Positionally independent and exchangeable late budding functions of the Rous sarcoma virus and human immunodeficiency virus Gag proteins

“Other retroviruses, including murine leukemia virus (MLV) [58,59], RSV [60-62], Mason-Pfizer monkey virus (M-PMV) [63,64], and Human T-cell leukemia/lymphoma virus type 1 (HTLV-1) [65-67], use a PPXY late domain (where X refers any amino acid) for virus budding.”

“Viral L domains have been found to be functionally interchangeable between different viruses and can exert their activity when positioned at different locations within Gag [98,100,103,105].”

“Although the amino acid sequences of the L domains are not conserved among retroviruses, they are functionally equivalent and interchangeable between different members of the Retroviridae (36).”

“some retroviral Gag proteins (46), the context/position dependence of these motifs has also been documented (see, for example, references 39 and 42).”

“In addition to the consensus retrovirus Gag proteins, the gag genes of retroviruses also encode proteins with a late budding function that is required for the efficient release of budding virions from the plasma membrane of infected cells (8, 10, 14, 17, 25, 30).”

“T10c (18), which contains the M domain of RSV along with the I and L domains of EIAV (Fig.”

“Forty-eight hours of transfection of 293T cells with 300 ng of NL p6 was carried out, and the indicated quantities (in nanograms) of pGag p6, pGag p6-p6, pGag p6-PTAP and/or pGag p6-p6 (15-51) are indicated.”

“studies on various retroviral Gag polyproteins have also suggested that conserved proline-rich motifs serve as docking sites for the cellular protein(s) to mediate the budding process (14, 19, 33, 34, 53).”

“(18, 41, 43, 61, 64, 66), but analogous domains have also been”

“(50) that L domains of two unrelated Gag proteins (HIV-1 and RSV) are exchangeable and can function in a positionally independent manner.”

“The differences in the transformation abilities between v-Src and AD1-Src suggest that the chimera might have a slight change in substrate accessibility, possibly due to a subtle change in subcellular localization in vivo.”

“location (38, 41, 54, 55, 74) and cease to function when the NC”

“Coordination of retroviral assembly is directed by three functional domains within the Gag polyprotein: the membrane-binding domain mediates the selective targeting to and stable binding of the plasma membrane, the interaction domains facilitate multimerization of Gag proteins and RNA binding, and the late domain recr…”

“First, late domains retain activity even when moved to atypical locations within Gag (26, 32).”

“Once at the plasma membrane, the late (L) domain coordinates the final release of the particle from the cell surface (14, 22, 45).”

“1), in which the p2b domain of RSV Gag has been replaced with the L domain from p6 of HIV Gag (27).”

“Functional equivalents of the M, I, and L domains have also been found in the human immunodeficiency virus type 1 (HIV-1) Gag protein (1, 26, 55).”

“While it is not clear how this mutant Gag excludes Env, it is important to appreciate that p6 is required for efficient budding, as it harbors the L domain for HIV-1 (18, 25, 47, 50).”

“It has been reported previously that L domains of different viruses are functionally interchangeable (19, 27, 30, 49) although there seems to exist a certain context or position dependence, at least in some cases (25).”

“Previously published data have shown that the HIV-1 L domain, located near the beginning of the p6 sequence, is a small proline-rich motif (P(7)-T(8)-A(9)-P(10)) required late in budding (20, 37).”

“Budding of M proteins is often directed by late (L)-domain motifs, which are most frequently PT/SAP, PPxY, YxxL or FPIV (Bouamr et al., 2003; Burleigh et al., 2005; Ciancanelli & Basler, 2006; Gottlinger et al., 1991; Harty et al., 1999, 2000; Huang et al., 1995; Irie et al., 2007; Jasenosky et al., 2001; Jayakar et a…”

“However, enhancement of particle release has previously been ascribed to the HIV-1 ancillary protein Vpu (16, 28, 47, 51, 57, 59) and to the HIV-1 carboxy-terminal Gag component p6 (17, 46).”

“tic Rous sarcoma virus late-budding domain (22, 38).”

“There are three functional assembly domains in Gag (29); each domain is responsible for a separate function in the assembly process: the membrane-binding domain (M domain), the late domain (L domain), and the interaction domain (I domain).”

“Finally, the PPxY motif, (where x can be any amino acid, usually a proline), was initially identified as a late domain in the p2b portion of RSV Gag [51].”

“However, the PTAP motif of HIV can be interchanged with other retroviral PPxY motifs without these viruses losing their ability to bud [11,12].”

“and can act autonomously in many contexts (30, 44, 47).”

“RSV p10 is just C terminal to the sequence PPPY, comprising the core of the late domain, which plays a key role in a late step in budding (29, 36), probably by interacting with one of the members of the cellular family of WW proteins (13).”

“There are three functional assembly domains in Pr (31).”

“We and others have shown that short peptide motifs in retroviral Gag precursor proteins are essential for proper release of assembled virions from infected cells in culture; these motifs are therefore referred to as “late” (L) domains (6, 13, 15, 23, 26, 43, 47, 55, 67, 72).”

“The lentivirus PS/TAPP motif region to which SnRV shows similarity to is an assembly domain required late in the virus particle budding process (22, 26, 42).”

“Motifs of the L domains in HIV-1 p6 (PTAPP), equine infectious anemia virus p9 (YXXL), and Rous sarcoma virus p2 (PPPPY) have been shown to be functionally interchangeable with regard to their effects on particle release (30).”

“While the EIAV and HIV-1 L-domains are distinct, we have shown that the YPDL and PTAP motifs are functionally interchangeable and positionally independent in Gag budding assays (39), perhaps indicating different entry points into a common cellular process that facilitates viral budding.”

“Since TIP47-binding region in MA overlaps a region implicated in Gag anchoring and binding at the plasma membrane (41,42), we could not exclude that the MA WE-AA mutation inhibiting Gag/TIP47 interaction may modulate Gag-binding affinity for the plasma membrane, and thus redistribute the MA labelling at or close to th…”

“Finally, the L domain, located in p6, is necessary for the pinching off of HIV-1 virions from virus-producing cells (17, 30).”

“Analysis of Gag-mediated budding led to the identification of three independent assembly motifs common to all retroviruses: the membrane binding domain (M) in MA, which targets Gag to the plasma membrane; the interaction (I) domains in the NC domain that mediate Gag-Gag and Gag-RNA interactions; and the late (L) domai…”

“The region in retroviral Gag harboring the MVB machinery interaction motifs was designated the late domain (LD) (40), as it controls the late step of virus budding in the infectious cycle.”

“Previous studies have shown that other viruses are able to use alternative amino acid domains in order to bypass specific TSG101 interactions and allow for viral exit (47, 49).”

“Interestingly, by using RSV Gag chimeras, researchers showed that the L domain activity carried by p2b could be functionally replaced with the C-terminal HIV-1 p6 sequence, and these domains could function independently of their position in the precursor since they can be located either internally or at the C-terminus…”

“This observation suggests that the function of Nedd4 in ASV release can be replaced by Tsg101 and most likely explains the welldocumented functional exchangeability of the HIV-1 and ASV L domains (Parent et al., 1995).”

“An equivalent domain in the lentiviruses has been found near the carboxy terminus of the Gag precursor (34).”

“…the impact of the L-domain region in FIV VLP production and since it has been reported that L-domains are transferable between unrelated viruses (Parent et al., 1995), we wanted to investigate the particle release efficiency of a FIV Gag chimera characterized by the Ebola virus L-domain…”

“T10C [25]), which lacks L domains needed for budding, is shown in lane 17.”

“The region within NC that promotes Gag interaction has been termed the I domain (Wills and Craven, 1991), which subsequently has been more narrowly defined as the region responsible for the assembly of particles of normal density (Bennett et al., 1993; Garnier et al., 1998; Parent et al., 1995).”

“The interchangeability of different classes of late domains demonstrated their main roles are to serve as docking sites for host factor recruitment [4,70,79].”

“These late domain motifs initiate assembly of the machinery through the binding of the E3-ubiquitin ligase NEDD4 and the ESCRT adaptor protein ALIX, respectively [19–25].”

“Mutational analysis mapped the RSV late domain to the PPPY motif in the small spacer peptide p2b of Gag (41, 54, 56).”

“The L-domains are short conserved sequence domains present in the matrix proteins of several enveloped viruses and were first identified as indispensable domains for budding of HIV, present in the HIV matrix Gag protein (12-15).”

“When p6 PTAP motif was inserted into Gag of other retroviruses, these mutants conserved their release activity if the HIV-1 NC was associated with p6 or if the PTAP sequence was inserted in proximity to the NC [363,364].”

“Three domains have been shown to be important for assembly of avian retroviral particles (50).”

“To date there are four classes of late domains identified: P(S/T)AP, originally found in the human immunodeficiency virus 1 Gag protein [22, 23]; YPX(1,3)L, originally discovered in the Gag protein of equine infectious anemia virus Gag [24]; PPXY, found first in the Rous sarcoma virus Gag protein [25]; and FPIV, found…”

“Functional studies that preceded detailed structural studies revealed an important contribution of NC to multimerization and assembly, a contribution termed the “I” or interaction domain (Derdowski et al., 2004; Parent et al., 1995; Sandefur et al., 1998, 2000; Weldon and Wills, 1993).”

“Experiments were performed using chemically transformed QT6 quail fibroblast cells (obtained from John Wills and Rebecca Craven, Penn State College of Medicine), which were maintained as described previously and transfected using calcium phosphate (32, 33, 48).”

“M proteins generally contain core consensus amino acid motifs called late (L) domains, which are essential for efficient viral budding (15, 16).”

“4B) that is required late in infection for release of budding particles from the cell (44, 62, 64); this sequence is therefore referred to as the L sequence.”

“A third type of retroviral L domain, first identified within the p2b region of Rous sarcoma virus (RSV) Gag (16), is defined by a PPXY motif that is thought to recruit members of the NEDD4 family of HECT ubiquitin ligases by providing a ligand for their WW domains (17).”

“The canonical sequences of the L domains are defined as P(T/S)AP, PPXY, and YPXL and are often interchangeable between species (28, 29).”

“This second cell-dependent defect is consistent with a number of previous studies that have suggested a role for host cell factors in the completion of the viral budding event (8, 20, 33, 37, 61).”

“domain sequences is required for the late membrane fission event and the final pinching off of the budding virus [4-6].”

“One interesting clue to the nature of viral budding is that L domains can be interchanged between different viruses: HIV p6, EIAV p9, or a portion of p6 that includes PTAP can provide L-domain function for RSV Gag (38).”

“A wellcharacterized example is the late domain function that has been mapped to a PTAP tetrapeptide region within p6 of human immunodeficiency virus type 1 (HIV-1) Gag (12, 16), a PPPY motif within p2b of Rous sarcoma virus (RSV) Gag (41, 43), and a YPDL sequence within p9 of equine infectious anemia virus (EIAV) Gag…”

“L domains are functionally exchangeable between unrelated viruses (6, 11, 15, 18) and can also be moved to ends of Gag opposite their normal location, indicating that they are positionally independent, too (15, 27).”

“One L-domain motif substitutes for another in promoting virion release, indicating that the different motifs share a common functio”

“The L domain, responsible for pinching off viral particles from the membrane, is located at either the C-terminal domain of Gag or the MA-CA junction (16, 37).”

“Pr2 (23, 40), which contains the very same deletion (Fig.”

“Intriguingly, the RNA viral L-domains were found to be interchangeable and movable while retaining their function in recruiting host proteins to promote virus egress (47-50).”

“It is unlikely that this duplication would affect assembly, since similar duplications constructed with RSV and HIV sequences efficiently released particles from cells (34).”

“nonspecific interactions with RNA (3, 9, 10, 25, 43, 65, 67).”

“stomatitis virus (11), Ebola virus (12), and influenza A virus (13).”

“The observation that some viral L-domains are functionally interchangeable between heterologous viruses has led to the identification of new viral L-domain sequences and motif binding partners [35,36].”

“Pr2, containing an inactive PR (27), was used to replace the corresponding fragment in pSV.”

“[80] L.”