HIV infection is influenced by dynamin at 3 independent points in the viral life cycle

Abstract

CD4 T cells are important cellular targets for HIV-1, yet the primary site of HIV fusion remains unresolved. Candidate fusion sites are either the plasma membrane or from within endosomes. One area of investigation compounding the controversy of this field, is the role of the protein dynamin in the HIV life cycle. To understand the role of dynamin in primary CD4 T cells we combined dynamin inhibition with a series of complementary assays based on single particle tracking, HIV fusion, detection of HIV DNA products and active viral transcription. We identify 3 levels of dynamin influence on the HIV life cycle. Firstly, dynamin influences productive infection by preventing cell cycle progression. Secondly, dynamin influences endocytosis rates and increases the probability of endosomal fusion. Finally, we provide evidence in resting CD4 T cells that dynamin directly regulates the HIV fusion reaction at the plasma membrane. We confirm this latter observation using 2 divergent dynamin modulating compounds, one that enhances dynamin conformations associated with dynamin ring formation (ryngo-1-23) and the other that preferentially targets dynamin conformations that appear in helices (dyngo-4a). This in-depth understanding of dynamin's roles in HIV infection clarifies recent controversies and furthermore provides evidence for dynamin regulation specifically in the HIV fusion reaction.

Resolving the Sites of HIV Entry: Dynamin Networks Hold the Key

“…For instance, small molecule inhibitor dyngo-4a can block HIV infection at a time consistent with transcriptional silencing but not endocytosis. Similarly, ryngo-1-23 can influence oligomeric states of dynamin to block HIV fusion but not endocytosis [29,49].These observations were further supported by work of Jones and colleagues [28] that indicates that different pools of dynamin oligomers are required for HIV fusion as opposed to the process of endocytosis. We will discuss this at length later in this review with respect to alternate hypotheses of how HIV fusion at the plasma membrane is dynamin dependent.…”

“…Like certain enveloped viruses such as Herpes Simplex virus-1 and Sendai virus, HIV entry was shown to be independent of pH and/or presence of lysosomal enzymes thus suggesting that virus entry into endolysosomal compartments was not required [19][20][21][22][23][24][25][26][27][28][29][30][31]. Studies with CD4 truncation mutations supported this view as mutations that tethered CD4 at the membrane did not impede HIV entry [20,22,23].…”

“…For CD4 T cells, the situation is more complex with evidence varying across a continuum of T cell subsets, T cell lines, through to activated and resting T cells. In addition, it is only of late that investigators have recognised and observed HIV entry in subsets of resting CD4 T cells derived in vivo [29]. For instance, T cell lines can be engineered to express a dominant negative version of dynamin, thus lacking constitutive endocytosis and yet being permissive to HIV fusion [27].…”

“…From our own investigations across CD4 T cells, we would readily conclude that the timing of action for fusion peptides is simply consistent with the time they act on the HIV fusion reaction (post chemokine receptor binding) and there is limited evidence both by ourselves and others [26] that the virus can escape the action of these compounds by entering into endosomes. Recently using a spectrum of approaches including small molecule inhibitors that target dynamin, endocytosis, viral fusion and replication pathways, both ourselves and others have demonstrated that HIV fusion in both activated and resting T cell subsets not only proceeds primarily through plasma membrane and but that this fusion reaction is dependent on dynamin [27][28][29].…”

“…The incorporation of different dynamin modulators [29] was key to resolving many of the controversies around the role of dynamin in HIV entry and infection. Here we will outline two dynamin compounds, ryngo-1-23 and dyngo-4a, their mechanism of action and the key observations when used to study dynamin's role in HIV entry/ fusion.…”