HUMAN IMMUNODEFICIENCY VIRUS TYPE I AS A TARGET FOR GENE THERAPY

Magnús Gottfredsson and Paul R. Bohjanen

Division of Infectious Diseases and International Health, Department of Medicine, Duke University Medical Center, Durham, NC 27710

Received 11/17/97 Accepted 11/24/97

3. HIV-1 LIFE CYCLE

It has recently become apparent that even during asymtomatic HIV-1 infection viral replication occurs at a very rapid rate. High-level virus replication occurs primarily in the lymph nodes, producing over 1-10 billion virions every day and destroying a similar number of T cells in the process (4, 5). A detailed understanding of the viral replication cycle (figure 1) and the functions of the viral gene products (figure 2) is needed in order to design effective antiviral strategies.

Figure 1. The HIV-1 life cycle.

A schematic presentation of the replicative cycle of HIV-1 is shown in figure 1. The replicative cycle starts by binding of the virus envelope glycoprotein gp120 to CD4 on the cell surface (6-8). The viral envelope protein gp41 then fuses with the cell membrane, a process which requires a cellular coreceptor. Recent studies have identified the chemokine receptor molecules CCR-5 (9-13) and CXCR-4 (14, 15) as the major coreceptors required for entry of HIV-1. Other coreceptors have been identified (10, 12), which may also play roles in HIV-1 infection. Macrophage-tropic strains of HIV-1, which constitute the vast majority of virus present in newly infected individuals seem to use CCR-5, while T-cell tropic strains which generally appear late in the course of infection use CXCR-4 (16-18).

After fusion of the viral envelope with the cell membrane, the viral core enters the cytoplasm where viral RNA is immediately transcribed by the viral enzyme reverse transcriptase (RT) into double-stranded cDNA. RT has three activities essential for retrovirus replication; it is an RNA-dependent DNA polymerase (i.e. reverse transcriptase), but also has DNA-dependent DNA polymerase activity (for synthesis of the second strand of the proviral DNA) and an RNase H activity. After synthesis of the viral cDNA, a nucleoprotein complex (or preintegration complex) containing the viral cDNA, matrix protein, integrase and other viral proteins, is translocated to the cell nucleus through connection with the cell nuclear import pathway (19). The Vpr accessory protein mediates transfer of the preintegration complex to the cell nucleus (19), making it possible for HIV to infect nondividing cells (20-22). The proviral DNA is then integrated into the host genome into regions of active transcription by the action of viral integrase (23). The integration process requires a host chromosomal protein in addition to the viral integrase (24).

The integrated provirus is flanked by long terminal repeats (LTRs) at each 5' and 3' end of the genome (figure 2A). The LTRs consist of repeated segments which are termed U3, R and U5. The LTRs contain the promoter sequences and the transcript polyadenylation signal (25). The viral genome contains structural and regulatory genes that are necessary for viral replication. The HIV-1 provirus uses the host cell's transcription machinery to produce the RNA transcripts necessary for viral replication. HIV-1 transcription is regulated in large part by the viral promoter which is located in the 5' long terminal repeat of the viral genome (see figure 2B). Host cellular RNA polymerase II transcription complexes are recruited to the HIV-1 promoter by specific interactions with promoter sequences. The HIV-1 promoter is similar to endogenous cellular promoters found within host cells in that it contains a canonical RNA polymerase II TATA box as well as binding sites for cellular transcription factors (for a review see reference 26). In addition, the HIV-1 promoter contains a non-conventional initiator element that appears to be unique to HIV-1 but is similar to cellular initiator elements (26, 27). The core promoter consists of the TATA box, initiator element, and three tandem binding sites for the constitutively expressed cellular transcription factor Sp1, and all of the elements necessary for transcription initiation in vitro are found in this core region (27). An upstream enhancer contains two binding sites for the inducible cellular transcription factor NF-kB and binding sites for a number of other cellular transcription factors including LEF, Ets-1, USF, NF-AT, and AP-1 (26, 27).

Figure 2. The HIV-1 genome. A. A schematic representation of the HIV-1 gene products encoded by the HIV-1 genomic sequence. B. A schematic representation of the HIV-1 promoter which is located within the 5' LTR of the viral genome. The core promoter consists of the initiator region, the TATA box, and three Sp1 sites. An upstream enhancer contains binding site for several cellular transcription factors including NF-kB, LEF, Ets-1, USF, NF-AT, and AP-1.

Although the cellular transcription machinery can sustain a low basal level of HIV-1 transcription, the viral protein Tat is required for high level HIV-1 transcription and activates HIV-1 transcription by greater than 100-fold (28-30). In the absence of Tat, most of the RNA transcripts produced from the HIV-1 promoter are short transcripts resulting from early transcription termination, whereas in the presence of Tat, the number of long full-length HIV-1 transcripts increases (31-34). Several cellular proteins have been shown to bind to Tat in vitro, including several components of the transcription machinery (32, 35-39). Tat has also been shown to bind to the RNA polymerase II holoenzyme (40, 41). The functional significance of these interactions is not clear, but it is possible that transcription complexes that contain Tat are capable of elongation competence, whereas transcription complexes lacking Tat produce predominantly short transcripts. Activation of HIV-1 transcription by Tat depends on an intact trans-activation response (TAR) RNA element. TAR is a 59 nucleotide RNA stem-loop structure that forms the 5' end of all HIV-1 transcripts (see figure 3). The TAR structure consists of two stems, a four nucleotide bulge, and a six nucleotide loop (42-46). Tat binds to the TAR bulge region in vitro (47-49) and may bind to the same region in vivo since mutations in the TAR bulge that inhibit Tat binding in vitro also inhibit Tat-mediated transcriptional activation in vivo (30, 50, 51). Interestingly, mutations in the TAR loop that have little or no effect on Tat binding in vitro abolish Tat-mediated transcriptional activation in vivo (52-55), suggesting that a cellular factor that recognizes the TAR loop is essential for Tat to activate HIV-1 transcription. Although the mechanism by which TAR RNA regulates Tat-mediated activation of HIV-1 transcription is still largely unknown, it has been suggested that RNA polymerase II transcription complexes that contain Tat bind to newly synthesized TAR RNA as it forms, and this binding activates the transcription complexes such that they become more elongation competent (56). This activation of RNA polymerase II transcription complexes may be mediated by a Tat-associated kinase which phosphorylates components of the transcription machinery (37, 57-61). A model for the regulation of HIV-1 transcription by Tat and TAR is depicted in figure 4.

Figure 3. The HIV-1 TAR sequence and structure.

Figure 4. A model for regulation of HIV-1 transcription by Tat and TAR. RNA polymerase II transcription complexes are recruited to the HIV-1 promoter (top panel). These complexes then begin transcripton of TAR RNA (middle panel). The newly synthesized TAR RNA interacts with Tat and cellular factors (bottom panel) and this interaction activates the transcription complex such that it becomes more elongation competent.

Early on in the viral replicative cycle, newly transcribed mRNA is spliced multiple times by the cellular splicing machinery. The Tat, Rev and Nef proteins are produced from these multiply spliced transcripts and subsequently start to accumulate. Tat activates transcription as described above and the Rev protein regulates transport of HIV-1 transcripts from the nucleus to the cytoplasm. Rev is a 116 amino-acid protein which localizes to the nuclei and nucleoli of infected cells (62). Rev binds to a major groove in the Rev-response element (RRE), a 210 nucleotide RNA sequence found in HIV-1 transcripts (63, 64). As Rev levels reach a critical level of expression, a progressive shift in viral mRNA production from multiply spliced (encoding Tat, Rev and Nef) to singly spliced and unspliced transcripts occurs (62). The Rev protein is required for transport of singly spliced and unspliced viral mRNA from the nucleus to the cytoplasm where these transcripts are subsequently used to synthesize structural proteins and viral enzymes (62, 65). A nuclear export signal (NES) has been identified in the Rev protein (66, 67). Presumably, the Rev-NES recognizes and binds to the nuclear export factor exportin 1 (68, 69, reviewed in 70) which subsequently shuttles the Rev-mRNA complex from the nucleus to the cytoplasm (figure 5). Rev-dependent transport of RRE-containing mRNA also involves other cellular factors, including the Rev/Rex activation domain-binding (Rab) protein (71) and possibly the Rev-interacting protein (Rip1p), a small nucleoporin-like protein (72, 73). Presumably, the interaction between Rev and these cellular factors directs the transport of RRE-containing transcripts to the nuclear pore (figure 5).

Figure 5. Rev-mediated HIV-1 mRNA export. Rev binds to a loop in the RRE sequence. Exportin 1, a cellular export factor, recognizes the nuclear export signal (NES) on Rev and the viral mRNA is subsequently transported by this complex from the cell nucleus through the nuclear pore to the cytoplasm where translation into proteins occurs. In addition to exportin 1, other cellular factors probably participate in this process.

The Nef protein is important for maintenance of high viral loads and for the development of AIDS (74). HIV infection leads to downregulation of surface expression of major histocompatibility complex class I (MHC-I) antigens (75, 76) and CD4 (77, 78) by endocytosis of these molecules from the cell surface. It has recently been shown that these effects are mediated by the Nef protein (78, 79). The stimulation of endocytosis by Nef could therefore represent a viral mechanism for evading the immune response because downregulation of MHC-I molecules makes HIV-infected cells less susceptible to lysis by cytotoxic T lymphocytes (75, 76, 80).

Once in the cytoplasm the unspliced viral mRNA is translated into a Gag-Pol polyprotein by a process that involves ribosomal frame shifting. The Gag-Pol polyprotein is cleaved by the viral protease into the mature virion structural proteins (matrix, capsid, and nucleocapsid) as well as the virion enzymes (protease, reverse transcriptase and integrase) (81). The viral envelope proteins are synthesized as the gp160 precursor polyprotein which is subsequently cleaved by cellular proteases into the external surface protein gp120 and the transmembrane envelope protein gp41. Viral assembly seems to be regulated at least in part by the accessory proteins Vpu and Vif (82, 83). Efficient packaging of genomic RNA of retroviruses into viral particles requires a specific nucleotide sequence, termed the y (psi) packaging sequence (84, 85). Packaging of HIV-1 RNA into virus particles then takes place, a process which involves an interaction between y and the zinc binding domains of the nucleocapsid protein (23, 86). Subsequently new virions are released from the surface of the host cell by budding and a new cycle can start.