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lördag 7 december 2013

Kertausta HIV-1 virusrakenteeswta Gag-Pro-Pol

Tarkista:  Gag-Pol fusion proteins
Kuva 5.(Fig. 5).  
Figure 5. Organization of retroviral Gag, Pro, and Pol proteins.
SITAATTI kuvan tekstistä ( näistä retroviruksista HIV on ihmisvirus)

 Organization of retroviral Gag, Pro, and Pol proteins. All Gag proteins contain poorly conserved MA, CA, and NC sequences and differ further in the number and positions of numerous other cleavage products, as shown. Translation past the 3′end of the Gag-coding sequence results in the synthesis of Gag-Pro-Pol fusion proteins. This occurs by either ribosome frameshifting (fs) or in-frame suppression of a stop codon (is). Note the special case of spumaviruses, where there is little processing and Pro-Pol is expressed from a spliced mRNA. The number of amino acids contained within the Gag and Gag-Pro-Pol proteins (Gag and Pro-Pol in the case of HFV) is indicated at the end of each molecule. Myristate is represented by the wavy lines. (SP) Spacer peptide; (TF) transframe protein; (DU) dUTPase; (PPPY) a proline-rich sequence required later in budding; (MHR) the major homology region of CA.


Amino-terminal Modifications

The Gag proteins of most retroviruses (as well as their Gag-Pro-Pol proteins) are cotranslationally modified at their amino termini by the addition of myristate, which is therefore also present at the amino terminus of MA (Fig. 5). This rare 14-carbon fatty acid is always attached to a terminal glycine, which is encoded by the second codon of gag and exposed after removal of the initiator methionine (Henderson et al. 1983; Wilcox et al. 1987; Schultz et al. 1988).
 Myristate is required for the binding of Gag to the plasma membrane. Replacement of the critical glycine with another amino acid prevents myristylation, and such mutants are invariably defective for particle formation (see, e.g., Rein et al. 1986; Rhee and Hunter 1987; Bryant and Ratner 1990). It should be emphasized, however, that the mere presence of myristate at the amino terminus of a Gag protein is not sufficient for membrane binding (Rhee and Hunter 1990b; J.W. Wills et al. 1991); therefore, flanking amino acid residues must make an essential contribution (Resh 1994).

 Direct evidence for this has been obtained from experiments in which myristylated peptides were used to assess membrane interactions in vitro. These studies demonstrated that the 14 carbons of myristate are not sufficient for strong membrane association, in contrast to palmitylated peptides, which contain 16 carbons that are sufficient for strong membrane association (Peitzsch and McLaughlin 1993). Consequently, myristate is especially well suited for proteins that need to cycle on and off membranes in response to additional factors that affect their binding. Indeed, there are now many examples of myristylated proteins whose release from the membrane is induced by phosphorylation, including the MARCKS protein and pp60c-src (Taniguchi and Maneti 1993; Walker et al. 1993; Resh 1994; McLaughlin and Aderem 1995). Hence, the presence of myristate does not necessarily prevent MA proteins from leaving the membrane to play additional parts in replication when the virus infects a new cell (Chapter 5.

The importance of elements other than myristate for membrane binding is best illustrated by the many retroviruses that replicate without it. These include bovine immunodeficiency virus (BIV; Tobin et al. 1994), equine infectious anemia virus (EIAV; Henderson et al. 1987), ASLV (Schwartz et al. 1983), and visna virus (Sonigo et al. 1985). Among these, the Gag protein of BIV is the only one for which no modification of any type has been found at the amino terminus (Tobin et al. 1994). The others are all blocked for amino-terminal sequencing, and therefore are modified in some way. In the case of ASLV, an acetyl group is added to the amino-terminal methionine (Palmiter et al. 1978), but this 2-carbon fatty acid is too short to provide a stable membrane interaction.

Replacement of acetate, and the methionine to which it is attached, with myristate (i.e., by substituting glycine at the second position to create a site for myristylation) does not interfere with budding or infectivity (Erdie and Wills 1990). In contrast, the structural proteins of certain yeast viruses (e.g., L-A double-stranded virus) exhibit an absolute requirement of N-acetyltransferase for their intracytoplasmic assembly and infectivity (Tercero and Wickner 1992; Tercero et al. 1993). Thus, it remains possible that some retroviruses will be found that require acetylation of Gag."

  • Thomas J Hope 2000 yksityiskohta:, jossa mainitaan GAG-POL FUUSIOPROTEIINIT

 Gag-Pol Precursor

The viral protease(Pro), Integrase (IN) RNase H and reversetranscriptase8 (T) are always  expressed within context of Gag-Pol fusion protein.
The Gag-Pol recursor( p160) is generated by a ribosomal frame shift event, which is triggered by a specific cis-acting RNA motif ( a heptanucleotide secuence p6 followed by s short stem loop in the distal region ogf the Gag RNA). When the ribosomes encounter this motif, they shift approximately 5 % of the time to the pol reading frame without interrupting translation. The frequence of ribosomal frameshicting explains why the Gag and the Gag-Pol precursor are produced at a ratio of approximately 20:1.
During viral maturation, the virally encoded protease (PR) cleaves the Pol polypeptide away from Gag and further digest it to separate the protease (p10), RT (p50), RNase H (p15), and integrase (IN, p31). These cleavages do not all occur "efficiently", for ecample, roughly 50% of RT protein remains linked ti RNase H as a single polypeptide (p65).

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