HIV Lifecycle: History
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Contributor:
Joseph Hokello
,
Kratika Tyagi
,
Richard Oriko Owor
,
Adhikarimayum Lakhikumar Sharma
,
Alok Bhushan
,
Rene Daniel
,
Mudit Tyagi

The theory of immune regulation involves a homeostatic balance between T-helper 1 (Th1) and T-helper 2 (Th2) responses. The Th1 and Th2 theories were introduced in 1986 as a result of studies in mice, whereby T-helper cell subsets were found to direct different immune response pathways. Subsequently, this hypothesis was extended to human immunity, with Th1 cells mediating cellular immunity to fight intracellular pathogens, while Th2 cells mediated humoral immunity to fight extracellular pathogens. Several disease conditions were later found to tilt the balance between Th1 and Th2 immune response pathways, including HIV infection, but the exact mechanism for the shift from Th1 to Th2 cells was poorly understood. 

  • HIV
  • HIV life cycle
  • viral transmission

1. Entry and Reverse Transcription

HIV specifically infects cells that express CD4 molecules on their surface, including macrophages, helper T cells and microglial cells. During entry, HIV interacts with both the CD4 receptor [1] and co-receptor, either CCR5 [2] or CXCR4, through its envelope glycoproteins (gp120) [3]. On the virus surface, the HIV gp120 forms trimers, with each monomer consisting of the gp120 and gp41 subunits. The gp120 domain interacts with the CD4 molecule and undergoes conformational changes on target cells that allow gp120 to subsequently interact with viral CCR5 or CXCR4 co-receptors [4]. Trimolecular complex formation stabilizes the binding of the virus and triggers additional changes in conformation in the gp41, which promotes fusion with the membrane and entry into the cell cytoplasm [4]. Following fusion events, the capsid core of HIV-1 gets degraded in a process referred to as uncoating, which was previously thought to occur within the cytoplasm to release a high molecular weight reverse transcription complex (RTC) [5]. However, recent reports have revealed that uncoating occurs in the nucleus of the target cell [6][7]. Apart from the CD4 molecule and the chemokine co-receptors, other HIV receptors have been reported. For instance, Dendritic cell (DC)-specific intracellular adhesion molecule (ICAM)-3-grabbing non-integrin (DC-SIGN), which is a c-type lectin, is important in disseminating HIV-1 to T cells by DCs, especially during HIV-1 mucosal transmission [8][9]. Similarly, Langerhans cells (LCs), particularly immature LCs, which are the first DC subsets that encounter HIV in the mucosa, are reported to inhibit the transfer of HIV-1 to T cells through interaction with a receptor called langerin, a c-type lectin [10][11][12]. Cicala et al. also reported that high levels of integrin α4β7, which is a gut-homing receptor, are expressed by mucosal CD4+ T cells. They observed that in the mucosa, the HIV-1 envelope first interacts with α4β7 on CD4+ T cells in close association with CD4 and CCR5 molecules and that this initial interaction of the envelope with α4β7 promotes efficient virus capture and facilitates efficient HIV infection of T cells [12]. Uniquely, retroviruses, including HIV, have the ability to convert their genomic RNA into cDNA following entry into a target cell, a reaction catalyzed by the reverse transcriptase (RT) enzyme of the virus [13]. The viral RT catalyzes the conversion of the single-strand HIV genomic RNA into complementary cDNA. The RNA template strand is then degraded by the RNase H enzyme; the DNA-dependent DNA polymerase activity of RT then converts the single-stranded cDNA into double-stranded DNA, referred to as the provirus. The heterodimeric RT enzyme consists of two subunits: a larger 66 KDa and a smaller 51 KDa subunit. Reserve transcription occurs within the RTC initiated by cellular tRNA, which, after binding to the primer binding site (pbs), leads to the generation of an RNA–DNA hybrid molecule. The U5 region within the 5′ LTR of the viral genome harbors the pbs. The RNase H enzyme, which is part of the RT holoenzyme, then degrades the RNA strand within the RNA–DNA hybrid, creating the DNA minus-strand strong stop [13]. In a process referred to as first-strand transfer, the DNA minus-strand strong stop then shifts to the 3′ ends from the 5′ ends of the RNA to prime the synthesis of the minus strand of the viral cDNA. Fragments of RNA resulting from minus-strand synthesis then bind to purine-rich sequences called polypurine tract (PPT) to initiate the synthesis of the viral plus-strand cDNA, resulting in double-stranded proviral cDNA. Recent studies have, however, demonstrated that there is a dynamic interplay between viral uncoating, reverse transcription and nuclear import [14][15][16]. For instance, a study involving single HIV-1 infection dynamics revealed that the CA protein enables the core docking of the pre-integration complex (PIC) at the nuclear envelope, suggesting that complete uncoating does not occur immediately following the entry of the viral core into the cytoplasm [14]. Similarly, other reports indicated that HIV reverse transcription is intimately linked to CA disassembly, whereby reverse transcription mechanically initiates CA disassembly [15][16].

2. Integration

Following the process of reverse transcription, HIV cDNA enters the nucleus as PIC with the help of Vpr [5][13]. Nuclear entry of the PIC is mediated by the Vpr through interactions with the host cell nuclear import machinery. The enzyme integrase (IN) within the PIC subsequently catalyzes the integration reaction of HIV cDNA into the chromosome of the host cell. Like all retroviruses and lentiviruses, HIV-1 requires the integration of its cDNA into the host cell DNA. Therefore, the host cell’s metabolic state influences the activity of the integrated provirus [17]. Integration of the HIV cDNA into the host chromosome is a very important step for retrovirus multiplication. For HIV-1, a 32 KDa viral integrase (IN) enzyme catalyzes the integration of viral cDNA into the host chromosomal DNA [5]. In a reaction referred to as 3′ end processing, viral cDNA integration begins with the clipping of several nucleotides from the 3′ end of both strands of the HIV cDNA, generating a cDNA with double strands with 3′-recessed ends. Similarly, IN also cleaves cellular genomic DNA at the sites of integration, after which viral cDNA is ligated to the cleaved cellular DNA ends in a process referred to as strand transfer. The resultant integrated viral cDNA is referred to as the provirus, which essentially behaves like a host gene. The nucleotide gaps between the newly integrated provirus and the host chromosome are repaired by the cellular enzymes to complete the integration process [13]. The HIV provirus now becomes the template for viral mRNA species synthesis, which codes for a full complement of structural, accessory and regulatory proteins of the virus required for replication and virulence [18][19].

3. Transcription

The HIV-1 LTR contains multiple cis-regulatory elements and serves as the site of HIV-1 transcriptional initiation and regulation. The HIV LTR consists of three distinct regions, namely the unique 3′ end (U3), the repeated (R) sequence and the unique 5′ (U5) domains. The promoter region is present within the U3 elements. The promoter mediates the binding of RNA polymerase II (RNAP II) and other crucial components of the transcription machinery. The TATA-box is located at the −28 nucleotide position upstream of the +1 transcription start site. Other important transcription factors, including Sp1 (3 sites) and NF-ĸB (2 sites), are located at the 5′ end of the TATA box [18][19][20]. The binding of a highly conserved 38 KDa TATA box-binding protein (TBP) to the TATA box initiates HIV-1 transcription from the LTR promoter. Additional transcription factors are recruited as a result of the TBP binding to form the pre-initiation complex known as the TBP-associated factors (TAFs). The resultant complex formed is made up of multiple proteins comprising TBP and TAF, referred to as TFIID, which, along with three SP1 binding sites, constitutes the minimal transcription complex that can induce basal HIV LTR promoter transcription. However, for efficient HIV LTR promoter-mediated transcription, it requires the TFIID interaction with upstream enhancer binding factors such as NF-ĸB, AP-1 or NFAT [13][21][22][23][24][25][26][27][28][29]. Transcription factor II H (TFIIH) exhibits kinase activity (CDK7) required for promoter clearance by phosphorylating the C-terminal domain (CTD) of RNAP II, whose recruitment to the HIV LTR promoter is reported to be the major determinant in HIV transcription, especially HIV-1 transcription initiation [19][30][31].
Without the viral Tat protein in the system, transcription of the HIV provirus is not efficient, and transcribing RNAP II that initiates HIV-1 gene expression disengages after a few nucleotides following transcription initiation. However, complete synthesis of HIV-1 mRNA increases when viral Tat is present due to increased efficiency in transcription elongation mediated by Tat, resulting in the synthesis of full-length viral mRNA transcripts [32][33][34]. Tat functions by unusually binding to an RNA element known as the transcription response region (TAR), formed by the first 59 nucleotides. TAR, a stem-loop structure, is present at the 5′ end of all nascent viral mRNA transcripts [19]. When viral Tat binds to TAR, Tat brings P-TEFb, a critical cellular factor that plays a vital role during the transcriptional elongation of cellular genes. P-TEFb predominantly comprises cyclin T1 and the kinase subunit, the cyclin-dependent kinase 9 (CDK9). This kinase component of P-TEFb, CDK9, catalyzes several events, including hyperphosphorylation of the CTD of the largest RNAP II subunit, which makes RNAP II more processive. The processive RNAP II subsequently leads to the generation of complete HIV genomic transcripts required to form new HIV progenies [13].
Transcription from the HIV-1 LTR promoter results in the generation of three categories of viral mRNA transcripts: small, multiply spliced mRNAs (~2 Kb) known to code for regulatory proteins Tat, Nef and Rev of HIV-1; singly spliced mRNAs (~5 Kb) known to code for Env, Vif, Vpu and Vpr proteins; and unspliced RNA (~9 Kb), which acts as mRNA to encode Gag and Gag-Pol polyprotein precursors, in addition to serving as full-length genomic mRNA (gRNA) for packaging into new HIV-1 virion [13][19]. In eukaryotes, mRNAs are spliced in the nucleus before their export for translation in the cytoplasm. Export of large singly spliced or unspliced HIV-1 RNA to the cytoplasm for translation is not usually efficient. To improve export efficiency, HIV-1 possesses a protein called Rev, which binds specifically to the Rev response element (RRE), a cis-acting RNA element that mediates the export of singly spliced and unspliced HIV-1 RNA out of the nucleus [13]. The Rev response element spans about 250 nucleotides located within the env gene and folds into a series of smaller stem-loop structures within one central bubble [13]. The RRE is present in all singly spliced as well as unspliced viral RNA transcripts to promote their export into the cytoplasm. Through cooperative protein–RNA and protein–protein interactions, multimers of Rev molecules bind to the RRE within partially spliced or unspliced RNAs to enhance their nuclear export to the cytoplasm.

4. Translation

Three of the HIV-1 ORF codes for Gag, Pol and Env polyprotein precursors, which are then proteolytically cleaved to form functional individual viral proteins. Unspliced HIV mRNA encodes the Gag (Pr-55 Gag) and Gag-Pol (Pr-160Gag-Pol) precursor polyproteins. Gag Pr-55Gag polyprotein is proteolytically cleaved into p17 or MA, p24 or CA, p7 or NC, and p6 structural viral proteins [5][13]. However, Pr-160Gag-Pol fusion precursor polypeptide is similarly processed to form p10 or PR, p66/51 or RT, and p33 or IN, all of which are the gene products of the pol gene [5][13]. Pr-55Gag and Pr-160Gag-Pol polyproteins are both recognized and processed to form functional viral proteins by the enzyme PR. The gp120 and the gp41 are proteolytic products encoded by the env gene. Other proteins of HIV, including virus accessory and regulatory proteins, are translational products of singly and multiply spliced viral mRNAs.

5. New Viral Progeny

An initial step in the formation of the nucleoprotein complex during virion assembly begins with the recognition and NC binding to the packaging signal, denoted as ψ, located near the gag gene initiation codon [5]. The packaging signal is necessary for the generation of the proper core viral nucleoprotein complex consisting of full genomic RNA, a necessity to form new HIV particles. Given that the packaging signal is removed during splicing, only unspliced complete genomic mRNA (gRNA) is packaged, resulting in new HIV particles. Another component of the nucleoprotein complex is the CA, which assembles in a tabular form and stabilizes the nucleoprotein complex formation [5]. The nucleoprotein core complex consisting of virus gRNA, NC and CA proteins then migrates to the cellular plasma membrane, where it assembles with the Env coat through interactions with N-terminus myristylated MA molecules within the nucleoprotein core. Myrstilation confers hydrophobicity to the nucleoprotein core and promotes its interaction with the lipid bilayer of the cellular cell membrane [5]. The final step in the virion assembly process involves the budding and egress of the new virion particles through the cellular plasma membrane. During budding, the virion acquires a portion of the cell membrane containing viral gp120 and gp41 required for the subsequent infection of the target cell. Gag molecules within new HIV-1 virions then undergo maturation to become fully infectious HIV particles.

This entry is adapted from the peer-reviewed paper 10.3390/life14010104

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