Monday, July 09, 2012

Hey "SAMHD1", You better stay out of nucleus

Hello fellows,

        "Science" is at the top of the world in past few weeks. Thats what i can say in brief. The  jury of H1N5 had just been settled, and a news wooped up claiming the virus is just a few mutations away, getting into humans. (Ooooh.... Didn't scare me though; Link). Scientists working at CERN finally give a verdict that Higgs Boson is a truth (Considered as a ground shaking discovery!; Link) Some serious evidence of dark matter (Huh, the news of Higgs boson blew this news away; Link) O my, i just could go with the list...... By the way, If you are thinking that I'm about to talk about any of these above you are wrong. I just couldn't resist saying this. The Photo posted on the left is that of a Large Hadron Collider at CERN (Image Source). And i cannot talk about Higgs boson (Or any of the other boson for that matter), cause i know only know as much as you know.

      Alright am not going to bluff more. So what do i have in blog pocket today. Hmmm, let me see. Oh yeah, Till the moment i was about to ink this blog, i was very much about to talk about  "Purinergic receptors and its important role in Human macrophage infection" influenced by the paper Hazleton JE etal publication in Journal of Immunology (Published in May2012; Link). The second paper that heavily caught my attention was a review article by Robert A. Britton and Vincent B. Young titled "Interaction between the intestinal microbiota and host in Clostridium difficile colonization resistance" (Link). But then another paper on HIV caught my attention.

For a background reading on HIV please refer to my previous post on HIV (Link), and have a look at the ppt below. So, the paper under discussion here is from BMC- Retrovirology Journal. 


    SIV stands for Simian immunodeficiency virus, or more interestingly known as African Green Monkey virus is thought to be parental type of HIV. Most of our understanding of HIV comes from experiments with this virus. The vast literature of HIV suggests that human cells have four major restriction factors to attack retroviruses. But as you would expect, the virus has armour evolved to protect itself. This is summarized in below table.

Photo: African Green Monkey (Source)

Table 1: Restriction factors against HIV/SIV (Source).

        APOBEC3G/F is a cytidine deaminase known to involve in antiretroviral immunity. Vif recruits cellular Elongin B/C-Cullin5 E3 ubiquitin ligase to target the host antiviral protein APOBEC3G (A3G) for proteasomal degradation. TRIM interacts with capsid of the HIV in cytoplasm. Tetherin ( Bst-2 CD317 ) is a cell surface protein, induced by IFNα, which can inhibit the HIV viral particle HIV overcomes this restriction by deploying vpu. The action is speculated to be mediated by recruiting SCF-E3 ubiquitin ligase complex, through an interaction between the β-TrCP protein and conserved phospho-serine residues in Vpu's cytoplasmic tail. If that is one part of the story, SAMHD1 is the other part of the story.

Fig 1: Vpx, SAMHD1 and viral replication (Source).

        SAMHD1 is a short version of SAM domain and HD domain 1, located in 20 pter-q12. The function of this protein is not well understood but is often speculated to possess a role in immunity and and cerebral vascular hemeostasis (Read more). Biochemically, it has a deoxynucleoside triphosphate triphosphohydrolase activity. Mutations with the function of this gene is known to be associated with Aicardi-Goutieres syndrome. (Click here to learn more about gene SAMHD1). It is a known fact that HIV-1 replicates poorly in human dendritic cells, monocytes and, and to an extent in macrophages. Th restriction factor was then traced to SAMHD1, the arch enemy of reverse transcriptase. And the counter force is applied by vpx. By the way, SAMHD1 localizes to the nucleus in human fibroblasts.

Fig 2: Ribbon representation of the SAMHD1 dimer with the major lobe (blue), minor lobe (grey) and C-terminal region (red). (Source)

Fig 3: HeLa cells expressing the indicated GFP4 fusion constructs
  The first experiment done by the authors was to fuse the SAMHD1 to a GFP4. HeLa cells expressing the GFP fusion constructs (green) was imaged by fluorescence microscopy. The cellular nuclei were stained by using DAPI (blue). By doing the same experiment, fusing the GFP at various sites, they were able to show that the Nuclear localization signal (NLS) was present in first 150 amino acids. A co-localization with fibrilarin suggested that SAMHD1 is in the nucleoplasm, but not in the nucleolus.

        With a site directed mutagenesis experiment, they further nailed down the location of NLS to peptide 11 KRPR 14 starting at position 11. To further prove that 11 KRPR 14 was indeed the NLS, they incorporated the implicated peptide sequence to N terminus of a GFP protein fused to the viral protein muNS. The muNS which is from avian reovirus that localizes to the cytoplasm. IF the KRPR is the signal then the muNS which is found in cytoplasm  is now expected to move into nucleus. And the results (Shown below, fig 4) was obvious. It moved.

Fig 4: Transferrability of NLS

      Now, lets see what happens in the virus vs SAMHD1 in battle field. It is stated that nuclear localized SAMHD1 blocks retroviral infection before reverse transcription. I have never understood this concept. My understanding is that reverse transcription happens in cytoplasm (Remember! RT interfered by APOBEC3G/F. So how exactly a person sitting in nucleus is messing with reverse transcription?). But answer seems to be there here. The authors went on to test, if SAMHD1 needs to sit in nucleus to cause effect? And they found answer is no. Am relieved.

       Now the question is what's the counter force of virus? Its shown that Vpx assembles with the CUL4A-DDB1 ubiquitin ligase through DCAF1 recruitment. This was presumed to cause erosion of restriction.

Fig 5: Presumed mode of action of HIV-2 Vpx in macrophage infection (Source).

      The authors hypothesized that nuclear localization of SAMHD1 is required for the ability of Vpx to induce its degradation and overcome restriction. To answer the question they just measured the ability of nuclear and cytoplasmic Vpx proteins to induce degradation of SAMHD1 variants that localized to the cytoplasm. They found that Vpx nuclear alleles required a  nuclaer localized SAMHD1 so as to induce its degradation and to overcome restriction.

       So I conclude second important thing. (the first important thing was 11 KRPR 14  is the NLS). The SAMHD1 should be present in cytoplasm to attack reverse transcriptase but for the vpx to attack SAMHD1, should be in nucleus. That means, if the SAMHD1 stays in cytoplasm cell has competitive edge. But if the SAMHD1 goes to nucleus, now the virus has the say. Ooops, thats wonderful isn't it. It should be noted that the vpx encounters the SAMHD1 in nucleus (O come on what do you expect, SAMHD1 to just sit and wait for a retrovirus to come when it has got the NLS to get into nucleus). Now note that if SAMHD1 is brought to cytoplasm for proteosomal degradation then virus is in trouble. So its safe to assume that the virus degrades the SAMHD1 at least partially, brings it to cytoplasm and then targets it to proteosomal degradation. Now for a min assume that the virus may have a mechanism to increase the SAMHD1 going to nucleus (Pathogens always possess these wooing to grave yard kind of strategy. Just guessing anyway).

       So what is the take home message. I would like to say i want to exploit this. Tell the SAMHD1 to be there in cytoplasm. Block the NLS...
Alberto Brandariz-Nuñez, Jose Carlos Valle-Casuso, Tommy E White, Nadine Laguette, Monsef Benkirane, Jurgen Brojatsch, & Felipe Diaz-Griffero (2012). Role of SAMHD1 nuclear localization in restriction of HIV-1 and SIVmac Retrovirology DOI: 10.1186/1742-4690-9-49

Further reading:
  1. Nadine Laguette etal. SAMHD1 is the dendritic- and myeloid-cell-specific HIV-1 restriction factor counteracted by Vpx. Nature 474, 654–657. Link
  2. Kasia Hrecka etal. Vpx relieves inhibition of HIV-1 infection of macrophages mediated by the SAMHD1 protein. Nature 474, 658–661. Link

No comments:

Post a Comment