ptap motif
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2018 ◽  
Vol 293 (30) ◽  
pp. 11687-11708 ◽  
Author(s):  
Shilpee Sharma ◽  
Prabhu S. Arunachalam ◽  
Malini Menon ◽  
Viswanath Ragupathy ◽  
Ravi Vijaya Satya ◽  
...  
Keyword(s):  

2015 ◽  
Vol 90 (2) ◽  
pp. 768-779 ◽  
Author(s):  
Angelica N. Martins ◽  
Abdul A. Waheed ◽  
Sherimay D. Ablan ◽  
Wei Huang ◽  
Alicia Newton ◽  
...  

ABSTRACTHIV-1 uses cellular machinery to bud from infected cells. This cellular machinery is comprised of several multiprotein complexes known as endosomal sorting complexes required for transport (ESCRTs). A conserved late domain motif, Pro-Thr-Ala-Pro (PTAP), located in the p6 region of Gag (p6Gag), plays a central role in ESCRT recruitment to the site of virus budding. Previous studies have demonstrated that PTAP duplications are selected in HIV-1-infected patients during antiretroviral therapy; however, the consequences of these duplications for HIV-1 biology and drug resistance are unclear. To address these questions, we constructed viruses carrying a patient-derived PTAP duplication with and without drug resistance mutations in the viral protease. We evaluated the effect of the PTAP duplication on viral release efficiency, viral infectivity, replication capacity, drug susceptibility, and Gag processing. In the presence of protease inhibitors, we observed that the PTAP duplication in p6Gagsignificantly increased the infectivity and replication capacity of the virus compared to those of viruses bearing only resistance mutations in protease. Our biochemical analysis showed that the PTAP duplication, in combination with mutations in protease, enhances processing between the nucleocapsid and p6 domains of Gag, resulting in more complete Gag cleavage in the presence of protease inhibitors. These results demonstrate that duplication of the PTAP motif in p6Gagconfers a selective advantage in viral replication by increasing Gag processing efficiency in the context of protease inhibitor treatment, thereby enhancing the drug resistance of the virus. These findings highlight the interconnected role of PTAP duplications and protease mutations in the development of resistance to antiretroviral therapy.IMPORTANCEResistance to current drug therapy limits treatment options in many HIV-1-infected patients. Duplications in a Pro-Thr-Ala-Pro (PTAP) motif in the p6 domain of Gag are frequently observed in viruses derived from patients on protease inhibitor (PI) therapy. However, the reason that these duplications arise and their consequences for virus replication remain to be established. In this study, we examined the effect of PTAP duplication on PI resistance in the context of wild-type protease or protease bearing PI resistance mutations. We observe that PTAP duplication markedly enhances resistance to a panel of PIs. Biochemical analysis reveals that the PTAP duplication reverses a Gag processing defect imposed by the PI resistance mutations in the context of PI treatment. The results provide a long-sought explanation for why PTAP duplications arise in PI-treated patients.


2012 ◽  
Vol 5 ◽  
pp. IDRT.S10652
Author(s):  
Takashi Irie ◽  
Elena Carnero ◽  
Adolfo García-Sastre ◽  
Ronald N. Harty

The M40 VSV recombinant was engineered to contain overlapping PTAP and PPxY L-domain motifs and flanking residues from the VP40 protein of Ebola virus. Replication of M40 in cell culture is virtually indistinguishable from that of control viruses. However, the presence of the Ebola PTAP motif in the M40 recombinant enabled this virus to interact with and recruit host Tsg101, which was packaged into M40 virions. In this brief report, we compared replication and the pathogenic profiles of M40 and the parental virus M51R in mice to determine whether the presence of the Ebola L-domains and flanking residues altered in vivo characteristics of the virus. Overall, the in vivo characteristics of M40 were similar to those of the parental M51R virus, indicating that the Ebola sequences did not alter pathogenesis of VSV in this small animal model of infection.


2007 ◽  
Vol 81 (17) ◽  
pp. 9193-9201 ◽  
Author(s):  
Stefanie Jäger ◽  
Eva Gottwein ◽  
Hans-Georg Kräusslich

ABSTRACT Ubiquitin is important for the release of human immunodeficiency virus 1 (HIV-1) and several other retroviruses. All major domains of the HIV-1 Gag protein are monoubiquitinated, but the modifying machinery and the function of HIV-1 Gag ubiquitination remain unclear. Here, we show that the induction of a late budding arrest by mutation of the HIV-1 PTAP motif or by specific inhibition of selected ESCRT components leads to an increase of Gag-ubiquitin conjugates in cells, which coincides with an accumulation of detergent-insoluble, multimerized Gag at the plasma membrane. Membrane flotation experiments revealed that ubiquitinated Gag is highly enriched in membrane-bound fractions. Based on these findings, we propose that a blocking of virus release results in increased Gag ubiquitination as a consequence of its prolonged membrane association. Consistent with this, ubiquitination of a membrane-binding-defective (G2A)Gag mutant was dramatically reduced and the ubiquitination levels of truncated Gag proteins correlated with their abilities to bind to membranes. We therefore propose that membrane association and multimerization of HIV-1 Gag proteins, rather than a specific motif within Gag, trigger recognition by the cellular ubiquitination machinery.


2006 ◽  
Vol 80 (7) ◽  
pp. 3634-3643 ◽  
Author(s):  
Irene J. Dorweiler ◽  
Susan J. Ruone ◽  
Huating Wang ◽  
Richard W. Burry ◽  
Louis M. Mansky

ABSTRACT Human T-cell leukemia virus type 1 (HTLV-1) Gag is targeted to the plasma membrane for particle assembly and release. How HTLV-1 Gag targeting occurs is not well understood. The PPPY and PTAP motifs were previously shown to be involved in HTLV-1 particle release with PTAP playing a more subtle role in virus budding. These L domains function through the interaction with host cellular proteins normally involved in multivesicular body (MVB) morphogenesis. The plasma membrane pathway rather than the MVB pathway was found to be the primary pathway for HTLV-1 particle release in HeLa cells. Intriguingly, disruption of the PTAP motif led to a defect in the targeting of Gag from the plasma membrane to CD63-positive MVBs. Particles or particle buds were observed to be associated with MVBs by electron microscopy, implying that Gag targeting to the MVB resulted in particle budding. Blocking clathrin-dependent endocytosis was found not to influence localization of the HTLV-1 Gag PTAP mutant, indicating that Gag did not reach the MVBs through clathrin-dependent endocytosis. Our observations imply that the interaction between Gag and TSG101 is not required for Gag targeting to the MVB. Overexpression of dynamitin p50 increased particle release, suggesting that there was an increase in the intracellular transport of MVBs to the cell periphery by the utilization of the dynein-dynactin motor complex. Intriguingly, virus particle release with this mutant was reduced by 20-fold compared to that of wild type in HeLa cells, which is in marked contrast to the less-than-twofold defect observed for particle production of the HTLV-1 Gag PTAP mutant from 293T cells. These results indicate that the role of the PTAP motif in L domain function is cell type dependent.


2004 ◽  
Vol 78 (12) ◽  
pp. 6636-6648 ◽  
Author(s):  
Gisela Heidecker ◽  
Patricia A. Lloyd ◽  
Kristi Fox ◽  
Kunio Nagashima ◽  
David Derse

ABSTRACT Three late assembly domain consensus motifs, namely PTAP, PPPY, and LYPXL, have been identified in different retroviruses. They have been shown to interact with the cellular proteins TSG101, Nedd4, and AP2 or AIP, respectively. Human T-cell leukemia virus type 1 (HTLV-1) has a PPPY and a PTAP motif, separated by two amino acids, located at the end of MA, but only the PPPY motif is conserved in the deltaretrovirus group. Like other retroviral peptides carrying the late motif, MA is mono- or di-ubiquitinated. A mutational analysis showed that 90% of PPPY mutant particles were retained in the cell compared to 15% for the wild-type virus. Mutations of the PTAP motif resulted in a 20% decrease in particle release. In single-cycle infectivity assays, the infectious titers of late motif mutants correlated with the amounts of released virus, as determined by an enzyme-linked immunosorbent assay. We observed binding of MA to the WW domains of the Nedd4 family member WWP1 but not to the amino-terminal ubiquitin E2 variant domain of TSG101 in mammalian two-hybrid analyses. The binding to WWP1 was eliminated when the PPPY motif was mutated. However, MA showed binding to TSG101 in the yeast two-hybrid system that was dependent on an intact PTAP motif. A dominant-negative (DN) mutant of WWP1 could inhibit budding of the intact HTLV-1 virus. In contrast, DN TSG101 only affected the release of virus-like particles encoded by Gag expression plasmids. Electron and fluorescent microscopy showed that Gag accumulates in large patches in the membranes of cells expressing viruses with PPPY mutations. Very few tethered immature particles could be detected in these samples, suggesting that budding is impaired at an earlier step than in other retroviruses.


2004 ◽  
Vol 78 (3) ◽  
pp. 1503-1512 ◽  
Author(s):  
Huating Wang ◽  
Nicholas J. Machesky ◽  
Louis M. Mansky

ABSTRACT In retroviruses, the late (L) domain has been defined as a conserved motif in the Gag polyprotein precursor that, when mutated, leads to the emergence of virus particles that fail to pinch off from the plasma membrane. These domains have been observed to contain the PPXY, PTAP, or YXXL motifs. The deltaretroviruses, which include bovine leukemia virus (BLV) and human T-cell leukemia virus type 1 (HTLV-1) and HTLV-2, have a conserved PPPY motif in the C-terminal region of the matrix (MA) domain of Gag, while HTLV-1 also encodes a PTAP motif in MA. In this study, we analyzed the roles of the PPPY and PTAP motifs in the C terminus of MA in HTLV-1 particle release. Mutation of either motif (i.e., PPPY changed to APPY or PTAP changed to PTRP) reduced budding efficiencies. Particle buds and electron-dense regions of plasma membrane were observed by electron microscopy. When the locations of PPPY and PTAP were switched, particle release was eliminated. Intriguingly, the replacement of the PTAP motif with either the PPPY or YPDL motifs did not influence the release of virus particles, but the replacement of the PPPY motif with either PTAP or YPDL eliminated particle production. This indicates that the role that PPPY plays in HTLV-1 budding cannot be replaced with either PTAP or YPDL. A similar observation was made with the BLV PPPY motif. Finally, HTLV-1 particle release was found to be sensitive to proteasome inhibitors, implicating a role for ubiquitin in HTLV-1 budding. In summary, our observations indicate that (i) the PPPY motif plays a crucial role in virus budding and (ii) the PTAP motif plays a more subtle role in HTLV-1 particle release. Each of these motifs may play an important role in virus release from specific cell types and therefore be important in efficient virus spread and transmission.


2003 ◽  
Vol 77 (22) ◽  
pp. 12373-12377 ◽  
Author(s):  
Juan Martin-Serrano ◽  
Paul D. Bieniasz

ABSTRACT Human immunodeficiency virus type 1 (HIV-1) encodes a PTAP motif within the p6 domain of Gag that recruits Tsg101 and associated factors to facilitate virion budding. In this study, we use trans-complementation assays to demonstrate that the PTAP motif acts synergistically with additional p6 sequences to mediate the formation of infectious extracellular HIV-1 virions. These studies suggest that Tsg101 recruitment is necessary but not sufficient to account for late-budding activity exhibited by HIV-1 p6.


2003 ◽  
Vol 77 (17) ◽  
pp. 9474-9485 ◽  
Author(s):  
Eva Gottwein ◽  
Jochen Bodem ◽  
Barbara Müller ◽  
Ariane Schmechel ◽  
Hanswalter Zentgraf ◽  
...  

ABSTRACT Late (L) domains are required for the efficient release of several groups of enveloped viruses. Three amino acid motifs have been shown to provide L-domain function, namely, PPXY, PT/SAP, or YPDL. The retrovirus Mason-Pfizer monkey virus (MPMV) carries closely spaced PPPY and PSAP motifs. Mutation of the PPPY motif results in a complete loss of virus release. Here, we show that the PSAP motif acts as an additional L domain and promotes the efficient release of MPMV but requires an intact PPPY motif to perform its function. Examination of HeLaP4 cells expressing PSAP mutant virus by electron microscopy revealed mostly late budding structures and chains of viruses accumulating at the cell surface with little free virus. In the case of the PPPY mutant virus, budding appeared to be mostly arrested at an earlier stage before induction of membrane curvature. The cellular protein TSG101, which interacts with the human immunodeficiency virus type 1 (HIV-1) PTAP L domain, was packaged into MPMV in a PSAP-dependent manner. Since TSG101 is crucial for HIV-1 release, this result suggests that the Gag-TSG101 interaction is responsible for the virus release function of the MPMV PSAP motif. Nedd4, which has been shown to interact with viral PPPY motifs, was also detected in MPMV particles, albeit at much lower levels. Consistent with a role of VPS4A in the budding of both PPPY and PTAP motif-containing viruses, the overexpression of ATPase-defective GFP-VPS4A fusion proteins blocked both wild-type and PSAP mutant virus release.


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