User:Kinkreet/HIV assembly and release

HIV assembly and release is an integral process of the infectious cycle for HIV. This process occurs at the latter stages of the infectious cycle, after attachment, fusion and entry, internalization and integration, and splicing and translation of viral RNAs.

Overview
Human immunodeficiency virus (HIV) is a ssRNA reverse transcribing viruses in the retrovirus family and lentivirus genus. It attaches to the host cell via a combination of weak binding to host receptors using its SU glycoprotein (gp120), and a stronger interaction with chemokine receptors. TM glycoprotein (gp41) on the virus then mediates fusion with the host cell, internalizing the virion and leads to the release of the ssRNA genetic material into the cytoplasm of the host cell. HIV (as all other retroviruses) contain its own reverse transcriptase which reverse transcribe the (+)ssRNA genome into dsDNA. The viral dsDNA then migrates into the nucleus, where viral integrase, another enzyme contained within the virion, randomly integrate the viral dsDNA into the host genome; this integrated viral dsDNA is called the provirus. The provirus is transcribed using DNA polymerase II to give RNA. This RNA can be spliced, incompletely-spliced, or unspliced, and the three different populations are used to produce different viral proteins. The spliced RNA are used to produce tat, rev, and nef proteins. The incompletely-spliced RNAs encode for env, which, after being exported using Rev, is subsequently cleaved into SU and TM envelope proteins (used for attachment and fusion), and the accessory proteins vif, vpu, and vpr. The unspliced full-length viral RNA is translated into Env, Gag and Gag-Pol polyproteins. The unspliced RNA can also act as the genomic RNA (gRNA) after being used in translation, or without being processed at all. These different viral proteins and gRNA must then be assembled into a functional virion, be budded and released from the host cell so as to infect more cells. After released, viral protease is used for the maturation of the virion.

Gag polyprotein
The human immunodeficiency virus type 1 structural polyprotein Pr55Gag is a central component in the capture of the gRNA (to be packaged), the assembly and budding of the virus. Pr55Gag is a 55kDa polyprotein, which means it is a protein with multiple domains, which when cleaved, becomes individual polypeptides, with individual functions. Pr55Gag and its frameshifted Gag-(pro)pol is synthesized through the translation of unspliced full-length viral RNA. Pr55Gag is made up of four major subdomains - Matrix (MA), Capsid (CA), Nucleocapsid (NC) and p6. The NC domain is flanked by two peptide - SP1 (p2) and SP2 (p1).

The CA protein is the major structural protein of the HIV capsid. When linked with NC, CA is able to assemble into capsid-like structures in vitro without other factors present, but becomes more alike the wildtype viral capsid with the presence of lipid membranes and nucleic acid.

Genmoic RNA capture and transport
After the provirus is transcribed and the mRNAs translated, all the components of a new sets of virion are ready to be assembled. This requires the assembly of the viral proteins, as well as capturing and packaging of the RNA into this protein shell. The viral mechanism must distinguish between the cellular and viral RNA population, and only include the viral genome. The different subdomains of the Pr55Gag polyprotein is responsible for this process; it is important to remember that all subdomains are physically linked to each other, as the polyprotein is not yet cleaved at this stage.

The specificity of binding of the virus for gRNA is mediated through the Gag protein using two compensatory mechanisms. As with other retroviruses, HIV packages two copies of its RNA genome. Dimeric genomic RNA (gRNA) binds to two zinc finger motifs of the NC subdomain of different Gag proteins; these zinc finger motifs are responsible for the encapsidation and specificity of binding of the viral RNA. The other interaction is mediated by the matrix (MA) subdomain. The MA subdomain contains a highly-basic region, which binds non-specifically to the RNA. Thus, the MA subdomain primarily provides the affinity of binding, and the NC subdomain provides the specificity.

The capture of the gRNA can occur at the same time as the RNA is being translated, where the parts of the RNA which have been translated is captured as soon as it is available, or without prior translation,, although co-translational capture have been implied as being more efficient. Capture occurs in the cytoplasm and give rise to monomeric or dimers of Gag proteins; the formation of higher-ordered multimers requires the interaction of the Gag protein with the plasma membrane.

The captured gRNA must then be transported to the plasma membrane, where other viral proteins are transported to, to allow it to be packaged and buds off the host cell. However, the Pr55Gag polyprotein should have gRNA attached in order to ensure the genome is transported to the virion; therefore, only bound Pr55Gag polyproteins should be transported. The gRNA (or other RNAs) must bind to the highly polybasic region of MA in order to mask its charge, only then will the MA be hydrophobic enough to be localized at lipid rafts on the plasma membrane. Also note that only a small number of Gag proteins migrate with the captured gRNA to the budding site at the plasma membrane and mutlimerizes there.

A large range of factors have been implemented to be involved the Gag-RNA interaction and trafficking, including UPF1, UPF3b,Staufen, ABCE1, and DDX6. In Drosophila, Staufen and ESCRT proteins interacts with RNA and has been implicated that this interaction is required for their subcellular trafficking. . However, the importance of these factors are unclear.

Once transported, the C-terminal domain of the Capsid subdomain (CA-CTD) binds to the plasma membrane and brings the gRNA near the plasma membrane. The MA subdomain also has an affinity for phosphatidylinositol(4,5)diphosphate (PIP2), such as at the plasma membrane, as oppose to other cellular membranes.

The interaction of the Gag protein with the plasma membrane is a critical step in the assembly of the HIV virion. The N terminus of the MA domain is covalently linked to a saturated myristic acid to give the myristoylated matrix protein, myr-MA. A model has been proposed where as the Pr55Gag interacts with the PIP2 on the plasma membrane, and along with Gag multimerising, displaces the RNA from MA, and releases the myristic acid which inserts into lipid rafts on the membrane and anchors the Pr55Gag onto the plasma membrane.

Packaging and budding
The precursor Gag polyprotein forms a rough spherical shell with ~5000 Gag proteins. The polyprotein is then proteolytically cleaved to produce the four domains. 1572 CA proteins reform into a conical structure in a hexameric lattice, with spacing of ~10 nm. This CA shell encloses the RNA. The number of CA in the mature shell is less than half the number of Gag proteins in the immature virus particle, meaning not all the cleaved CA proteins are integrated into the virion shell.

The MA protein is released from the membrane when the Gag precursor polyprotein is cleaved into its subdomains.

Dimerization of the RNA genome is mechanistically coupled with packaging of the genome. This coupling is achieved through interaction between the NC subdomain of the Pr55Gag and the 5' untranslated region (UTR) of the RNA genome.

Endosomal sorting complex required for transport (ESCRT)
The endosomal sorting complex required for transport (ESCRT) system consists of four different cellular proteins - ESCRT-0, I-III.

Recruitment of the Endosomal sorting complex required for transport (ESCRT) system by HIV allows vesicle budding into endosomal compartments (multi-vesicular bodies (MVB)), cell division and cytokinesis.

The requirement for ESCRT-II in the budding process of HIV have been shown to be integral, but also that it is not required; no consensus has been reached.

The p6 domain is located near the C-terminus of Pr55Gag. The late (L) domains of the p6 domain is essential in the budding of HIV. Two L domains have been identified - P-T/S-A-P and YPXnL (where X is any amino acid and n = 1~3 residues). P-T/S-A-P has shown to be very sensitive, as conservative mutations in this motif led to defects in release of the virion. . P-T/S-A-P is also converesed in HIV-1 and HIV-2, other retroviruses, and Filoviridae, Arenaviridae and Hepeviridae. The YPXnL late domain binds to apoptosis-linked gene 2-interacting protein X (ALIX, an associated protein of the ESCRT pathway) and promotes budding.

Viral maturation
Mutations in the p6Gag protein prevented the budded virus from being released.