Functions of Early (AP-2) and Late (AIP1/ALIX) Endocytic Proteins in Equine Infectious Anemia Virus Budding

Abstract

The proline-rich L domains of human immunodeficiency virus 1 (HIV-1) and other retroviruses interact with late endocytic proteins during virion assembly and budding. In contrast, the YPDL L domain of equine infectious anemia virus (EIAV) is apparently unique in its reported ability to interact both with the 2 subunit of the AP-2 adaptor protein complex and with ALG-2-interacting protein 1 (AIP1/Alix) protein factors involved in early and late endosome formation, respectively. To define further the mechanisms by which EIAV adapts vesicle trafficking machinery to facilitate virion production, we have examined the specificity of EIAV p9 binding to endocytic factors and the effects on virion production of alterations in early and late endocytic protein expression. The results of these studies demonstrated that (i) an ϳ300-residue region of AIP1/Alix-(409 -715) was sufficient for binding to the EIAV YPDL motif; (ii) overexpression of AIP1/Alix or AP-2 2 subunit specifically inhibited YPDL-mediated EIAV budding; (iii) virion budding from a replication-competent EIAV variant with its L domain replaced by the HIV PTAP sequence was inhibited by wild type or mutant 2 to a level similar to that observed when a dominant-negative mutant of Tsg101 was expressed; and (iv) overexpression or siRNA silencing of AIP1/Alix and AP-2 revealed additive suppression of YPDL-mediated EIAV budding. Taken together, these results indicated that both early and late endocytic proteins facilitate EIAV production mediated by either YPDL or PTAP L domains, suggesting a comprehensive involvement of endocytic factors in retroviral assembly and budding that can be accessed by distinct L domain specificities. Equine infectious anemia virus (EIAV)2 is a member of the lentivirus subfamily of retroviruses, which also includes human immunodeficiency virus 1 (HIV-1). The EIAV genome is the simplest among the lentivirus family and encodes three major structural proteins (Gag, Pol, and Env) along with three accessory gene products (Tat, Rev, S2). Like all retroviruses, EIAV Gag protein is synthesized as a polyprotein that upon virion maturation is processed by virus-encoded protease to yield four major structural proteins: matrix, capsid, nucleocapsid, and p9 protein. All four structural proteins of Gag polyprotein play critical and distinctive roles in retrovirus assembly and budding. Matrix proteins direct targeting of Gag polyproteins to cell membranes for viral assembly and budding (1, 2). Both capsid and nucleocapsid have been shown to mediate Gag-Gag interactions during viral assembly, with capsid responsible for the dimerization of homologous Gag polyproteins (3, 4) and nucleocapsid essential for multimerization of Gag molecules (5, 6). The EIAV p9 protein contains a YPDL late (L) domain that is critical for virion release during the late stage of virus budding. Proline-rich L domains such as PTAP and PPPY with similar functions have also been identified from HIV-1, Rous sarcoma virus, and a variety of other enveloped viruses (7).L domains appear to...

Involvement of host cellular multivesicular body functions in hepatitis B virus budding

“…AIP1 also is thought to have an important role in generating membrane curvature (52,54). For some viruses, AIP1 provides primary or accessory access to the MVB machinery by serving as a binding target for viral late domains of the YPXL/LXXLF class (51,55). Budding by the lentivirus equine infectious anemia virus (EIAV), e.g., strongly depends on interaction of its Gag subunit p9 with AIP1 (55).…”

“…For some viruses, AIP1 provides primary or accessory access to the MVB machinery by serving as a binding target for viral late domains of the YPXL/LXXLF class (51,55). Budding by the lentivirus equine infectious anemia virus (EIAV), e.g., strongly depends on interaction of its Gag subunit p9 with AIP1 (55). The p9 YPDL domain that binds AIP1 also binds the clathrinassociated AP2 adaptor complex that sorts cargo proteins into coated pits for endocytosis, and this interaction is important for EIAV budding (55).…”

“…Budding by the lentivirus equine infectious anemia virus (EIAV), e.g., strongly depends on interaction of its Gag subunit p9 with AIP1 (55). The p9 YPDL domain that binds AIP1 also binds the clathrinassociated AP2 adaptor complex that sorts cargo proteins into coated pits for endocytosis, and this interaction is important for EIAV budding (55). Interestingly, HBV envelope proteins S and L were recently reported to interact with, respectively, AP2 (10) and ␥2-adaptin, a protein related to the large subunits of vesicle adaptor complexes such as AP2 (9,56).…”

The Central Role of the Matrix Protein in Nipah Virus Assembly and Morphogenesis

“…Over-expression of AIP1/Alix has been shown to enhance SeV budding (108) and HIV-1 VLP (120) release, but decrease EIAV release (21). As a consequence of these observations, we sought to determine whether AIP1/Alix might play a role in NiV M protein release.…”

“…As discussed earlier, the cellular protein AIP1/Alix is a component of the MVB machinery that facilitates budding of the retroviruses equine infectious anemia virus (EIAV) and human immunodeficiency virus (HIV-1) (21,38,79,87,120,131), as well as the paramyxovirus SeV (108). Over-expression of AIP1/Alix has been shown to enhance SeV budding (108) and HIV-1 VLP (120) release, but decrease EIAV release (21).…”