV assembly and release

For full viral assembly, HIV transcription and subsequent translation must reach a critical threshold for viral assembly to occur. 106 One common misconception with respect to HIV latency is the complete lack of HIV transcription and translation in all latently infected cells. For instance, latency in resting CD4 T cells can also exist where low levels of detectable viral RNA and proteins are present, but at a level that does not lead to assembly and production of fully assembled and infectious virions.

Once a threshold of viral protein production is reached, viral proteins that are destined for virions are recruited and assembled at the plasma membrane. 106 Not all virally produced proteins are incorporated with assembling virions, with the dominant virion-associated proteins expressed as either a fusion protein with the structural protein HIV Gag (i.e. HIV Gag-Pol) or are able to non-covalently associate with HIV Gag (e.g. HIV Vpr). For post translational events, HIV Gag exists as a monomer and oligomerisation of HIV Gag triggers the exposure of a myristoylation motif that binds to the membrane-enriched lipid called phosphatidylinositol 4,5-bisphosphate. While HIV Gag oligomerisation can proceed when expressed in isolation, it can also be triggered by viral RNA binding largely through the NC domain of Gag, 107 thus ensuring viral inocula have a greater propensity to bud from the cell membrane with its genetic cargo.

Oligomerisation of HIV Gag at the plasma membrane leads to the formation of what is known as the immature viral shell, primarily consisting of repeating Gag polyprotein hexamers. 108 A partially intact spherical shell is hypothesised to leave the membrane, with the aid of scission from the membrane through the Gag-specific recruitment of cellular proteins involved in the endosomal sorting complexes required for transport (ESCRT) complex, that effectively acts as molecular scissors to liberate virions from the membrane.

Shortly after leaving the membrane, HIV protease is activated and culminates in the sequential cleavage of Gag and Gag-Pol proteins. This process, commonly referred to as viral maturation, is marked by condensation of a large pool of HIV capsid, that leads to encapsulating many viral proteins including HIV RNA-bound NC within the conical shaped viral core.

While the above description outlines the specific role of the major structural protein, HIV Gag, other viral proteins perform a variety of roles to subvert normal cellular function and facilitate viral replication (Table 1). Much about these processes remains poorly understood. Vpr acts to alter host-cell transcription and arrests infected cells at the G2/M phase of cell division. 109 Nef induces downregulation of the CD4 receptor and MHC class I molecules.55, 57, 110 Vpu promotes degradation of CD4 in the endoplasmic reticulum and counteracts an interferon-induced restriction factor known as tetherin, which acts by tethering and preventing the release of newly formed HIV particles from the plasma membrane. 68 Vif is necessary for subsequent efficient infectivity of the newly produced viral particles 70, 111 and counteracts cytidine deaminases (enzymes present especially in macrophages and T cells) that are naturally occurring host defence mechanisms against retroviruses. These proteins include APOBEC3G and APOBEC3F and are degraded by HIV. 111

HIV transmission and biomedical prevention

Recent failures and successes of biomedical prevention in conjunction with the studies of HIV discordant couples (i.e. where one partner is positive and the other negative and thus at risk of acquisition from a known donor), have led to seminal contributions which give us a greater understanding of how HIV is spread person to person. Biomedical prevention in the form of topical microbicides (essentially lubricants that can kill or inhibit HIV) over the past decade has evolved from using formulations consisting of spermicidal detergents through to gel formulations containing the same or similar ingredients as current antiretroviral therapies.112 The first clinical trials using the spermicidal detergent noxanol-9 (N-9) showed increased incidence of HIV transmission 113 and following trials using polyanion-based gels showed either enhanced or limited effects.67, 114 However, these trials did provide important evidence about how the virus was spread. For instance, the use of N-9 and its deleterious effects on the genital mucosa illustrated how primary mucosal barriers were essential for blocking the incoming virions. The combined knowledge of the early microbicide (including polyanions) trials also demonstrated that the maintenance of good reproductive health further strengthened these primary barriers and thus limited the numbers of people contracting HIV through sexual transmission. The more recent studies using microbicides with antiretrovirals, 115 combined with studies on pre-exposure antiretroviral treatment for recipients and treatment as prevention in HIV positive donors also emphasised that there is a clear window during HIV transmission that can be targeted at the recipient and donor level. 116 One of the most successful trials in this latter context was HPTN 052. In this trial of HIV serodiscordant couples, the treatment of the partner with infection led to a reduction in HIV transmission events of 96%. 116 Thus successful control of viral load in the donor, as is the case with limiting mother-to-child HIV transmission, 117 is indeed paramount in limiting the spread between people.

While these prevention strategies gave broad insights into HIV transmission and its prevention, the studies of early HIV acquisition in HIV discordant couples provided the viral genetic footprint that is needed for a virus to cross hosts. The study of the genetic makeup of HIV strains that are the first detected during infection pointed to an extreme bottleneck where only the fittest virus is transmitted. 118 Curiously, the genetic diversity of transmitting strains is reflected in the ease with which the virus can infect. For instance there is greater genetic diversity in HIV founder strains that are transmitted from male to female compared to female-to-male transmitting viruses. In addition those recipients with additional risk factors such as sexually transmitted infection (STI) further relax the genetic bottleneck of the virus and allow a more diverse viral population to pass rather than a stringently defined transmitting strain. 118, 119 While the genetic makeup of the incoming virions in the early stages of HIV transmission is now mapped in large cohorts at fine detail with near whole HIV genomes, understanding the actual biological phenotype of the transmitting strain is still under consideration. 118, 120-127 Recent studies by Carlson and colleagues point to the transmitting strain being the fittest virus, 118 that is, the virus that makes it across the mucosa and has the ability to sustain early and robust viral replication before the mounting of the acquired immune response. While most researchers point to changes in the HIV envelope, it is rather the overall genetic makeup of the virus that dictates the fitness of virus in vivo. As to whether the HIV envelope in a transmitting strain has a preference for HIV targets, in vitro assays suggest the founder viruses rely on high levels of CD4 and thus on having a preference for CD4 T cells. 120, 125, 128 That said, the appearance of HIV within the brain is largely mediated through macrophage infections and thus the clear-cut distinction that transmitting strains preferentially infect CD4 T cells rather than other known HIV targets like dendritic cells and macrophages is still a topic of debate. Even if founder strains did preferentially target CD4 T cells, we must note that other HIV reservoirs, like macrophages and dendritic cells, do exist in vivo and must be taken into context for the overall pathogenesis in vivo (whether that be during the acute or chronic stages of disease).

The taxonomy of HIV and the primate immunodeficiency viruses

HIV is subdivided into two very broad types: HIV-1 and HIV-2 (Figure The molecular phylogeny of primate immunodeficiency viruses). HIV-1 is by far the most common and broadly distributed HIV type, accounting for most of HIV infections worldwide. HIV-2 is genetically closer to SIV strains than HIV-1 and as a consequence HIV-2 can be refractory towards certain HIV-1 reverse transcriptase and HIV protease inhibitors. The pathogenicity of HIV-2 in vivo is far less than HIV-1.129 HIV-2 also has a limited global prevalence (estimated between 1 to 2 million cases worldwide) with restriction primarily to areas within West Africa.

The differences between HIV types 1 and 2 reflect their distinct zoonotic origin.130 HIV-1 is most similar to SIV strains isolated from chimpanzees and HIV-2 to those from the sooty mangabey. HIV-2 results in a less virulent infection than HIV-1, with generally lower viral loads, lower rates of vertical transmission and slower progression of the disease in an individual with the infection. 130-133

1.3
Figure The molecular phylogeny of primate immunodeficiency viruses

Note: An unrooted phylogenetic tree estimated using an alignment of the envelope gene (gp160). HIV is classified into types 1 and 2, the latter co-segregating with simian immunodeficiency virus (SIV ) strains from sooty mangabey and the former with those from chimpanzee indicating probable distinct zoonotic origins. HIV forms three groups: M, N and O. Group M contain the nine subtypes and further circulating recombinant forms that contribute to the overwhelming majority of HIV infections worldwide. (In this analysis of envelope, CRF01_AE is the only circulating recombinant strain included. The scale bar indicates 10% nucleotide sequence divergence.

HIV-1 lineages-groups

HIV-1 is divided into three quite distinct lineages: groups M, N and O. Again, the worldwide distribution of these groups is not equal: group M (for Main) strains are substantially more common in the global epidemic than the group O (Outlier) strains, which are largely confined to Africa, with sporadic cases reported elsewhere. The group N (non-M, non-O) strains have only been isolated in Cameroon. 134

Subtypes and circulating recombinant forms

There are currently nine subtypes that are consistently identified as different subtypes regardless of the genomic region analysed (Figure 1.4). Additionally, several recombinant forms of the virus, the genomes of which are made up of different regions from distinct subtypes, have been separately classified as circulating recombinant forms (CRFs).

1.4
Figure Phylogeny of HIV subtype M envelope gene

Note: A tree based on an alignment of envelope gene sequences (gp120 portion) from reference-subtype strains currently recommended by the Los Alamos National Laboratory (USA) HIV Sequence Database. Group M strains are clearly segregated into subtypes A to K (without I and with the further subdivision of subtype F into sub-subtypes F1 and F2). The tree topology is star shaped, indicating a shared common ancestor and roughly equal divergence times for these strains.

The subtypes and CRFs show strong patterns of distribution in the global pandemic. Western countries, including Australia, continue to have an epidemic that is almost exclusively subtype B in all risk groups. Thailand originally showed a sharp segregation of subtypes between risk groups, with heterosexual transmission largely due to CRF01-AE, and injecting drug use (IDU) transmission due to subtype B; since then CRF01-AE has come to predominate in all risk groups.135-137 India and South Africa are experiencing explosive epidemics of subtype C.138, 139 All HIV subtypes described to date have also been detected in sub-Saharan Africa.

HIV subtype A/E was first described in Thailand as a new subtype E. Subsequent analysis of the entire genome of this form showed it to be a recombinant between subtypes A (that is most prevalent in Africa) and subtype E (that is unique). Isolates of this strain were then termed subtype A/E. As this strain is so prevalent in Asia, it was among the first to be renamed under the new nomenclature of CRFs. It is now referred to as HIV CRF01-AE.

Sub-subtype classification

A further refinement in the subtype classification has been made recently to distinguish strains that form distinct groups within subtypes, but are not sufficiently different to warrant classification as a novel subtype. A detailed description of the most recent HIV taxonomy has been published by the Los Alamos National Laboratory (USA) HIV Sequence Database and is continuously updated on the Laboratory’s database website.

The global pandemic of HIV

The number of individuals with the infection at the end of 2013 reached 35.3 million of which 17.7 million were women and 3.3 million were children under the age of 15 years. The improved access to antiretroviral treatment (in 2012, around 9.7 million people living with HIV had access to antiretroviral therapy in low- and middle-income countries), the implementation of prevention programs and the development of low-cost testing for early detection appear to be having an impact on the HIV epidemic in many countries. For instance total new HIV infections fell by 33% in 2012 compared to estimates in 2001. New HIV infections among adults and adolescents decreased by 50% or more in 26 countries between 2001 and 2012. In addition new HIV infections among children have declined by 52% over the same 2001 to 2012 period. AIDS-related deaths have fallen by 30% since the peak in 2005.140

Figure1.5a-Adults-and-children-estimated-living with HIV 2013Source: Joint United Nations Programme on HIV/AIDS 2014

Figure1.5b Global summary of AIDS epidemic 2013.jpgSource: Joint United Nations Programme on HIV/AIDS 2014

North America and Western and Central Europe

The number of people living with HIV in North America and Western and Central Europe reached an estimated 2.3 million in 2013. Approximately 54% of these cases are from the USA. The majority of new infections in 2011 were attributed to the USA (approximately 50 000 of a total of 88 000 newly diagnosed cases). In the USA, sexual activity among men who have sex with men (MSM) is still the main mode of HIV transmission accounting for approximately 63% of new HIV infections in the USA, while IDU and unprotected paid sex represent the minority of new transmission cases.

The rates of diagnosed HIV cases doubled between 2000 and 2009 in Bulgaria, the Czech Republic, Hungary, Lithuania, Slovakia and Slovenia. In contrast, the number of people newly diagnosed with HIV decreased by more than 20% in Latvia, Portugal and Romania. Over the same period there was an increase by more than 50% in Germany and the United Kingdom; antiretroviral treatment within this region remains a challenge. In the USA the late diagnosis of HIV, poor treatment adherence and high levels of early treatment discontinuation contribute to avoidable HIV-related deaths. Whereas in western and central Europe people at risk are not being tested for HIV. Lack of testing is problematic in terms of treatment, and for the potential effect on increased levels of HIV transmission.

Asia, the Pacific and Australia

There were an estimated 350 000 (220 000 – 550 000) new HIV infections in Asia and the Pacific in 2012, a decline of 26% since 2001. In most countries in Asia and the Pacific, sex workers and their clients, MSM, transgender people and people who inject drugs represent the populations most affected by the epidemic. In 2012, 1.25 million were accessing antiretroviral treatment (51% [43-63%]). In the Asia-Pacific region,12 countries account for more than 90% of people living with HIV and more than 90% of new HIV infections: Cambodia, China, India, Indonesia, Malaysia, Myanmar, Nepal, Pakistan, Papua New Guinea, the Philippines, Thailand and Vietnam. Australia’s closest neighbour Papua New Guinea, although with a total of 32 000 people with HIV infection, has observed a decline in new HIV infections of over 50% since 2001 and a 31% decrease between 2005 to 2013. Presently over 40% have access to antiretroviral treatment.

In Australia 1236 cases of HIV infection were newly diagnosed in 2013, similar to levels in 2012 when the number of cases was the highest in Australia since the early 1990s. The annual number of new HIV diagnoses has gradually increased over the past 14 years, from 724 diagnoses in 1999. As in the USA, the dominant form of transmission is MSM accounting for over 70% of new cases in 2013. In 2013 between 13 200 and 19 500 were receiving antiretroviral treatment and had undetectable levels of HIV, corresponding to 49–73% of all people with HIV and 57–84% of people with diagnosed HIV infection. Australian figures cited and respective updates can be found at: https://kirby.unsw.edu.au/surveillance/Annual-Surveillance-Reports.

Sub-Saharan Africa

Around 24.7 million or nearly 70% of all people living with HIV worldwide live in Sub-Saharan Africa (58% of them women). There were 1.5 million [1.3 million–1.6 million] new HIV infections in sub-Saharan Africa in 2013. However, new infections are declining. There was a 33% drop in new HIV infections among all ages in the region between 2005 and 2013. Previously this area also had the lowest prevalence of antiretroviral treatment. Fortunately, successive increases in antiretroviral treatment have occurred, especially over the last 3 years, with 2012 recording an increase in 1.7 million people on treatment. Collectively 37% people in this region now have access to treatment. In addition 68% were receiving effective drug regimens to prevent mother-to child transmission in 2013. Access to antiretroviral treatment has related directly to a reduction in HIV-related deaths in this region. For instance, in South African over the period 2005 to 2013 there was a 48% reduction of HIV-related deaths. The limited uptake of antiretroviral treatment in Nigeria has unchanged the rate of HIV-related deaths in 2005 compared to 2013.

Move towards an HIV cure

Present limitations on therapeutic driven cures

The persistence of HIV in the face of antiretroviral treatment reflects the complex nature of the target: the human immune system. Our acquired immune system relies on the persistence of long-lived memory CD4 T cells that can be activated upon future pathogenic exposure. Analogous to an acquired immune response, HIV can establish a latent reservoir within this latter CD4 memory T cell type and thus persist for long periods of time (estimated to be several decades with current half life estimates of 44 months).141

Cure strategies aimed at purging HIV reservoirs

Several approaches to eradicate reservoirs of HIV by clinical treatment have attempted to use latency reversing agents (LRAs) in an HIV cure strategy known as ‘shock and kill’. That is, in patients undergoing antiretroviral treatment, the dormant virus in resting memory CD4 T cells would be rendered ‘awake’ and while these cells are replicating virus they would succumb to the action of virus-specific cytolytic T lymphocytes, but would not further spread virus due to the presence of effective antiretroviral therapy. The most prominent and current LRAs being used in clinical trials are derived from the class of histone deacetylase inhibitors (summarised at http://www.hdacis.com/index.html). The LRA approach has yet to definitively demonstrate functional or complete HIV cure and is still the focus of current clinical research.

The Berlin patient and HIV gene therapy

The only known example of a person to be cured of HIV is the Berlin patient.142, 143 In this setting, the patient had HIV infection for over 10 years and underwent treatment for acute myeloid leukaemia. The treatment consisted of chemotherapy, antithymocyte globulin, total body irradiation and finally a haematopoietic stem cell transplantation from a donor with a homozygous delta 32 mutation in the HIV coreceptor CCR5 (this mutation is well known to confer protection against R5 HIV isolates). Currently 5 years off therapy, the Berlin patient still has undetectable levels of HIV using current detection methodology. Given the complicated nature of treatment for this patient, it has been difficult to assess what level of treatment led to this phenotype. Two other patients, known as Boston patient A and Boston patient B, have undergone similar haematopoietic stem cell transplantation regimes, but with donors that carry the wild type form of CCR5. 144 Both Boston patients had rebound viral loads at 84 days and 225 days post HIV therapy interruption.145 Thus the process of stem cell transplantation may indeed need a donor with a homozygous delta 32 mutation or mimic to create an analogous situation to that of the Berlin patient.

The low frequency of donors who carry homozygous delta 32 mutation in the HIV coreceptor CCR5 has made the Berlin patient approach difficult in the context of overall feasibility.146 The hope is that gene therapy treatment of cells within patients with HIV infection might render cells ex vivo resistant to HIV infection, thereby not only mimicking those of the stem cell donor used in the Berlin patient, but also dealing with issues of graft versus host disease (as the genetically manipulated cells are derived from the same patient). Advancement in gene therapy techniques that can render donor cells resistant to HIV range from: lowering CCR5 expression levels; 147-152 expression of peptides that inhibit the HIV fusion reaction; 147, 152, 153 and post entry manipulation that can transcriptionally silence the virus and thus keep it permanently in a dormant state. 154-156 Treatment may also consist of combination approaches, for example, current clinical trials are underway that use both reduction CCR5 and expression of HIV fusion inhibiting peptides (http://www.businesswire.com/news/home/20150527005387/en#.VZDZGUZfcoS). While the latter gene therapies have great potential, delivery of the genetic information to the right cells still provides issues in terms of feasibility. Peripheral CD4 T cells for instance are notoriously difficult at taking on the genetic information using existing transduction techniques. 157

Footnote: Robert Oelrichs is a staff member of the International Bank for Reconstruction and Development/The World Bank. The findings, interpretations and conclusions presented in this article do not necessarily reflect the views of the Executive Directors of the World Bank or the Governments they represent.

References

[1] Barre-Sinoussi F, Chermann JC, Rey F, Nugeyre MT, Chamaret S, Gruest J et al. Isolation of a T-lymphotropic retrovirus from a patient at risk for acquired immune deficiency syndrome (AIDS). Science 1983;220:868-71.

[2] Gallo RC, Sarin PS, Gelmann EP, Robert-Guroff M, Richardson E, Kalyanaraman VS, et al. Isolation of human T-cell leukemia virus in acquired immune deficiency syndrome (AIDS). Science 1983;220:865-7.

[3] Levy JA, Hoffman AD, Kramer SM, Landis JA, Shimabukuro JM, Oshiro LS. Isolation of lymphocytopathic retroviruses from San Francisco patients with AIDS. Science 1984;225:840-2.

[4] Levy JA, Mitra G, Mozen MM. Recovery and inactivation of infectious retroviruses from factor VIII concentration. Lancet 1984;2:722-3.

[5] Clavel F, Guetard D, Brun-Vezinet F, Chamaret S, Rey MA, Santos-Ferreira MO, et al. Isolation of a new human retrovirus from West African patients with AIDS. Science 1986;233:343-6.

[6] Sharp PM, Hahn BH. Origins of HIV and the AIDS pandemic. Cold Spring Harb Perspect Med 2011;1:a006841.

[7] Zhu T, Korber BT, Nahmias AJ, Hooper E, Sharp PM, Ho DD. An African HIV-1 sequence from 1959 and implications for the origin of the epidemic. Nature 1998;391:594-7.

[8] Worobey M, Gemmel M, Teuwen DE, Haselkorn T, Kunstman K, Bunce M, et al. Direct evidence of extensive diversity of HIV-1 in Kinshasa by 1960. Nature 2008;455:661-4.

[9] Chiu IM, Yaniv A, Dahlberg JE, Gazit A, Skuntz SF, Tronick SR, et al. Nucleotide sequence evidence for relationship of AIDS retrovirus to lentiviruses. Nature 1985;317:366-8.

[10] Wain-Hobson S, Alizon M, Montagnier L. Relationship of AIDS to other retroviruses. Nature 1985;313:743.

[11] Arthur LO, Bess JW Jr, Sowder RC 2nd, Benveniste RE, Mann DL, Chermann JC, et al. Cellular proteins bound to immunodeficiency viruses: implications for pathogenesis and vaccines. Science 1992;258:1935-8.

[12] Gelderblom H, Reupke H, Winkel T, Kunze R, Pauli G. MHC-antigens: constituents of the envelopes of human and simian immunodeficiency viruses. Z Naturforsch1987;42:1328-34.

[13] Orentas RJ, Hildreth JE. Association of host cell surface adhesion receptors and other membrane proteins with HIV and SIV. AIDS Res Hum Retroviruses 1993;9:1157-65.

[14] Kwong PD, Wyatt R, Robinson J, Sweet RW, Sodroski J, Hendrickson WA. Structure of an HIV gp120 envelope glycoprotein in complex with the CD4 receptor and a neutralizing human antibody. Nature 1998;393: 648-59.

[15] Wyatt R, Kwong PD, Desjardins E, Sweet RW, Robinson J, Hendrickson WA, et al. The antigenic structure of the HIV gp120 envelope glycoprotein. Nature 1998;393:705-11.

[16] Carlson LA, Briggs JA, Glass B, Riches JD, Simon MN, Johnson MC, et al. Three-dimensional analysis of budding sites and released virus suggests a revised model for HIV-1 morphogenesis. Cell Host Microbe 2008;4:592-9.

[17]Wiegers K, Rutter G, Kottler H, Tessmer U, Hohenberg H, Krausslich HG. Sequential steps in human immunodeficiency virus particle maturation revealed by alterations of individual Gag polyprotein cleavage sites. J Virol 1998;72:2846-54.

[18] Ganser BK, Li S, Klishko VY, Finch JT, Sundquist WI. Assembly and analysis of conical models for the HIV-1 core. Science 1999;283:80-3.

[19] Li S, Hill CP, Sundquist WI, Finch JT. Image reconstructions of helical assemblies of the HIV-1 CA protein. Nature 2000;407:409-13.

[20] Forshey BM, Aiken C. Disassembly of human immunodeficiency virus type 1 cores in vitro reveals association of Nef with the subviral ribonucleoprotein complex. J Virol 2003;77:4409-14.

[21] Malim MH, Bohnlein S, Hauber J, Cullen BR. Functional dissection of the HIV-1 Rev trans-activator--derivation of a trans-dominant repressor of Rev function. Cell 1989;58:205-14.

[22] Laspia MF, Rice AP, Mathews MB. HIV-1 Tat protein increases transcriptional initiation and stabilizes elongation. Cell 1989;59:283-92.

[23] Paillart JC, Shehu-Xhilaga M, Marquet R, Mak J. Dimerization of retroviral RNA genomes: an inseparable pair. Nature reviews. Microbiology 2004;2: 461-72.

[24] Ono A, Ablan SD, Lockett SJ, Nagashima K, Freed EO. Phosphatidylinositol (4,5) bisphosphate regulates HIV-1 Gag targeting to the plasma membrane. Proc Natl Acad Sci USA 2004;101:14889-94.

[25] Chukkapalli V, Hogue IB, Boyko V, Hu WS, Ono A. Interaction between the human immunodeficiency virus type 1 Gag matrix domain and phosphatidylinositol-(4,5)-bisphosphate is essential for efficient gag membrane binding. J Virol 2008;82:2405-17.

[26] Dorfman T, Mammano F, Haseltine WA, Gottlinger HG. Role of the matrix protein in the virion association of the human immunodeficiency virus type 1 envelope glycoprotein. J Virol 1994;68:1689-96.

[27] Yu X, Yuan X, Matsuda Z, Lee TH, Essex M. The matrix protein of human immunodeficiency virus type 1 is required for incorporation of viral envelope protein into mature virions. J Virol 1992;66:4966-71.

[28] Gamble TR, Yoo S, Vajdos FF, von Schwedler UK, Worthylake DK, Wang H, et al. Structure of the carboxyl-terminal dimerization domain of the HIV-1 capsid protein. Science 1997;278:849-53.

[29] Berkowitz RD, Luban J, Goff SP. Specific binding of human immunodeficiency virus type 1 gag polyprotein and nucleocapsid protein to viral RNAs detected by RNA mobility shift assays. J Virol 1993;67:7190-200.

[30] Burniston MT, Cimarelli A, Colgan J, Curtis SP, Luban J. Human immunodeficiency virus type 1 Gag polyprotein multimerization requires the nucleocapsid domain and RNA and is promoted by the capsid-dimer interface and the basic region of matrix protein. J Virol 1999;73:8527-40.

[31] Cimarelli A, Luban J. Human immunodeficiency virus type 1 virion density is not determined by nucleocapsid basic residues. J Virol 2000;74:6734-40.

[32] Garrus JE, von Schwedler UK, Pornillos OW, Morham SG, Zavitz KH, Wang HE, et al. Tsg101 and the vacuolar protein sorting pathway are essential for HIV-1 budding. Cell 2001;107:55-65.

[33] Paxton W, Connor RI, Landau NR. Incorporation of Vpr into human immunodeficiency virus type 1 virions: requirement for the p6 region of gag and mutational analysis. J Virol 1993;67:7229-37.

[34] Martin-Serrano J, Zang T, Bieniasz PD. HIV-1 and Ebola virus encode small peptide motifs that recruit Tsg101 to sites of particle assembly to facilitate egress. Nat Med 2001;7:1313-9.

[35] Dalgleish AG, Beverley PC, Clapham PR, Crawford DH, Greaves MF, Weiss RA. The CD4 (T4) antigen is an essential component of the receptor for the AIDS retrovirus. Nature 1984;312):763-7.

[36] Klatzmann D, Barre-Sinoussi F, Nugeyre MT, Danquet C, Vilmer E, Griscelli C, et al. Selective tropism of lymphadenopathy associated virus (LAV) for helper-inducer T lymphocytes. Science 1984;225:59-63.

[37] Klatzmann D, Champagne E, Chamaret S, Gruest J, Guetard D, Hercend T, et al. T-lymphocyte T4 molecule behaves as the receptor for human retrovirus LAV. Nature 1984;312:767-8.

[38] Maddon PJ, Dalgleish AG, McDougal JS, Clapham PR, Weiss RA, Axel R. The T4 gene encodes the AIDS virus receptor and is expressed in the immune system and the brain. Cell 1986;47:333-48.

[39] Chan DC, Fass D, Berger JM, Kim PS. Core structure of gp41 from the HIV envelope glycoprotein. Cell 1997;89:263-73.

[40] Weissenhorn W, Dessen A, Harrison SC, Skehel JJ, Wiley DC. Atomic structure of the ectodomain from HIV-1 gp41. Nature 1997;387:426-30.

[41] Baltimore D. RNA-dependent DNA polymerase in virions of RNA tumour viruses. Nature 1970;226:1209-11.

[42] Temin HM, Mizutani S. RNA-dependent DNA polymerase in virions of Rous sarcoma virus. Nature 1970;226:1211-3.

[43] Bowerman B, Brown PO, Bishop JM, Varmus HE. A nucleoprotein complex mediates the integration of retroviral DNA. Genes Dev 1989;3:469-78.

[44] Desfarges S, Salin B, Calmels C, Andreola ML, Parissi V, Fournier M. HIV-1 integrase trafficking in S. cerevisiae: a useful model to dissect the microtubule network involvement of viral protein nuclear import. Yeast 2009;26:39-54.

[45] McDonald D, Vodicka MA, Lucero G, Svitkina TM, Borisy GG, Emerman M, et al. Visualization of the intracellular behavior of HIV in living cells. J Cell Biol 2002;159:441-52.

[46] Arhel N, Genovesio A, Kim KA, Miko S, Perret E, Olivo-Marin JC, et al. Quantitative four-dimensional tracking of cytoplasmic and nuclear HIV-1 complexes. Nat Methods 2006;3:817-24.

[47] Wlodawer A, Miller M, Jaskolski M, Sathyanarayana BK, Baldwin E, Weber IT, et al. Conserved folding in retroviral proteases: crystal structure of a synthetic HIV-1 protease. Science 1989;245:616-21.

[48] Navia MA, Fitzgerald PM, McKeever BM, Leu CT, Heimbach JC, Herber WK, et al. Three-dimensional structure of aspartyl protease from human immunodeficiency virus HIV-1. Nature 1989;337:615-20.

[49] Muesing MA, Smith DH, Capon DJ. Regulation of mRNA accumulation by a human immunodeficiency virus trans-activator protein. Cell 1987;48:691-701.

[50] Berkhout B, Silverman RH, Jeang KT. Tat trans-activates the human immunodeficiency virus through a nascent RNA target. Cell 1989;59:273-82.

[51] Dayton AI, Sodroski JG, Rosen CA, Goh WC, Haseltine WA. The trans-activator gene of the human T cell lymphotropic virus type III is required for replication. Cell 1986;44:941-7.

[52] Fisher AG, Feinberg MB, Josephs SF, Harper ME, Marselle LM, Reyes G, et al. The trans-activator gene of HTLV-III is essential for virus replication. Nature 1986;320:367-71.

[53] Hadzopoulou-Cladaras M, Felber BK, Cladaras C, Athanassopoulos A, Tse A, Pavlakis GN. The rev (trs/art) protein of human immunodeficiency virus type 1 affects viral mRNA and protein expression via a cis-acting sequence in the env region. J Virol 1989;63:1265-74.

[54] Aiken C, Konner J, Landau NR, Lenburg ME, Trono D. Nef induces CD4 endocytosis: requirement for a critical dileucine motif in the membrane-proximal CD4 cytoplasmic domain. Cell 1994;76:853-64.

[55] Garcia JV, Miller AD. Serine phosphorylation-independent downregulation of cell-surface CD4 by nef. Nature 1991;350:508-11.

[56] Rhee SS, Marsh JW. Human immunodeficiency virus type 1 Nef-induced down-modulation of CD4 is due to rapid internalization and degradation of surface CD4. J Virol 1994;68(8):5156-63.

[57] Schwartz O, Marechal V, Le Gall S, Lemonnier F, Heard JM. Endocytosis of major histocompatibility complex class I molecules is induced by the HIV-1 Nef protein. Nat Med 1996;2:338-42.

[58] Greenberg M, DeTulleo L, Rapoport I, Skowronski J, Kirchhausen T. A dileucine motif in HIV-1 Nef is essential for sorting into clathrin-coated pits and for downregulation of CD4. Curr Biol 1998;8:1239-42.

[59] Fackler OT, Luo W, Geyer M, Alberts AS, Peterlin BM. Activation of Vav by Nef induces cytoskeletal rearrangements and downstream effector functions. Molecular cell 1999;3:729-39.

[60] Saksela K, Cheng G, Baltimore D. Proline-rich (PxxP) motifs in HIV-1 Nef bind to SH3 domains of a subset of Src kinases and are required for the enhanced growth of Nef+ viruses but not for down-regulation of CD4. EMBO J 1995;14:484-91.

[61] He J, Choe S, Walker R, Di Marzio P, Morgan DO, Landau NR. Human immunodeficiency virus type 1 viral protein R (Vpr) arrests cells in the G2 phase of the cell cycle by inhibiting p34cdc2 activity. J Virol 1995;69:6705-11.

[62] Re F, Braaten D, Franke EK, Luban J. Human immunodeficiency virus type 1 Vpr arrests the cell cycle in G2 by inhibiting the activation of p34cdc2-cyclin B. J Virol 1995;69:6859-64.

[63] Rogel ME, Wu LI, Emerman M. The human immunodeficiency virus type 1 vpr gene prevents cell proliferation during chronic infection. J Virol 1995;69:882-8.

[64] Jowett JB, Planelles V, Poon B, Shah NP, Chen ML, Chen IS. The human immunodeficiency virus type 1 vpr gene arrests infected T cells in the G2 + M phase of the cell cycle. J Virol 1995;69:6304-13.

[65] Popov S, Rexach M, Ratner L, Blobel G, Bukrinsky M. Viral protein R regulates docking of the HIV-1 preintegration complex to the nuclear pore complex. J Biol Chem 1998;273:13347-52.

[66] Heinzinger NK, Bukinsky MI, Haggerty SA, Ragland AM, Kewalramani V, Lee MA, et al. The Vpr protein of human immunodeficiency virus type 1 influences nuclear localization of viral nucleic acids in nondividing host cells. Proc Natl Acad Sci USA 1994;91:7311-5.

[67] Van Damme L, Govinden R, Mirembe FM, Guedou F, Solomon S, Becker ML, et al. Lack of effectiveness of cellulose sulfate gel for the prevention of vaginal HIV transmission. N Engl J Med 2008;359:463-72.

[68] Neil SJ, Zang T, Bieniasz PD. Tetherin inhibits retrovirus release and is antagonized by HIV-1 Vpu. Nature 2008;451:425-30.

[69] Willey RL, Maldarelli F, Martin MA, Strebel K. Human immunodeficiency virus type 1 Vpu protein induces rapid degradation of CD4. J Virol 1992;66:7193-200.

[70] Sheehy AM, Gaddis NC, Choi JD, Malim MH. Isolation of a human gene that inhibits HIV-1 infection and is suppressed by the viral Vif protein. Nature 2002;418:646-50.

[71] Wiegand HL, Doehle BP, Bogerd HP, Cullen BR. A second human antiretroviral factor, APOBEC3F, is suppressed by the HIV-1 and HIV-2 Vif proteins. EMBO J 2004;23:2451-8.

[72] Zheng YH, Irwin D, Kurosu T, Tokunaga K, Sata T, Peterlin BM. Human APOBEC3F is another host factor that blocks human immunodeficiency virus type 1 replication. J Virol 2004;78(11):6073-6.

[73] LaBranche CC, Hoffman TL, Romano J, Haggarty BS, Edwards TG, Matthews TJ, et al. Determinants of CD4 independence for a human immunodeficiency virus type 1 variant map outside regions required for coreceptor specificity. J Virol 1999;73:10310-9.

[74] Puffer BA, Pohlmann S, Edinger AL, Carlin D, Sanchez MD, Reitter J, et al. CD4 independence of simian immunodeficiency virus Envs is associated with macrophage tropism, neutralization sensitivity, and attenuated pathogenicity. J Virol 2002;76:2595-605.

[75] Alkhatib G, Combadiere C, Broder CC, Feng Y, Kennedy PE, Murphy PM, et al. CC CKR5: a RANTES, MIP-1alpha, MIP-1beta receptor as a fusion cofactor for macrophage-tropic HIV-1. Science 1996;272:1955-8.

[76] Choe H, Farzan M, Sun Y, Sullivan N, Rollins B, Ponath PD, et al. The beta-chemokine receptors CCR3 and CCR5 facilitate infection by primary HIV-1 isolates. Cell 1996;85:1135-48.

[77] Deng H, Liu R, Ellmeier W, Choe S, Unutmaz D, Burkhart M, et al. Identification of a major co-receptor for primary isolates of HIV-1. Nature 1996;381:661-6.

[78] Doranz BJ, Rucker J, Yi Y, Smyth RJ, Samson M, Peiper SC, et al. A dual-tropic primary HIV-1 isolate that uses fusin and the beta-chemokine receptors CKR-5, CKR-3, and CKR-2b as fusion cofactors. Cell 1996;85:1149-58.

[79] Dragic T, Litwin V, Allaway GP, Martin SR, Huang Y, Nagashima KA, et al. HIV-1 entry into CD4+ cells is mediated by the chemokine receptor CC-CKR-5. Nature 1996;381:667-73.

[80] Feng Y, Broder CC, Kennedy PE, Berger EA. HIV-1 entry cofactor: functional cDNA cloning of a seven-transmembrane, G protein-coupled receptor. Science 1996;272:872-7.

[81] Berger EA, Murphy PM, Farber JM. Chemokine receptors as HIV-1 coreceptors: roles in viral entry, tropism, and disease. Ann Rev Immunol 1999;17:657-700.

[82] Eckert DM, Kim PS. Mechanisms of viral membrane fusion and its inhibition. Ann Rev Biochem 2001;70:777-810.

[83] Kilby JM, Hopkins S, Venetta TM, DiMassimo B, Cloud GA, Lee JY, et al. Potent suppression of HIV-1 replication in humans by T-20, a peptide inhibitor of gp41-mediated virus entry. Nat Med 1998 4:1302-7.

[84] Rizzuto CD, Wyatt R, Hernandez-Ramos N, Sun Y, Kwong PD, Hendrickson WA, et al. A conserved HIV gp120 glycoprotein structure involved in chemokine receptor binding. Science 1998;280:1949-53.

[85] Groenink M, Fouchier RA, Broersen S, Baker CH, Koot M, van't Wout AB, et al. Relation of phenotype evolution of HIV-1 to envelope V2 configuration. Science 1993;260(:1513-6.

[86] Margolis L, Shattock R. Selective transmission of CCR5-utilizing HIV-1: the 'gatekeeper' problem resolved? Nature reviews. Microbiology 2006;4:312-7.

[87] Schuitemaker H, van 't Wout AB, Lusso P. Clinical significance of HIV-1 coreceptor usage. J Transl Med 2011;9 Suppl 1:S5.

[88] Berkowitz RD, Beckerman KP, Schall TJ, McCune JM. CXCR4 and CCR5 expression delineates targets for HIV-1 disruption of T cell differentiation. J Immunol 1998;161:3702-10.

[89] Bleul CC, Wu L, Hoxie JA, Springer TA, Mackay CR. The HIV coreceptors CXCR4 and CCR5 are differentially expressed and regulated on human T lymphocytes. Proc Natl Acad Sci USA 1997;94:1925-30.

[90] de Roda Husman AM, Blaak H, Brouwer M, Schuitemaker H. CC chemokine receptor 5 cell-surface expression in relation to CC chemokine receptor 5 genotype and the clinical course of HIV-1 infection. J Immunol 1999;163:4597-603.

[91] Rossio JL, Esser MT, Suryanarayana K, Schneider DK, Bess JW Jr, Vasquez GM, et al. Inactivation of human immunodeficiency virus type 1 infectivity with preservation of conformational and functional integrity of virion surface proteins. J Virol 1998;72:7992-8001.

[92] Fatkenheuer G, Nelson M, Lazzarin A, Konourina I, Hoepelman AI, Lampiris H, et al. Subgroup analyses of maraviroc in previously treated R5 HIV-1 infection. N Engl J Med 2008;359:1442-55.

[93] Gulick RM, Lalezari J, Goodrich J, Clumeck N, DeJesus E, Horban A et al. Maraviroc for previously treated patients with R5 HIV-1 infection. N Engl J Med 2008;359:1429-41.

[94] De Clercq E, Yamamoto N, Pauwels R, Balzarini J, Witvrouw M, De Vreese K, et al. Highly potent and selective inhibition of human immunodeficiency virus by the bicyclam derivative JM3100. Antimicrob Agents Chemother 1994;38: 668-74.

[95] Schols D, Struyf S, Van Damme J, Este JA, Henson G, De Clercq E. Inhibition of T-tropic HIV strains by selective antagonization of the chemokine receptor CXCR4. J Exp Med 1997;186:1383-8.

[96] Moyle G, DeJesus E, Boffito M, Wong RS, Gibney C, Badel K, et al. Proof of activity with AMD11070, an orally bioavailable inhibitor of CXCR4-tropic HIV type 1. Clin Infect Dis 2009;48:798-805.

[97] Gallay P, Hope T, Chin D, Trono D. HIV-1 infection of nondividing cells through the recognition of integrase by the importin/karyopherin pathway. Proc Natl Acad Sci USA 1997;94:9825-30.

[98] Bukrinsky MI, Sharova N, Dempsey MP, Stanwick TL, Bukrinskaya AG, Haggerty S, et al. Active nuclear import of human immunodeficiency virus type 1 preintegration complexes. Proc Natl Acad Sci USA 1992;89:6580-4.

[99] Stremlau M, Owens CM, Perron MJ, Kiessling M, Autissier P, Sodroski J. The cytoplasmic body component TRIM5alpha restricts HIV-1 infection in Old World monkeys. Nature 2004;427:848-53.

[100] Sakuma R, Noser JA, Ohmine S, Ikeda Y. Rhesus monkey TRIM5alpha restricts HIV-1 production through rapid degradation of viral Gag polyproteins. Nat Med 2007;13:631-5.

[101]  Stremlau M, Perron M, Lee M, Li Y, Song B, Javanbakht H, et al. Specific recognition and accelerated uncoating of retroviral capsids by the TRIM5alpha restriction factor. Proc Natl Acad Sci USA 2006;103:5514-9.

[102] Ryoo J, Choi J, Oh C, Kim S, Seo M, Kim SY, et al. The ribonuclease activity of SAMHD1 is required for HIV-1 restriction. Nat Med 2014;20:936-41.

[103] Bushman FD, Fujiwara T, Craigie R. Retroviral DNA integration directed by HIV integration protein in vitro. Science 1990;249:1555-8.

[104] Craigie R, Bushman FD. HIV DNA integration. Cold Spring Harb Perspect Med 2012;2:a006890.

[105] Nekhai S, Jeang KT. Transcriptional and post-transcriptional regulation of HIV-1 gene expression: role of cellular factors for Tat and Rev. Future Microbiol 2006;1:417-26.

[106] Pace MJ, Graf EH, Agosto LM, Mexas AM, Male F, Brady T, et al. Directly infected resting CD4+T cells can produce HIV Gag without spreading infection in a model of HIV latency. PLoS Pathog 2012;8(7):e1002818.

[107] O'Carroll IP, Soheilian F, Kamata A, Nagashima K, Rein A. Elements in HIV-1 Gag contributing to virus particle assembly. Virus Res 2013;171:341-5.

[108] Briggs JA, Krausslich HG. The molecular architecture of HIV. J Mol Biol 2011;410:491-500.

[109] Poon B, Grovit-Ferbas K, Stewart SA, Chen IS. Cell cycle arrest by Vpr in HIV-1 virions and insensitivity to antiretroviral agents. Science 1998;281:266-9.

[110] Greenway AL, McPhee DA, Grgacic E, Hewish D, Lucantoni A, Macreadie I, et al. Nef 27, but not the Nef 25 isoform of human immunodeficiency virus-type 1 pNL4.3 down-regulates surface CD4 and IL-2R expression in peripheral blood mononuclear cells and transformed T cells. Virology 1994;198:245-56.

[111] Malim MH, Bieniasz PD. HIV Restriction Factors and Mechanisms of Evasion. Cold Spring Harb Perspect Med 2012;2:a006940.

[112] Morris GC, Lacey CJ. Microbicides and HIV prevention: lessons from the past, looking to the future. Curr Opin Infect Dis 2010;23:57-63.

[113] Van Damme L, Ramjee G, Alary M, Vuylsteke B, Chandeying V, Rees H, et al. Effectiveness of COL-1492, a nonoxynol-9 vaginal gel, on HIV-1 transmission in female sex workers: a randomised controlled trial. Lancet 2002;360:971-7.

[114] Skoler-Karpoff S, Ramjee G, Ahmed K, Altini L, Plagianos MG, Friedland B, et al. Efficacy of Carraguard for prevention of HIV infection in women in South Africa: a randomised, double-blind, placebo-controlled trial. Lancet 2008;372:1977-87.

[115] Abdool Karim Q, Abdool Karim SS, Frohlich JA, Grobler AC, Baxter C, Mansoor LE, et al. Effectiveness and safety of tenofovir gel, an antiretroviral microbicide, for the prevention of HIV infection in women. Science 2010;329:1168-74.

[116] Cohen MS, Chen YQ, McCauley M, Gamble T, Hosseinipour MC, Kumarasamy N, et al. Prevention of HIV-1 infection with early antiretroviral therapy. N Engl J Med 2011;365:493-505.

[117] Connor EM, Sperling RS, Gelber R, Kiselev P, Scott G, O'Sullivan MJ, et al. Reduction of maternal-infant transmission of human immunodeficiency virus type 1 with zidovudine treatment. Pediatric AIDS Clinical Trials Group Protocol 076 Study Group. N Engl J Med 1994;331:1173-80.

[118] Carlson JM, Schaefer M, Monaco DC, Batorsky R, Claiborne DT, Prince J, et al. HIV transmission. Selection bias at the heterosexual HIV-1 transmission bottleneck. Science 2014;345:1254031.

[119] Shaw GM, Hunter E. HIV transmission. Cold Spring Harb Perspect Med 2012;2: pii: a006965.

[120] Parrish NF, Gao F, Li H, Giorgi EE, Barbian HJ, Parrish EH, et al. Phenotypic properties of transmitted founder HIV-1. Proc Natl Acad Sci USA 2013;110:6626-33.

[121] Shen R, Kappes JC, Smythies LE, Richter HE, Novak L, Smith PD. Vaginal myeloid dendritic cells transmit founder HIV-1. J Virol 2014;88:7683-8.

[122] Mlcochova P, Watters SA, Towers GJ, Noursadeghi M, Gupta RK. Vpx complementation of 'non-macrophage tropic' R5 viruses reveals robust entry of infectious HIV-1 cores into macrophages. Retrovirology 2014;11:25.

[123] Fenton-May AE, Dibben O, Emmerich T, Ding H, Pfafferott K, Aasa-Chapman MM, et al. Relative resistance of HIV-1 founder viruses to control by interferon-alpha. Retrovirology 2013;10:146.

[124] Liao HX, Tsao CY, Alam SM, Muldoon M, Vandergrift N, Ma BJ, et al. Antigenicity and immunogenicity of transmitted/founder, consensus, and chronic envelope glycoproteins of human immunodeficiency virus type 1. J Virol 2013;87:4185-201.

[125] Parker ZF, Iyer SS, Wilen CB, Parrish NF, Chikere KC, Lee FH, et al. Transmitted/founder and chronic HIV-1 envelope proteins are distinguished by differential utilization of CCR5. J Virol 2013;87:2401-11.

[126] Parrish NF, Wilen CB, Banks LB, Iyer SS, Pfaff JM, Salazar-Gonzalez JF, et al. Transmitted/founder and chronic subtype C HIV-1 use CD4 and CCR5 receptors with equal efficiency and are not inhibited by blocking the integrin alpha4beta7. PLoS Pathog 2012;8:e1002686.

[127] Wilen CB, Parrish NF, Pfaff JM, Decker JM, Henning EA, Haim H, et al. Phenotypic and immunologic comparison of clade B transmitted/founder and chronic HIV-1 envelope glycoproteins. J Virol 2011;85:8514-27.

[128] Salazar-Gonzalez JF, Salazar MG, Keele BF, Learn GH, Giorgi EE, Li H, et al. Genetic identity, biological phenotype, and evolutionary pathways of transmitted/founder viruses in acute and early HIV-1 infection. J Exp Med 2009;206:1273-89.

[129] Marlink R, Kanki P, Thior I, Travers K, Eisen G, Siby T, et al. Reduced rate of disease development after HIV-2 infection as compared to HIV-1. Science 1994;265:1587-90.

[130] Gao F, Bailes E, Robertson DL, Chen Y, Rodenburg CM, Michael SF, et al. Origin of HIV-1 in the chimpanzee Pan troglodytes troglodytes. Nature 1999;397:436-41.

[131] Ariyoshi K, Jaffar S, Alabi AS, Berry N, Schim van der Loeff M, Sabally S, et al. Plasma RNA viral load predicts the rate of CD4 T cell decline and death in HIV-2-infected patients in West Africa. AIDS 2000;14:339-44.

[132] Berry N, Ariyoshi K, Balfe P, Tedder R, Whittle H. Sequence specificity of the human immunodeficiency virus type 2 (hiv-2) long terminal repeat u3 region in vivo allows subtyping of the principal hiv-2 viral subtypes a and b. AIDS research and human retroviruses 2001;17:263-7.

[133] O'Donovan D, Ariyoshi K, Milligan P, Ota M, Yamuah L, Sarge-Njie R, et al. Maternal plasma viral RNA levels determine marked differences in mother-to-child transmission rates of HIV-1 and HIV-2 in The Gambia. MRC/Gambia Government/University College London Medical School working group on mother-child transmission of HIV. AIDS 2000;14:441-8.

[134] Simon F, Mauclere P, Roques P, Loussert-Ajaka I, Muller-Trutwin MC, Saragosti S, et al. Identification of a new human immunodeficiency virus type 1 distinct from group M and group O. Nat Med 1998;4:1032-7.

[135] McCutchan FE, Viputtigul K, de Souza MS, Carr JK, Markowitz LE, Buapunth P, et al. Diversity of envelope glycoprotein from human immunodeficiency virus type 1 of recent seroconverters in Thailand. AIDS Research Hum Retroviruses 2000;16:801-5.

[136] Mastro TD, Kunanusont C, Dondero TJ, Wasi C. Why do HIV-1 subtypes segregate among persons with different risk behaviors in South Africa and Thailand? AIDS 1997;11(:113-6.

[137] Ou CY, Takebe Y, Weniger BG, Luo CC, Kalish ML, Auwanit W, et al. Independent introduction of two major HIV-1 genotypes into distinct high-risk populations in Thailand. Lancet 1993;341:1171-4.

[138] Van Harmelen JH, Van der Ryst E, Loubser AS, York D, Madurai S, Lyons S, et al. A predominantly HIV type 1 subtype C-restricted epidemic in South African urban populations. AIDS Research Hum Retroviruses 1999;15:395-8.

[139] Lole KS, Bollinger RC, Paranjape RS, Gadkari D, Kulkarni SS, Novak NG, et al. Full-length human immunodeficiency virus type 1 genomes from subtype C-infected seroconverters in India, with evidence of intersubtype recombination. J Virol 1999;73(1):152-60.

[140] Joint United Nations Programme on HIV/AIDS (UNAIDS). The Gap Report. Geneva: UNAIDS; 2014.

[141] Pierson T, McArthur J, Siliciano RF. Reservoirs for HIV-1: mechanisms for viral persistence in the presence of antiviral immune responses and antiretroviral therapy. Ann Rev Immunol 2000;18:665-708.

[142] Allers K, Hutter G, Hofmann J, Loddenkemper C, Rieger K, Thiel E, et al. Evidence for the cure of HIV infection by CCR5Delta32/Delta32 stem cell transplantation. Blood 2011;117:2791-9.

[143] Hutter G, Nowak D, Mossner M, Ganepola S, Mussig A, Allers K, et al. Long-term control of HIV by CCR5 Delta32/Delta32 stem-cell transplantation. N Engl J Med 2009;360:692-8.

[144] Henrich TJ, Hu Z, Li JZ, Sciaranghella G, Busch MP, Keating SM, et al. Long-term reduction in peripheral blood HIV type 1 reservoirs following reduced-intensity conditioning allogeneic stem cell transplantation. J infect Dis 2013;207:1694-702.

[145] Henrich TJ, Hanhauser E, Marty FM, Sirignano MN, Keating S, Lee TH, et al. Antiretroviral-Free HIV-1 Remission and Viral Rebound After Allogeneic Stem Cell Transplantation: Report of 2 Cases. Ann Intern Med 2014;161:319-27.

[146] Martinson JJ, Chapman NH, Rees DC, Liu YT, Clegg JB. Global distribution of the CCR5 gene 32-basepair deletion. Nat Genet 1997;16:100-3.

[147] Petit N, Dorgham K, Levacher B, Burlion A, Gorochov G, Marodon G. Targeting both viral and host determinants of human immunodeficiency virus entry, using a new lentiviral vector coexpressing the T20 fusion inhibitor and a selective CCL5 intrakine. Hum Gene Ther Methods 2014;25:232-40.

[148] Savkovic B, Nichols J, Birkett D, Applegate T, Ledger S, Symonds G, et al. A quantitative comparison of anti-HIV gene therapy delivered to hematopoietic stem cells versus CD4+ T cells. PLoS Comput Biol 2014;10:e1003681.

[149] Tebas P, Stein D, Tang WW, Frank I, Wang SQ, Lee G, et al. Gene editing of CCR5 in autologous CD4 T cells of persons infected with HIV. N Engl J Med 2014;370:901-10.

[150] Hofer U, Henley JE, Exline CM, Mulhern O, Lopez E, Cannon PM. Pre-clinical modeling of CCR5 knockout in human hematopoietic stem cells by zinc finger nucleases using humanized mice. J Infect Dis 2013;208 Suppl 2:S160-4.

[151] Didigu CA, Wilen CB, Wang J, Duong J, Secreto AJ, Danet-Desnoyers GA, et al. Simultaneous zinc-finger nuclease editing of the HIV coreceptors ccr5 and cxcr4 protects CD4+ T cells from HIV-1 infection. Blood 2014;123:61-9.

[152] Symonds GP, Johnstone HA, Millington ML, Boyd MP, Burke BP, Breton LR. The use of cell-delivered gene therapy for the treatment of HIV/AIDS. Immunol Res 2010;48:84-98.

[153] Younan PM, Polacino P, Kowalski JP, Peterson CW, Maurice NJ, Williams NP, et al. Positive selection of mC46-expressing CD4+ T cells and maintenance of virus specific immunity in a primate AIDS model. Blood 2013;122:179-87.

[154] Suzuki K, Hattori S, Marks K, Ahlenstiel C, Maeda Y, Ishida T, et al. Promoter targeting shRNA suppresses HIV-1 infection in vivo through transcriptional gene silencing. Mol Ther Nucleic Acids 2013;2:e137.

[155] Suzuki K, Ishida T, Yamagishi M, Ahlenstiel C, Swaminathan S, Marks K, et al. Transcriptional gene silencing of HIV-1 through promoter targeted RNA is highly specific. RNA Biol 2011;8:1035-46.

[156] Suzuki K, Kelleher AD. Transcriptional regulation by promoter targeted RNAs. Curr Top Med Chem 2009;9:1079-87.

[157] Pace MJ, Agosto L, O'Doherty U. R5 HIV env and vesicular stomatitis virus G protein cooperate to mediate fusion to naive CD4+ T Cells. J Virol 2011;85:644-8.