Luttge Benjamin G, Shehu-Xhilaga Miranda, Demirov Dimiter G, Adamson Catherine S, Soheilian Ferri, Nagashima Kunio, Stephen Andrew G, Fisher Robert J, Freed Eric O
Virus-Cell Interaction Section, HIV Drug Resistance Program, NCI-Frederick, Bldg. 535, Rm. 108, Frederick, MD 21702-1201, USA.
J Virol. 2008 Mar;82(5):2106-19. doi: 10.1128/JVI.02337-07. Epub 2007 Dec 19.
Infection of domestic cats with feline immunodeficiency virus (FIV) is an important model system for studying human immunodeficiency virus type 1 (HIV-1) infection due to numerous similarities in pathogenesis induced by these two lentiviruses. However, many molecular aspects of FIV replication remain poorly understood. It is well established that retroviruses use short peptide motifs in Gag, known as late domains, to usurp cellular endosomal sorting machinery and promote virus release from infected cells. For example, the Pro-Thr/Ser-Ala-Pro [P(T/S)AP] motif of HIV-1 Gag interacts directly with Tsg101, a component of the endosomal sorting complex required for transport I (ESCRT-I). A Tyr-Pro-Asp-Leu (YPDL) motif in equine infectious anemia virus (EIAV), and a related sequence in HIV-1, bind the endosomal sorting factor Alix. In this study we sought to identify and characterize FIV late domain(s) and elucidate cellular machinery involved in FIV release. We determined that mutagenesis of a PSAP motif in FIV Gag, small interfering RNA-mediated knockdown of Tsg101 expression, and overexpression of a P(T/S)AP-binding fragment of Tsg101 (TSG-5') each inhibited FIV release. We also observed direct binding of FIV Gag peptides to Tsg101. In contrast, mutagenesis of a potential Alix-binding motif in FIV Gag did not affect FIV release. Similarly, expression of the HIV-1/EIAV Gag-binding domain of Alix (Alix-V) did not disrupt FIV budding, and FIV Gag peptides showed no affinity for Alix-V. Our data demonstrate that FIV relies predominantly on a Tsg101-binding PSAP motif in the C terminus of Gag to promote virus release in HeLa cells, and this budding mechanism is highly conserved in feline cells.
家猫感染猫免疫缺陷病毒（FIV）是研究人类免疫缺陷病毒1型（HIV-1）感染的重要模型系统，因为这两种慢病毒在发病机制上有许多相似之处。然而，FIV复制的许多分子方面仍知之甚少。众所周知，逆转录病毒利用Gag中的短肽基序（称为晚期结构域）来篡夺细胞内体分选机制，并促进病毒从感染细胞中释放。例如，HIV-1 Gag的Pro-Thr/Ser-Ala-Pro [P(T/S)AP] 基序直接与Tsg101相互作用，Tsg101是转运所需内体分选复合体I（ESCRT-I）的一个组成部分。马传染性贫血病毒（EIAV）中的Tyr-Pro-Asp-Leu（YPDL）基序以及HIV-1中的相关序列与内体分选因子Alix结合。在本研究中，我们试图鉴定和表征FIV晚期结构域，并阐明参与FIV释放的细胞机制。我们确定，FIV Gag中PSAP基序的诱变、小干扰RNA介导的Tsg101表达敲低以及Tsg101的P(T/S)AP结合片段（TSG-5'）的过表达均抑制了FIV释放。我们还观察到FIV Gag肽与Tsg101的直接结合。相比之下，FIV Gag中潜在的Alix结合基序的诱变并不影响FIV释放。同样，Alix的HIV-1/EIAV Gag结合结构域（Alix-V）的表达也不会破坏FIV出芽，并且FIV Gag肽对Alix-V没有亲和力。我们的数据表明，FIV主要依赖于Gag C末端中与Tsg101结合的PSAP基序来促进病毒在HeLa细胞中的释放，并且这种出芽机制在猫科动物细胞中高度保守。
