Critical Role of the Human T-Cell Leukemia Virus Type 1 Capsid N-Terminal Domain for Gag-Gag Interactions and Virus Particle Assembly

Abstract

The retroviral Gag protein is the main structural protein responsible for virus particle assembly and release. Like human immunodeficiency virus type 1 (HIV-1) Gag, human T-cell leukemia virus type 1 (HTLV-1) has a structurally conserved capsid (CA) domain, including a β-hairpin turn and a centralized coiled-coil-like structure of six α helices in the CA amino-terminal domain (NTD), as well as four α-helices in the CA carboxy-terminal domain (CTD). CA drives Gag oligomerization, which is critical for both immature Gag lattice formation and particle production. The HIV-1 CA CTD has previously been shown to be a primary determinant for CA-CA interactions, and while both the HTLV-1 CA NTD and CTD have been implicated in Gag-Gag interactions, our recent observations have implicated the HTLV-1 CA NTD as encoding key determinants that dictate particle morphology. Here, we have conducted alanine-scanning mutagenesis in the HTLV-1 CA NTD nucleotide-encoding sequences spanning the loop regions and amino acids at the beginning and ends of α-helices due to their structural dissimilarity from the HIV-1 CA NTD structure. We analyzed both Gag subcellular distribution and efficiency of particle production for these mutants. We discovered several important residues (i.e., M17, Q47/F48, and Y61). Modeling implicated that these residues reside at the dimer interface (i.e., M17 and Y61) or at the trimer interface (i.e., Q47/F48). Taken together, these observations highlight the critical role of the HTLV-1 CA NTD in Gag-Gag interactions and particle assembly, which is, to the best of our knowledge, in contrast to HIV-1 and other retroviruses. Retrovirus particle assembly and release from infected cells is driven by the Gag structural protein. Gag-Gag interactions, which form an oligomeric lattice structure at a particle budding site, are essential to the biogenesis of an infectious virus particle. The CA domain of Gag is generally thought to possess the key determinants for Gag-Gag interactions, and the present study has discovered several critical amino acid residues in the CA domain of HTLV-1 Gag, an important cancer-causing human retrovirus, which are distinct from that of HIV-1 as well as other retroviruses studied to date. Altogether, our results provide important new insights into a poorly understood aspect of HTLV-1 replication that significantly enhances our understanding of the molecular nature of Gag-Gag interaction determinants crucial for virus particle assembly.

M-Sec induced by HTLV-1 mediates an efficient viral transmission

“…The protrusions of MT-2 or SLB-1 cells (Fig 2B ) appear to be smaller in length but larger in number than those of HIV-1-infected macrophages or U87 cells [8,9]. Furthermore, the ability of M-Sec to facilitate the clustering of Gag (Figs 8, S12, and 9) may be beneficial for viral transmission because Gag is the key driver for the formation of viral particles [31][32][33][34][35]. Consistent with this idea, an alanine-scanning mutagenesis analysis of Gag has identified several mutants that not only fail to form puncta but also produce lesser amounts of viral particles when compared to the wild type Gag [34].…”

“…However, these well-known functions of M-Sec might not be sufficient to explain the potent effect of M-Sec knockdown in the mouse model (Fig 6). Gag is a viral structural protein that localizes to the inner leaflet of the plasma membrane [12,[31][32][33][34][35]. In both MT-2 and SLB-1 cells, the signal of Gag did not overlap with that of Calnexin, an endoplasmic reticulum marker (S10 Fig) . The signal of Gag partially overlapped with that of GM130 (a Golgi marker) in MT-2 cells, but not in SLB-1 cells (S10 Fig) . Thus, Gag appears to mainly localize to the inner leaflet of the plasma membrane in these cells.…”

“…In both MT-2 and SLB-1 cells, the signal of Gag did not overlap with that of Calnexin, an endoplasmic reticulum marker (S10 Fig) . The signal of Gag partially overlapped with that of GM130 (a Golgi marker) in MT-2 cells, but not in SLB-1 cells (S10 Fig) . Thus, Gag appears to mainly localize to the inner leaflet of the plasma membrane in these cells. Gag is also known to form puncta, which is an indicator of their assembly requisite for the formation of viral particles [12,[31][32][33][34][35]. In fact, both control (S1 Video) and M-Sec knockdown MT-2 cells (S2 Video)…”

Human immunodeficiency virus type 2 capsid protein mutagenesis reveals amino acid residues important for virus particle assembly

“…Briefly, HeLa cells were cultured in six-well plates on no. 1.5 standard glass coverslips coated with poly-l-lysine, and experiments were performed as previously described [50]. HeLa cells were transiently transfected with eYFP-tagged Gag and untagged Gag expression plasmids at a 1:4 ratio using GenJet, ver II (SignaGen, Gaithersburg, MD).…”

Solution Conformation of Bovine Leukemia Virus Gag Suggests an Elongated Structure

“…On the other hand, the MA domains of Gag from BLV and HTLV-2 were both shown to play a critical role in the packaging of gRNA [57][58][59]. In HTLV-1, Gag-Gag interactions are critical for viral assembly and budding; interestingly, the key residues for these interactions reside in the N-terminal domain of CA [60,61]. This suggests similarity to HIV-1 in that residues of CA also play a role in assembly, but unlike HIV-1, occur in the absence of membrane phosphoinositides [24,25,35].…”