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Sean Hogan

PLOS Pathogens: Different Modes of Retrovirus Restriction by Human APOBEC3A and APOBEC3... - 22 views

  • One such family of restriction factors is the apolipoprotein B editing complex 3 (A3) cellular cytidine deaminases (CDA). While A3 genes are found in all mammals, their number differs from species to species. For example, humans have 7 A3 genes (A3A to A3H) while mice have only one gene. All proteins in this family contain at least one CDA domain that deaminates carbon 4 of cytidine in single-stranded DNA, resulting in a uracil that causes G to A transitions in the opposing strand [3].
    • alexridesducati
       
      Can these genes be exploited for antiviral therapy and if so, can it be done without harm to the host due to mutations?
  • viral cDNA accumulation
  • Packaging of A3G into virions is counteracted by HIV Vif (viral infectivity factor) protein. In virus-producer cells, Vif binds to A3G as well other A3 family members, and recruits cellular E3 ubiquitin ligase complexes, leading to ubiquitination and subsequent proteasomal degradation, thereby preventing packaging of A3G into budding virions [12]–[14]. Lentiviral Vif proteins show strong species-specificity. For example, HIV-1 Vif counteracts human A3G but only certain simian A3G homologues [15], [16]; it also does not interact with mouse A3 [17].
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  • Other members of the A3 family are believed to affect other exogenous viruses as well as endogenous retrovirus/retroelement movement within the genome. In particular, human A3A is a potent inhibitor of IAP and MusD and other retrotransposons such as LINE-1 and this inhibition is CDA-independent, at least in cultured cells [18]–[20]. A3A also inhibits adeno-associated virus replication, a nuclear-replicating parvovirus, via CDA-independent means [20]. In monocytes, A3A restricts HIV-1 infection and the decrease in A3A levels that occurs during monocyte-to-macrophage development is concomitant with increased susceptibility to HIV-1 infection [21]. A3A is not packaged into HIV virions and is thought to restrict infection by targeting incoming virus [22]–[24]. In contrast, A3A is packaged in human T-lymphotropic virus type-I virions and restricts infection, at least in transfected cells [25]. A3A preferentially deaminates cytidines that are in a TC motif [26].
  • Different A3 family members block infection by diverse retroviruses from different species, including HIV-2 [27], porcine endogenous retrovirus [28], [29], xenotropic, Friend (F-MLV) and Moloney murine leukemia virus (M-MLV) [30]–[32] and mouse mammary tumor virus (MMTV) [33]. Additionally, A3 proteins may restrict other virus families, including parvoviruses [20], [34], hepatitis B virus [35]–[37], papillomaviruses [38] and herpes simplex virus I [39]. Thus, it has been suggested that A3 proteins exist, at least in part, to prevent zoonotic transmission of viruses [40].
  • Here, we show that transgenic mice expressing the human A3A or A3G proteins restrict murine retrovirus infection in vivo in disparate ways. A3G was packaged into virions in vivo, leading to the deamination of both MLV and MMTV viral genomes. In contrast, A3A was not packaged, and appeared to restrict infection in a largely CDA-independent manner. Finally, we show that Vif/A3G interactions can be studied in this in vivo model, thus providing a potentially useful system for the analysis of small molecule inhibitors of A3 proteins and Vif.
  • To determine the level of transgene expression, we first isolated RNA from different tissues, including peripheral blood mononuclear cells (PBMCs), and performed reverse-transcribed real-time quantitative PCR (RT-qPCR). RNA from human H9 cultured cells and human and C57BL/6 mouse PBMCs served as controls. For each transgene, there was one high- (A3Ghigh, A3Ahigh) and one low- (A3Glow, A3Alow) expressing strain, defined by their relative expression in lymphoid tissues. The A3Ghigh strain expressed higher levels of the transgene than the endogenous mouse gene in spleen and thymus, but similar A3G levels in mouse and human PBMCs, while the A3Glow strain expressed approximately 10-fold lower levels in these tissues (Figure 1A). In contrast, the A3Ahigh strain expressed similar or lower levels than mouse A3; there was also about 2-fold lower expression of A3A in mouse PBMCs than in human PBMCs (Figure 1B). The A3Alow strain had very low but detectable levels of expression in several tissues. Since the β-actin regulatory region was used, transgene expression was seen in many tissues and in several at levels higher than endogenous mouse A3 (e.g. heart, brain and liver) (Figure 1A and 1B). We also performed western blots on different tissues from the 4 different mouse strains, using antiserum that detects both A3A and A3G. The relative protein expression levels were similar to that seen at the RNA level (Figure S1A and S1B).
  • We next determined if the in vivo-produced A3A and A3G proteins were functionally active. Extracts were prepared from primary splenocyte cultures and equal amounts (total protein concentration/volume) were incubated with FAM-labeled substrates containing the A3A- or A3G-preferred target sequence (S50-TTC and S50-CCC, respectively). As controls, we also performed these assays with extracts prepared from 293T cell lines transfected with A3A or A3G. Activity could be readily detected in transgenic mice expressing high levels of A3A or A3G. Further, in accord with the known specificity of the cytidine deaminases, extracts from the A3Ahigh mice deaminated the TTC- more efficiently than CCC-containing substrates, while those from A3Ghigh mice more efficiently deaminated the CCC substrate (Figure 2). For both A3Alow and A3G low, trace amounts of activity were detectable with the preferred substrates, while no activity was detectable with either endogenous mA3 or from mA3 knockout splenocytes. No deaminase activity was detected with WT mouse extracts, perhaps because the mouse protein has lower overall activity or expression. These data show that the transgenic mice expressed catalytically active human deaminases in these heterologous cells.
  • Humans have 7 APOBEC3 genes and determining how each specifically functions to inhibit retroviruses like HIV is complicated, because all 7 can be produced in a given cell type or tissue.
    • laceemarie
       
      What cell/tissue type(s) are these APOBEC3 genes naturally turned on in? 
  • To overcome this limitation, we made transgenic mice that express two of the human proteins, APOBEC3A and APOBEC3G in mice that do not express their own APOBEC3. These mice were able to effectively block infection by several mouse retroviruses
    • laceemarie
       
      What cell type(s) did they use? Does it matter which?
  • We were unable to perform similar assays with in vivo produced MMTV, because the only cell-free virus in mice is found in milk and mammary tumors and we have not yet established breeding colonies of virus-infected human A3 transgenic mice.
    • laceemarie
       
      Was this a screw up, or is it not that important to look at assays with in vivo produced MMTV. And by "not yet," does that mean they are going to? I feel like if your going to use this virus in your experimental studies, you should figure out ways to perform the assays, regardless of how you get the virus. It would appear that they knew this information before hand, so maybe an assay on MMTV is less relevant. 
    • laceemarie
       
      *you're
  • A3G and A3A inhibit retrovirus infection by different means.
    • becky214
       
      Since they use different means of inhibition, do they work together to prevent infection? Or is an and either/or type scenario?
  • Two A3A and A3G mouse strains each were generated, expressing levels of these proteins within the range or at levels lower than that seen in human cells. This likely has relevance to what occurs in individual humans, where non-coding region polymorphisms in A3 genes alter expression levels and may influence progression to HIV-induced disease
    • laceemarie
       
      Could this have something to do with how HIV works in the HIV controllers? Where they still exhibit virus particles, but at a lower amount, don't necessarily spread the virus as much, and don't exhibit as intense of HIV symptoms?
    • ameliaobert
       
      If A3A is less clear, but does not get packaged, where must the A3A involvement with the incoming virus be located (for myeloid cells)? As well as if it is known to use retroelement retotransposition and replication inhibiton in paraoviruses by nh2 to oh independent means, how does the A3A know to be signlle to change the structure of cystine?
  • An additional limitation of previous studies done on human A3 proteins is the reliance on transfecting constructs expressing A3 proteins, which may not reflect the endogenous levels of a protein expression found in vivo
    • Sean Hogan
       
      Couldn't they mimic the in vivo environment by overexpressing necessary proteins in the host?
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    Focus paper for retrovirus presentation.
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    This will be the focus paper for 11/14.
becky214

Targeting therapeutics to an exposed and conserved binding element of the HIV-1 fusion ... - 10 views

  • We report that the C-terminal region of the HIV-1 gp41 ectodomain (and gp160 precursor molecule) appears to be partially exposed and vulnerable to an antiviral agent before the receptor-mediated conformational changes that initiate membrane fusion.
    • laceemarie
       
      How long is this domain "exposed and vulnerable" before the membranes fuse? Would whatever antiviral treatment that targets this have to be able to hang around, so to speak, for a while in between HIV-host interactions, without being degraded or absorbed or moved to another place in or out of the body?
  • and those that can eliminate infected cells, thereby reducing persistent and latent reservoirs of the virus.
    • apopp10
       
      It would be interesting to see if there will be a drug that can eventually do this that will be low-cost and is manageable in dosage. The problem is how much the virus mutates to evade these drugs and the host immune system. Combination therapy with the RT inhibitor, protease inhibitor, the experimental design listed her and this drug may be effective at treating HIV in the future.
  • These studies do not determine whether 5-Helix interacts with the native conformation of Env or, rather, some misfolded conformation of gp41 and gp160 on the cell surface.
    • Sean Hogan
       
      What would cause the misfolding of Env? Wouldn't it be advantageous to interact with both in order to prevent HIV infection?
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  • Moreover, we demonstrate that this binding is sufficient to concentrate a recombinant toxin to selectively kill HIV-1-infected cells. Our results suggest that the gp41 C-peptide region is a viable target for development of antiviral therapeutics, neutralizing antibodies, and cytotoxic agents directed against infected cells.
    • becky214
       
      How could this help develop antibodies when it is a a toxin that will kill HIV-1-infected cells? 
  • Such a molecule, administered together with agents that induce the expression of dormant integrated provirus (e.g., cytokines or activators of protein kinase C), might help to reduce or possibly eliminate latent reservoirs of HIV-1
    • becky214
       
      I think this is really interesting that it can possibly eliminate HIV that is latent. I have not heard of any anti-viral agents that target latent cells, and this sounds like it could be revolutionary in research on HIV-1.
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    Focus paper for wednesday
apopp10

The Major Genetic Determinants of HIV-1 Control Affect HLA Class I Peptide Presentation - 11 views

  • Yet a small number of people demonstrate sustained ability to control HIV replication without therapy. Such individuals, referred to as HIV controllers, typically maintain stable CD4+ cell counts, do not develop clinical disease, and are less likely to transmit HIV to others (2).
    • laceemarie
       
      This is really cool! I have not heard of this until now. Have these people been studied to find out why this occurs? Is it because of the genetics of the person or a certain mutation in the virus? And the key words here are "less likely." That's a pretty broad statement - "less likely" meaning how likely?
    • slgoogin8981
       
      It is interesting to see that there are 3 allelic variants that correlate with disease prognosis, but how does this information benefit society? We can't change peoples genetic so to alter their prognosis. Is this just a benefit to know what medications would be most beneficial with the least amount of side effects?
  • Abstract
    • Sean Hogan
       
      Presentation Paper 11/14
  • ...2 more annotations...
  • Although variation in the entire HLA protein is involved in the differential response to HIV across HLA allotypes, the major genetic effects are condensed to the positions highlighted in this study, indicating a structural basis for the HLA association with disease progression that is probably mediated by the conformation of the peptide within the class I binding groove.
    • becky214
       
      If variation in the HLA protein results in different responses to HIV, does it also cause different responses to antiviral HIV drugs?
    • apopp10
       
      So it's only the amino acid position that accounts for the SNP and HLA association signals? It does not matter what amino acid you place there? Individual amino acid chemistry will have no affect on it?
Casey Finnerty

HIV Latency - 0 views

  • For HIV-1, the term latency was initially used in the clinical sense to describe the long asymptomatic period between initial infection and the development of AIDS. However, with the advent of sensitive RT-PCR assays for viremia (Piatak et al. 1993), it became clear that HIV-1 replicates actively throughout the course of the infection, even during the asymptomatic period. The major mechanism by which HIV-1 evades immune responses is not latency but rather through rapid evolution of escape mutations that abrogate recognition by neutralizing antibodies and cytolytic T lymphocytes (Bailey et al. 2004). Nevertheless, it has become clear that HIV-1 can establish a state of latent infection at the level of individual T cells
jiyoung yoon

Association of HIV Diversity and Survival in HIV-Infected Ugandan Infants - 10 views

    • Sarah Muncy
       
      Ha, so again, like an organism. Where a population has a great deal of genetic diversity, there are more traits onto which natural selection can act. The more diverse the HIV is at this given stage means a failure in this case though, as the host dies and cannot transmit the disease. Strange.
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    That's a really cool observation, Sarah! It's really all about transmission. Since HIV is (primarily) sexually transmitted, infection in infants (via transovarial transmission) may be considered a dead end for the virus. Perhaps vertical transmission is an aberrance. If HIV would evolve into a less virulent form, perhaps vertical transmission would become more important. Just sayin'...
Sarah Muncy

The HIV Virus: A Possible Cure for Leukemia? | Yahoo! Health - 0 views

  • It's important to note that the T-cells are removed from the patient before being bioengineered with the HIV virus
    • Sarah Muncy
       
      Why is it important to note that the disabled virus isn't injected into the patient? It's function isn't like chemotherapy at all- why even make the comparison?
    • Sarah Muncy
       
      Is there some reason they use T cells only? Are they trying to target T cells, but not dendritic cells or macrophages?
  • therapies that involve the reprogramming of a patient’s immune system, may also eventually be used to fight cancerous breast and prostate tumors.
    • Sarah Muncy
       
      What about lupus, or even allergies for that matter?
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  • Within hours
    • Sarah Muncy
       
      Within hours?!
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    Whoa. If someone is doing HIV- this may be helpful. The virus is being used as a delivery device for genetics to reprogram lymphocytes. They're like biological nanorobots.
sdahlseng10

Possible applications for replicating HIV 1 vectors - 0 views

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    Review Article; Journal club 1: This is a basic review article about HIV virus vectors and their potential uses in clinical science.
Casey Finnerty

Phys.org - News and Articles on Science and Technology - 0 views

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    HIV structure
Casey Finnerty

'Binding, bending and bonding': polypurine tract-primed initiation of plus-strand DNA s... - 0 views

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    This review describes nicely HIV replication and the role of the PPTs.
sdahlseng10

In Vivo Gene Delivery and Stable Transduction of Nondividing Cells by a Lentiviral Vector - 0 views

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    Supplemental Article to Lesch et al. (2011); Journal club 1: This article provides interesting support to the effectiveness of lentivirus vectors for gene therapy treatment. Not only is HIV or lentivirus efficient at transduction to rapidly dividing cells, it is also capable of transduction to nondividing cells; making it an exciting vector for gene therapy treatment. The range of diseases that could be treatable with a gene therapy vector that can target other cell types than hematopoietic cells could transform the science of gene therapy.
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