Retrovirus replication cycle

1.4.1 Early steps: Membrane fusion, Reverse Transcription and Integration

As mentioned above when the gp120 molecule binds to the CD4 receptor on a target cell and a conformational change exposes the binding sites for the co-receptors.

Engagement of the co-receptors triggers an irreversible conformation change exposing the fusion peptide and the initiation of the fusion of the viral envelope and the host cell plasma membrane (reviewed here [46, 47]). Once fusion is completed the capsid core is released into the host cell cytoplasm and reverse transcription takes place (Figure 1.2). Reverse transcription is primed by the cellular tRNA^Lys3 bound to the primer binding site (PBS), 18 nucleotides in the U5 region. RT first

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synthesizes the minus strand towards the 5’ end and at the same time the RNase H activity of RT degrades the RNA in the growing RNA/DNA hybrid. When the enzyme reaches the 5’ end of the genome (R) the process comes to a stop generating a DNA fragment named “minus-strand strong-stop DNA”. Since the R sequences are identical at both ends of the genome, the R DNA stretch from the 5’ end is used to prime synthesis from the 3’ end in a process called “first strand transfer” (first jump).

The RT then continues minus strand synthesis and degrades the plus strand RNA.

Two regions are resistant to RNase H degradation – the polypurine tract (PPT) and the central PPT (cPPT). The resulting short RNA sequences serve as primers for plus strand DNA synthesis. The tRNA primer is degraded by RT when it reaches the end of the minus strand and a second strand transfer occurs (second jump) to the 5’

end of the minus strand and plus strand DNA synthesis can be completed. In this process a cDNA is generated with the full LTR (U3-R-U5) (reviewed here [48-50]).

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The early steps after release of the capsid into the host cell are still poorly understood. The entire capsid or part of the CA molecules dissociated from the cytoplasmic viral complex by a process called uncoating taking place in the first hour after fusion is completed. Not much is known about this process but the stability of the capsid core is tightly linked to proper reverse transcription. Delay of reverse transcription by RT inhibitors can alter uncoating kinetics and mutations in the CA changing capsid stability lead to defects in reverse transcription [51-53]. Several cellular factors most importantly TRIM5α and Cyclophillin A (CypA) are known to influence core stability [51, 54, 55]. The fact that HIV-1 and other viruses from the genus lentivirus including SIV can infect non-dividing cells such as terminally differentiated macrophages or resting T cells shows that HIV-1 is able to actively penetrate the nucleus of target cells. This is in contrast to gammaretorviruses such as murine leukemia virus (MLV), which rely on a disassembled nucleus during cell division for integration [56-58].

Once the pre-integration complex (PIC) arrives in the nucleus the cDNA is integrated into the host cell genome via the integrase. The integrase creates a dinucleotide CA overhang at the 3’ ends of the HIV-1 cDNA, which is covalently attached to the host genome in a process termed strand transfer reaction ([59, 60]

and reviewed here [61]). The gap occurring in the host chromosome is repaired by cellular enzymes. Another outcome of nuclear import of the PIC is the dead-end process mediated by host cell nuclear ligases where either one LTR (1-LTR circles) or two LTR (2-LTR circles) cDNA molecules are circularized [62]. These circles can be detected by PCR and are exploited as a marker for nuclear import [63]. The selection of the integration site is not completely random and is associated with markers of active transcription [64, 65]. The HIV integrase associates with the cellular chromatin tethering factor LEDGF/p75 to find the preferred integration sites [66].

1.4.2 Late steps: Viral gene expression, capsid assembly, maturation and release

Once the reverse transcribed genome is integrated into the host chromosome it is called a provirus. The LTR sequence now serves as promoter for expression of viral mRNA and genomic RNA. The 5’ LTR contains an RNA Pol II TATA box and binding sites for the transcription factor nuclear factor of activated T cells (NFAT), nuclear factor-κB (NF-κB) and activator protein 1 (AP-1). The nature of the main transcription

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factors link HIV-1 expression to T cell activation, which is necessary to activate NFAT, NF-κB and AP-1. The processivity of the RNA Pol II transcribing the HIV-1 genome is quite low and the presence of Tat enhances the HIV-1 expression. Tat binds to the trans-activation response (TAR) element, a secondary stem loop structure in the nascent HIV-1 mRNA, which leads to fully transcribed HIV-1 mRNA [67].

To express multiple proteins from a single promoter HIV-1 uses a complicated splicing scheme. There are three general mRNA products: unspliced full length genomic RNA (9 kb), single spliced mRNA (4 kb) and fully spliced mRNA species (2.2 kb). Genomic RNA encode Gag and the Gag-Pol precursor, the single spliced mRNA code for the Env precursor protein, Vpu, Vif and Vpr while the multiply spliced mRNA code for Tat, Rev and Nef [68]. Using deep sequencing methods the most recent count of HIV-1 mRNA was found to be at least 109 different spliced mRNA variants [69].

Since cellular immature and unspliced mRNA is normally retained in the nucleus, HIV-1 has a mechanism to allow unspliced mRNA export. Rev is used to achieve that goal [70-72]. Rev contains a nuclear localization signal (NLS) and nuclear export signal (NES) and can thus shuttle between the nucleus and the cytoplasm. It binds the Rev responsive element (RRE) present in all unspliced and single spliced mRNA but not in the fully spliced mRNA coding for Tat, Rev and Nef. Rev uses cellular factors to transport mRNA species with the RRE present into the cytoplasm (reviewed here [73]). Rev also serves as a switch between expression of the regulator genes Tat, Nef, Rev, the accessory proteins and Env as well as Gag-Pol.

Once enough Rev accumulates, unspliced and single spliced mRNA is preferentially transported into the cytoplasm and the proteins for assembly of new viral particles are synthesized.

Once the Gag-pol open reading frame (ORF) is expressed, the MA is immediately localized to the plasma membrane [33] and recruits Gag-Pol and NC, which in turn mediates encapsidation of the viral genome via the packaging signal (ψ) [74, 75]. NC interacts specifically with dimerized RNA genomes, which is mediated by the dimer initiation site (DIS) and required for proper packaging of the genome [76]. Env is synthesized to some extent into the endoplasmatic reticulum (ER) and transported

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via the Golgi-apparatus – where it gets glycosylated – to the plasma membrane.

Once at the plasma membrane, the long cytoplasmic tail of the gp41 interacts with MA and forms “raft”-like domains. To bud off the host membrane HIV-1 recruits the cellular ESCRT pathway [77]. Once the immature viral particle is released from the host cell the PR cleaves the Pr55^gag and Pr160^gag-pol to generate the mature and infectious particle (reviewed here [78]).