This are the readings to answer the questions above: HIV Life Cycle: Overview Frank Kirchhoff Institute of Molecular Virology, Ulm University Medical Center, Ulm, Germany Definition The HIV life cycle defines the steps and changes the virus undergoes from its first contact with a target cell to the production of new infectious viral particles that can initiate the next round of replication. The combination of reverse transcription of viral RNA into DNA and integration of the latter into the host cell genome is a key feature of the retroviral replication cycle. Introduction The goal of this chapter is to provide a brief overview on the “life” cycle of HIV, which should perhaps be better referred to as the viral “replication” cycle, since viruses do not have their own metabolism and are thus usually not considered living organisms. Viruses can be considered as intracellular parasites that are strictly dependent on living host cells for reproduction. Understanding how HIV interacts with its target cells in order to replicate is of great interest because it may provide important clues for the generation of improved antiretroviral drugs and the development of novel strategies to control or even eliminate the virus. The main targets of HIV are CD4+ helper T cells, which are key regulators of the humoral and cellular immune responses. Thus, their destruction and depletion by mechanisms that are not fully understood render the body unable to defend itself against opportunistic pathogens. When HIV infects an activated CD4+ T cell, it hijacks and manipulates its transcriptional and translational machinery to reproduce itself. As briefly outlined below and specified in the following chapters, HIV has to utilize a multitude of cellular factors and to counteract the antiretroviral activity of others in order to complete its replication cycle. Theoretically, each interaction with cellular factors that are essential for virus replication (termed “virus-dependency” factors) or strengthening of antiretroviral (or “host restriction”) factors provides a potential means to interfere with the HIV life cycle. Although activated CD4+ helper T cells represent the main target cells for HIV replication, the virus can also infect other cell types, such as macrophages, immature dendritic cells, and more resting Teell subsets. The latter cell types are not important for the bulk of virus replication but most likely play important roles in innate and adaptive antiviral immune responses. Furthermore, they may harbor the virus in a silent integrated proviral form and thus contribute to the establishment and maintenance of viral reservoirs that prevent the eradication of HIV from the human body, even under optimized antiretroviral therapy (ART). Finding ways to activate these latent proviruses to eliminate the infected cells and to cure HIV infection is a main challenge in AIDS research. A better understanding of the steps in the viral life cycle, especially the regulation of proviral transcription, may help to achieve this HIV-1 provirus ROLTR tal D ma LTR wprovt RNA genome 2. PBS (RNAL” primer binding site) * PPT (Polypurine tract RRE TAR (Tat responsive dement Rev responsive element polyadenylation signal Representative transcripts Tat Rev, Net Net Early: RRE-Rev independent T Gag-Pol Vi Late RRE-Rev dependent Vpr – Vpu, Eny Fig. 1 Overview of the organization and expression of the HIV-1 genome. Some cis-regulatory elements in the viral RNA genome and representative early and late RNAs are presented. Stars indicate splice sites HIV Structure The HIV genome contains the typical retroviral genes gag, pol, and env flanked by long terminal repeats (LTRS), which contain the viral promoter (Fig. 1, upper). Gag codes for the structural proteins capsid (CA), matrix (MA), and nucleocapsid (NC); pol encodes the enzymes reverse transcriptase (RT), protease (PR), and integrase (IN); and env encodes the glycoproteins gp120 and gp41 (Swanson and Malim 2008). In addition, HIV has six regulatory genes (tat, rev, nef, vif, vpr, and vpu) and is thus considered a “complex” retrovirus. Tat enhances proviral transcription, and Rev is essential for the export of incompletely or unspliced viral mRNAs into the cytoplasm. The remaining genes were named “accessory” because they are not absolutely required for replication in some cell culture systems (Kirchhoff 2010). However, they perform a multitude of activities facilitating viral immune evasion and antagonize a variety of specific antiretroviral cellular (host restriction) factors and are thus essential for viral spread in vivo ( Viral Auxiliary Proteins). HIV-I belongs to the Lentivirus genus (lentis = slow) of the Retroviridae family. The virion has a spherical shape and a diameter of 100-130 nm (or 1/10,000 mm)(HIV-1 Virion Structure). The viral envelope is composed of a lipid membrane, which is derived from the host cell and contains cellular proteins, as well as about 7-12 trimeric complexes of viral envelope (Env) protein (Fig. 2). Env consists of the external glycoprotein 120 (gp120) that mediates viral attachment and the transmembrane glycoprotein 41 (gp41) that is critical for viral fusion. Gp41 is associated with the viral p17 matrix protein and encompasses a conical capsid that consists of the viral Gag protein, p24. The capsid contains two single strands of viral RNA with positive polarity and a length of close to 10,000 nucleotides. The RNA is associated with the nucleocapsid proteins, as well as the RT and IN. The virions also contain some copies of the viral protease and the accessory Vif, Vpr, and Nef proteins, as well as some cellular factors, such as tRNA!, which is used as primer for reverse transcription HIV-1 provirus LTR 029 villa Ovpu Pr rev! revan ILTR p17 p24 MA CA . O p9 p. p11 p51/66 p32 gp120 NC PR RT IN VIf Vpr SU 16 O gp41 TM 1 transmembrane- protein, TM, gp41 surface envelope glycoprotein su, gp120 nucleocapsid, NC, p6 tRNA matrix, MA, P17 reverse transcriptase RT, p66, p51 capsid, CA, p24 protease, PR, P10 Cyclophylin A integrase, IN, p32 .p6 Vpr membrane viral RNA cellular protein Vit Fig. 2 Schematic presentation of the expression of viral proteins that are found in the viral particle (upper) and of the mature HIV virion (lower) Overview on the HIV Replication Cycle The HIV-1 life cycle is complex and can roughly be divided in an early and a late phase of replication. The early phase begins with the attachment of the virion at the cell surface and ends with the integration of the proviral DNA into the host genome (Fig. 3). The late phase of replication starts with the initiation of proviral transcription and ends with the release of fully infectious progeny virions. In highly activated CD4+ T cells, the HIV life cycle lasts just one to two days and is associated with the programmed death of both virally infected cells and uninfected bystander cells. The viral life cycle illustrates some of the challenges associated with HIV infection. The viral RT has a very high mutation rate (~1 error per 10,000 nucleotides), and the viral populations in an infected individual are not uniform but rather a collection of the so-called quasispecies. Together with the short generation time and massive virus production (up to 2 x 10′ virions per day), this allows HIV to rapidly adapt to its host environment and to develop resistance against antiretroviral drugs or immune responses. To generate a higher barrier for the evolution of resistances, combination therapies are currently used to treat HIV. Furthermore, proviral integration into the host genome allows the virus to hide in long-living cells. Finally, HIV infects and kills CD4+ helper T cells that are crucial for the maintenance of a functional immune system. It is important to consider, however, that other cell types can also be infected and that the time frame of viral replication and the fate of the infected cells vary. Macrophages, for example, may produce HIV over several weeks and store infectious virions intracellularly (Macrophage-Specific Aspects of HIV-1 Infection). Although a lot of exciting progress has been made, we are still just beginning to understand the multitude of interactions of HIV with its host cell. 1. Entry Env proteins, gp120. gp41 11. Maturation Protease, p10 10. Budding Gag 9. Assembly Gag 2. Reverse Transcription Reverse Transpptase, p66, p51 8. Translation 3. Uncoating Capsid, p24 4. Nuclear Import Capsid, p24 7. RNA Export Rev 5. Integration Integrase, p32 Pro Transcription Tat Fig. 3 Overview of the viral replication cycle Viral Attachment Cell-free HIV virions usually have a half-life of just about 20-30 min in infected individuals. Thus, the virus must find and infect a new target cell within a very short time frame. As described below, CD4 is the primary receptor, and the chemokine receptors CCR5 and CXCR4 are the main co-receptors of HIV entry (Fusion). These receptors are sufficient to render cells susceptible to HIV entry and thus determine the viral cell tropism. However, the densities of the Env trimers on the virions and of the CD4 receptor on the target cells are frequently low. Thus, viral attachment is often inefficient and a limiting step for HIV infection. Several receptors, such as poly-glycans, lectins, and others, can bind HIV virions in a more unspecific manner and may thereby greatly increase viral infection rates (Attachment/Binding). On the one hand, they concentrate virions at the cell surface and facilitate their interaction with CD4 and a co-receptor to allow virion fusion. Furthermore, they may trap viral particles at the cell surface to stabilize them and to mediate trans infection of susceptible T cells. For example, it has been suggested that dendritic cells (DCs) bind HIV virions at the site of genital exposure and transport them to the lymph nodes where they mediate both trans infection and stimulation of T cells that results in massive virus production (van Kooyk and Geijtenbeek 2003). Binding and Fusion Viral entry is a complex multi-step process that offers multiple possibilities for therapeutic inter- vention (Didigu and Doms 2012). Either directly or following unspecific binding of HIV to its target cell, the infection process is initiated by the interaction of the external viral glycoprotein gp120 with the cellular CD4 receptor (Fusion). CD4 binding induces conformational changes in the Eny Page 4 of 9 trimer that allow the interaction of gp120 with either the CXCR4 (X4) or CCR5 (R5) co-receptor. Usually, only R5 viruses are sexually transmitted and found during chronic infection. In comparison, X4 HIV strains emerge late during the course of infection and are associated with rapid progression to AIDS in the absence of antiretroviral therapy. Co-receptor interaction induces additional confor- mational changes that allow the gp41 transmembrane protein, which is usually hidden by the gp120, to insert its hydrophobic fusion peptide into the cell membrane to make the first direct contact between the virus and its target cell. Thereafter, the trimeric gp41 complex forms a helical bundle structure which pulls the cellular and viral membranes together, thus allowing virion fusion and the release of the contents of the virus particle into the cell. Drugs that block the CCR5 co-receptor or prevent viral fusion are already used to treat HIV infection in the clinic, and other HIV entry inhibitors are in preclinical development Reverse Transcription Once fusion is completed, the genetic information of the virus can enter into the cell. The HIV genome consists of two plus-stranded RNAs that are protected by the nucleocapsid. After fusion, the single- stranded viral RNAs are transcribed into linear double-stranded DNAs by a process called reverse transcription (RT). It is called “reverse” transcription because it reverses the order of events that take place during the regular transcription process, i.e., generation of messenger RNA from nuclear DNA followed by export into the cytoplasm and protein synthesis. It is performed by an enzyme called reverse transcriptase that is characteristic – although not unique – to retroviruses and involves a very complex series of events that are outlined in chapter Reverse Transcriptase-Catalyzed HIV-I DNA Synthesis. The very first drugs against HIV inhibited reverse transcription. Currently, there are two different classes: nucleoside and nucleotide analog reverse transcriptase inhibitors (NRTIS), which prevent further elongation of the DNA chain, and non-nucleoside reverse transcriptase inhibitors (NNRTIS) that bind to the enzyme and renderit inactive (cross-link to ART section). Uncoating and Nuclear Entry Uncoating refers to the disassembly of the viral capsid before import of the viral genome into the nucleus. Untimely uncoating due to point mutations in the capsid protein or interactions with the tripartite motif 5-alpha protein (TRIM5a) impairs viral infectivity (Uncoating and Nuclear Entry). Early studies suggested that uncoating may occur immediately after viral entry. More recent data, however, suggest that capsids may remain intact for several hours and that this stability is critical for HIV-I infection. Furthermore, it seems that uncoating is tightly associated with reverse transcription and accompanies the transition of the reverse transcription complex (RTCs) to the pre-integration complex (PIC) that is competent for integration into the host cell genome (Arhel 2010). However, the exact timing of uncoating is still poorly understood and subject to intense research. Lentiviruses have the unique ability to infect nondividing terminally differentiated cells, such as macrophages; thus, the viral genome must be transported through an intact nuclear membrane. HIV-I PICs must be actively transported through the nuclear pore since they are too large to cross them by passive diffusion. Originally, the viral matrix protein, Vpr, and integrase have been implicated in nuclear entry. However, recent studies suggest that none of them are essential for infection of nondividing cells and that the HIV-1 capsid protein may play a key role in this process, although the underlying mechanisms remain to be fully elucidated. Integration After successful generation of linear ds DNA and its transport across the nuclear membrane, HIV must insert its genome into that of the host cell for gene expression and productive infection (Fig. 3). Once the viral DNA is inserted into the cellular DNA by an enzyme called integrase ( Integration), the cell is usually infected for the remainder of its life span. As part of the host cell chromosome, the proviral DNA is replicated along with the host DNA. Thus, spread of infection can be achieved either by infection of new cells or by multiplication of cells already containing proviral DNA. Notably, several compounds that inhibit viral integration are now successfully used in the clinic (cross-link to ART section). In some long-living cells such as memory T cells, the integrated viral genome, which is referred to as a provirus, may remain silent for many years. This constitutes a main problem for viral eradication because as long as the provirus remains inactive, these cells are not recognized by the immune system and thus not eliminated. Sometimes the proviruses may become activated and produce infectious HIV that damages the immune system if ART is discontinued after many years. Whether or not a cell becomes latently or productively infected depends on the type and state of activation of the infected target cells during infection as well as subsequent exogenous stimulation (Colin and Van Lint 2009). The viral reservoirs are not fully defined, but quiescent CD4+ T cells seem to constitute an important part of them. Transcription In productively infected cells, the integrated HIV provirus serves as a template for the transcription of both viral messengers and genomic RNA by the cellular Pol II polymerase (Transcription (Initiation, Regulation, Elongation)). Proviral transcription is initiated by the viral promoter, which is located in the U3 region of the 5′ LTR and active in many cell types. Viral gene expression is strictly dependent on cellular transcription factors, such as NF-kB and NFAT. Initially, the tran- scriptional output is low because elongation of viral transcripts is very inefficient and the viral transactivator protein Tat is required for effective viral gene expression (Tat Expression and Function). Tat binds to a specific sequence in the R region of the 5′ LTR, named the trans-acting response (TAR) element, to increase transcriptional processivity (Fig. 1). This effect is dependent on the cellular factor pTEFb. Tat allows the efficient synthesis of full-length HIV transcripts, and more than 25 different mRNAs in three size classes are generated by alternative splicing: (i) unspliced RNA (9 kb) serving as genomic RNA or to produce the Gag and Gag-Pol precursors; (ii) singly spliced (4 kb) RNA encoding Vif, Vpr, Vpu, and Env; or (iii) fully spliced (2 kb) RNA expressing Tat, Rev, and Nef (Fig. 1). Transport of unspliced and partially spliced mRNAs from the nucleus to the cytoplasm is mediated by the viral Rev protein which interacts with the rev responsive element (RRE) in the viral RNA and the cellular export factor Crml to connect these viral RNAs to the export machinery (Rev Expression and Function). Translation and Assembly As mentioned above, the fully spliced viral RNAs that are initially generated encode for Tat, Rev, and Nef. Tat boosts viral transcription and RNA elongation, and Rev mediates the transport of unspliced and partially spliced viral RNAs to the cytoplasm. Nef performs a large number of functions and basically seems to make the infected cell less visible to the immune system by down-modulation of several surface receptors, such as CD4 and class I MHC, and manipulates the cells in a way that they become more effective producers of fully infectious viral particles. Synthesis of Tat and Rev allows the generation of full-length unspliced mRNA that expresses the Gag and Gag-Pol precursors which are then processed to major structural and enzymatic proteins. In parallel, the Vif, Vpr, Vpu, and Env proteins are synthesized from single spliced viral RNAs (Fig. 1). Viral assembly is a complex and highly ordered process (Virus Assembly). In brief, the Gag and Gag-Pol precursors multimerize via interactions between Gag proteins. Furthermore, both pre- cursors are N-terminally myristoylated in the matrix domain and thus concentrated in lipid rafts at the inner leaflet of the plasma membrane (Ganser-Pornillos et al. 2008). The viral Env glycoproteins are recruited to these building platforms through interactions with the matrix protein. Finally, two copies of genomic viral RNA are recruited to this complex through interactions of their stem loops packaging signal with the zinc fingers present in the NC domain of Gag (Fig. 3). Furthermore, the viral Vif protein, which antagonizes the restriction factor APOBEC3G, as well as some cellular factors, are also recruited to the sites of virion assembly and incorporated into viral particles. The accumulation of viral proteins and RNA at the plasma membrane induces first its curvature and subsequently the formation of a membrane-coated spherical particle. Budding The release of progeny virions from the infected cells is called budding. The late domain of the p6 part of Gag and the cellular Tsg101 protein are involved in this step which allows newly formed HIV to pinch off and enter into the circulation ( Budding). Notably, also this late step of the viral replication cycle is targeted by a restriction factor: tetherin (BST-2) tethers mature and infectious viruses to the cell surface and is counteracted by the HIV-1 Vpu and the Nef or Env proteins of other primate lentiviruses (Martin-Serrano and Neil 2011). Maturation The HIV particles are released in an immature and noninfectious form that is morphologically characterized by a thick layer of radially arranged Gag and Gag-Pol precursors (Maturation). During or shortly after budding, the viral protease becomes activated and cleaves the Gag and Gag-Pol precursors into their mature final components. As a consequence, the configuration of the proteins is reorganized to generate the characteristic electron-dense conical inner core and to render the virus infectious (Briggs and Kräusslich 2011). Drugs that block this last essential step of the viral life cycle by inhibiting the viral protease are a main component of effective ART. Viral Dependency and Host Restriction Factors It has long been known that viruses are strictly dependent on live host cells in order to replicate. Only recently, however, it has become clear that these interactions may be far more complex than previously appreciated. Several studies have used genome-wide knockout strategies in order to better assess the cellular factors that may be critical for effective replication of HIV. All of them found that HIV may utilize hundreds of cellular proteins in order to complete its life cycle. Similarly, a recent study performed elegant broad-based screens to clarify how many cellular proteins interact with viral proteins and identified about 500 of them (Jäger et al. 2011). Altogether, these studies provide an exciting first glimpse at the enormous complexity of interactions between HIV and the host cell. However, there are some caveats. For example, the overlap between the potential viral dependency factors in the genome-wide knockout studies was minimal (Bushman et al. 2009) most likely because of variations in the cell types, viral strains, and experimental conditions used. Furthermore, it is difficult to assess how many other cellular proteins may be affected by each individual knockdown. Similarly, most interaction studies cannot distinguish between direct inter- actions between viral and host cell proteins or interactions with larger protein complexes. Nonethe- less, these studies have provided exciting first insights into the enormous complexity of interactions between the virus and the host cell and may help to identify additional key factors involved in HIV replication. Although HIV hijacks the host cell and takes advantage of many cellular proteins and pathways, it has become clear that the cell is not a very friendly environment for the virus, because it encodes specific antiviral factors that may inhibit HIV at various steps of its life cycle and are counteracted by the viral accessory proteins ( Cellular Restriction Factors). In brief, the best characterized antiviral or host restriction factors are APOBEC3G which induces lethal hyper-mutation of the retroviral genome and affects reverse transcription, TRIMS, protein that induces untimely uncoating of incoming retroviral capsids, and tetherin that inhibits virion release. Unfortunately, HIV-1 evolved effective strategies to evade or counteract these antiviral host factors (Kirchhoff 2010). Target Cell Dependency The main target cells for viral replication in vivo are activated CD4+ T cells, and the viral life cycle illustrated in Fig. 3 provides a rough overview of the events in this cell type. Similarly, almost all studies on the HIV replication cycle have been performed using immortalized cell lines or fully activated T cells. In vivo, however, many T cells are minimally activated. This may not be very important for the bulk of virus replication but most likely highly relevant for the establishment of latent viral reservoirs and for the pathogenesis of AIDS. For example, some long-living memory T cells may return to a quiescent stage upon HIV infection and not show any gene expression for several years before they finally become activated and start to produce infectious HIV particles. Cells that carry the provirus but do not produce viral proteins cannot be recognized and eliminated by the immune system and constitute a major obstacle to virus elimination. Furthermore, it is important to consider that HIV does not only infect T cells and that the viral replication cycle may be somewhat different in other cell types. Macrophages, for example, are also productively infected by HIV and may contribute to the viral reservoirs and facilitate viral evasion of the blood-brain barrier (Macrophage-Specific Aspects of HIV-1 Infection). Furthermore, HIV may also infect immature dendritic cells, microglial cells, and (possibly) stem cells although the relevance of these and other potential target cells of HIV for viral spread and pathogenesis in vivo is currently largely unclear. Conclusions and Perspectives Although enormous progress has been made in understanding the complex events and interactions that are critical for the life cycle of HIV, a lot remains to be leamed. It will be essential to further clarify which host dependency factors are really critical for viral replication in the human host. Encyclopedia of AIDS DOI 10.1007/978-1-4614-9610-6_60-1 Springer Science-Business Media New York 2013 Currently, only a single cellular factor (CCR5) is targeted for antiviral treatment. Preventing the use of other host cell proteins that are obligatory for viral replication may lead to the development of novel antiretroviral strategies that make it more difficult for HIV to develop resistance. Furthermore, it seems likely that additional restriction factors will be discovered and strengthening them or weakening the viral antagonists may also allow to impair the viral replication cycle at different steps. It will also be important to further clarify why some T cells get stuck after the early stage of infection and become latently infected and how to stimulate them in order to eliminate the viral reservoirs. Furthermore, recent studies suggest that the protection of specific T cell subsets may play a major role in the lack of disease progression in some monkey species that are naturally infected with simian immunodeficiency viruses. Since the fate and type of the viral target cell determine the damage for the individual, it seems important to further study target cell-dependent differences in the viral replication cycle. Finally, it is important to consider that in vivo HIV is mainly replicating in lymphoid organs that are densely packed with different cell types and may spread through direct cell-cell contact References Arhel N. Revisiting HIV-1 uncoating. Retrovirology. 2010;7:96. Briggs JA, Kräusslich HG. The molecular architecture of HIV. J Mol Biol. 2011;410(4):491–500. Bushman FD, Malani N, Fernandes J, D’Orso I, Cagney G, Diamond TL, Zhou H, Hazuda DJ, Espeseth AS, König R, Bandyopadhyay S, Ideker T, Goff SP, Krogan NJ, Frankel AD, Young JA, Chanda SK. Host cell factors in HIV replication: meta-analysis of genome-wide studies. PLoS Pathog. 2009;5(5):e1000437. Colin L, Van Lint C. Molecular control of HIV-1 postintegration latency: implications for the development of new therapeutic strategies. Retrovirology. 2009;6:111. Didigu CA, Doms RW. Novel approaches to inhibit HIV entry. Viruses. 2012;4:309–24. Ganser-Pornillos BK, Yeager M, Sundquist WI. The structural biology of HIV assembly. Curr Opin Struct Biol. 2008;18(2):203-17. Jäger S, Cimermancic P, Gulbahce N, Johnson JR, McGover KE, Clarke SC, Shales M, Mercenne G, Pache L, Li K, Hernandez H, Jang GM, Roth SL, Akiva E, Marlett J, Stephens M, D’Orso I, Fernandes J, Fahey M, Mahon C, O’Donoghue AJ, Todorovic A, Morris JH, Maltby DA, Alber T, Cagney G, Bushman FD, Young JA, Chanda SK, Sundquist WI, Kortemme T, Hernandez RD, Craik Cs, Burlingame A, Sali A, Frankel AD, Krogan NJ. Global landscape of HIV-human protein complexes. Nature. 2011;481(7381):365–70. Kirchhoff F. Immune evasion and counteraction of restriction factors by HIV-1 and other primate lentiviruses. Cell Host Microbe. 2010;8(1):55-67. Martin-Serrano J, Neil SJ. Host factors involved in retroviral budding and release. Nat Rev Microbiol. 2011;9(7):519-31. Quashie PK, Sloan RD, Wainberg MA. Novel therapeutic strategies targeting HIV integrase. BMC
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