Quantcast

Subscribe to:
POZ magazine
E-newsletters
Join POZ: Facebook MySpace Twitter Pinterest
Tumblr Google+ Flickr MySpace
POZ Personals
Sign In / Join
Username:
Password:
Welcome, Guest. Please login or register.
July 22, 2014, 05:16:01 AM

Login with username, password and session length


Members
Stats
  • Total Posts: 631414
  • Total Topics: 47799
  • Online Today: 218
  • Online Ever: 585
  • (January 07, 2014, 02:31:47 PM)
Users Online
Users: 3
Guests: 183
Total: 186

Welcome


Welcome to the POZ/AIDSmeds Community Forums, a round-the-clock discussion area for people with HIV/AIDS, their friends/family/caregivers, and others concerned about HIV/AIDS.  Click on the links below to browse our various forums; scroll down for a glance at the most recent posts; or join in the conversation yourself by registering on the left side of this page.

Privacy Warning:  Please realize that these forums are open to all, and are fully searchable via Google and other search engines. If you are HIV positive and disclose this in our forums, then it is almost the same thing as telling the whole world (or at least the World Wide Web). If this concerns you, then do not use a username or avatar that are self-identifying in any way. We do not allow the deletion of anything you post in these forums, so think before you post.

  • The information shared in these forums, by moderators and members, is designed to complement, not replace, the relationship between an individual and his/her own physician.

  • All members of these forums are, by default, not considered to be licensed medical providers. If otherwise, users must clearly define themselves as such.

  • Forums members must behave at all times with respect and honesty. Posting guidelines, including time-out and banning policies, have been established by the moderators of these forums. Click here for “Am I Infected?” posting guidelines. Click here for posting guidelines pertaining to all other POZ/AIDSmeds community forums.

  • We ask all forums members to provide references for health/medical/scientific information they provide, when it is not a personal experience being discussed. Please provide hyperlinks with full URLs or full citations of published works not available via the Internet. Additionally, all forums members must post information which are true and correct to their knowledge.

  • Product advertisement—including links; banners; editorial content; and clinical trial, study or survey participation—is strictly prohibited by forums members unless permission has been secured from POZ.

To change forums navigation language settings, click here (members only), Register now

Para cambiar sus preferencias de los foros en español, haz clic aquí (sólo miembros), Regístrate ahora

Finished Reading This? You can collapse this or any other box on this page by clicking the symbol in each box.

Author Topic: Engineered Human Fusion Protein Inhibits HIV-1 Replication  (Read 2785 times)

0 Members and 1 Guest are viewing this topic.

Offline Rev. Moon

  • Member
  • Posts: 3,782
  • Smart ass faggot ©
Engineered Human Fusion Protein Inhibits HIV-1 Replication
« on: September 09, 2009, 01:29:39 PM »
I had read elsewhere on these boards about the New World owl monkeys and TRIM 5, but this was just posted at JCI, so I figured those of you who like to stay in the know would be interested. 

First is an excerpt from Science Daily and below is the abstract (it is quite a lengthy piece), discussion, and link to full article from the Journal of Clinical Investigation.

Engineered Human Fusion Protein Inhibits HIV-1 Replication

http://www.sciencedaily.com/releases/2009/09/090908193430.htm

Quote

ScienceDaily (Sep. 8, 2009) — In 2004, Jeremy Luban and colleagues from the University of Geneva, Switzerland, reported that New World owl monkeys (Aotus genus) make a fusion protein – AoT5Cyp – that potently blocks HIV-1 infection. The human genome encodes the equivalent of the 2 components of AoT5Cyp (i.e., TRIM5 and cyclophilin A), but humans unfortunately do not make the T5Cyp fusion protein.

In their new study in the Journal of Clinical Investigation, Luban et al. have engineered a human HIV-1 inhibitor modeled after AoT5Cyp, by fusing human cyclophilin A to human TRIM5 (hT5Cyp). The human fusion protein blocked HIV-1 infection of human macrophage and T cell lines, without disrupting normal cell function.

Mice engineered to lack B, T, and NK immune cells (to ensure that the animals do not reject grafts of human material) were then engrafted with human CD4+ T cells engineered to contain hT5Cyp. HIV-1 replication was potently inhibited in these animals.

The authors concluded that hT5Cyp is a robust inhibitor of HIV-1 replication and a promising anti–HIV-1 gene therapy candidate.

Journal reference:
Luban et al. Potent inhibition of HIV-1 by TRIM5-cyclophilin fusion proteins engineered from human components. Journal of Clinical Investigation, 2009; DOI: 10.1172/JCI39354


Potent inhibition of HIV-1 by TRIM5-cyclophilin fusion proteins engineered from human components

http://www.jci.org/articles/view/39354

Quote

Published September 8, 2009
Received for publication March 27, 2009, and accepted in revised form July 8, 2009.

Abstract

New World monkeys of the genus Aotus synthesize a fusion protein (AoT5Cyp) containing tripartite motif-containing 5 (TRIM5) and cyclophilin A (CypA) that potently blocks HIV-1 infection. We attempted to generate a human HIV-1 inhibitor modeled after AoT5Cyp, by fusing human CypA to human TRIM5 (hT5Cyp). Of 13 constructs, 3 showed substantial HIV-1–inhibitory activity when expressed in human cell lines. This activity required capsid binding by CypA and correlated with CypA linkage to the TRIM5a capsid-specificity determinant and the ability to form cytoplasmic bodies. CXCR4- and CCR5-tropic HIV-1 clones and primary isolates were inhibited from infecting multiple human macrophage and T cell lines and primary cells by hT5Cyp, as were HIV-2ROD, SIVAGMtan, FIVPET, and a circulating HIV-1 isolate previously reported to be AoT5Cyp resistant. The anti–HIV-1 activity of hT5Cyp was surprisingly more effective than that of the well-characterized rhesus TRIM5α, especially in T cells. hT5Cyp also blocked HIV-1 infection of primary CD4+ T cells and macrophages and conferred a survival advantage to these cells without disrupting their function. Extensive attempts to elicit HIV-1 resistance to hT5Cyp were unsuccessful. Finally, Rag2–/–γc–/– mice were engrafted with human CD4+ T cells that had been transduced by optimized lentiviral vectors bearing hT5Cyp. Upon challenge with HIV-1, these mice showed decreased viremia and productive infection in lymphoid organs and preserved numbers of human CD4+ T cells. We conclude that hT5Cyp is an extraordinarily robust inhibitor of HIV-1 replication and a promising anti–HIV-1 gene therapy candidate.


Quote
Discussion

Anti–HIV-1 gene therapies have generally modified host cell factors required for viral replication or inhibited essential viral elements (4). Here, a third approach was adopted: the exploitation of natural inhibitors that evolved over millions of years in primates in response to retroviral attack (14–16, 33). The potent block to HIV-1 observed with AoT5Cyp inspired the design of a human equivalent, hT5Cyp, that robustly blocks HIV-1 in vitro and in vivo. A distinct T5Cyp fusion gene was generated by an independent retrotransposition event in Old World macaques. In this case, restriction activity was detected against HIV-2 and FIV, but not against HIV-1 (39–43). The convergent evolution of T5Cyp fusion proteins with distinct retroviral specificities indicates a strong selection for these potent restriction factors. While the specific force behind selection remains to be elucidated for individual TRIM5 orthologs, in each case the selective pressure is likely to have been a retrovirus (15).

Because TRIM5α and T5Cyp are modular proteins, it was expected that the engineering of a restrictive hT5Cyp would not be difficult. Surprisingly, only 3 of 13 hT5Cyp fusions inhibited HIV-1 with results comparable to AoT5Cyp. No correlation was observed between protein levels and antiviral activity, and CA-binding activity was not sufficient to restrict HIV-1 (Figure 2, A and B). Anti–HIV-1 activity was observed when hCypA was fused at the apex of the hTRIM5α PRYSPRY domain in the structural model (Figure 2C). Analysis of nonsynonymous mutations indicates that this region undergoes some of the strongest selective pressure in the primate lineage, and functional experiments pinpoint it as a specificity determinant for CA recognition (15, 16, 44). CypA fusion to this site may have antiviral activity because this apical loop is uniquely situated for coordinating CA recognition and effector domains of TRIM5. Correlation between restriction activity and the ability of hT5Cyp fusion proteins to form cytoplasmic bodies suggests that activity requires particular spatial constraints or association with unknown cofactors. The strict structural requirements for engineering a functional hT5Cyp emphasize the remarkable nature of the fusion gene that was generated by retrotransposition in New World owl monkeys (7).

Optimal gene therapy candidates provide high efficacy and low antigenicity. Hematopoietic progenitors transduced with rhesus macaque TRIM5α or human-rhesus TRIM5α chimeras differentiate into HIV-1–resistant macrophages and T cells (34, 45). These proteins, however, are potentially antigenic, which could lead to the elimination of modified cells in vivo (22). Nonetheless, efforts have been taken to minimize potential sources of antigenicity. By substituting only critical amino acids within the hTRIM5α PRYSPRY domain (hTRIM5αR332P, hTRIM5αrh332-332) one can reduce the risk of antigenicity, but this also decreases anti–HIV-1 efficacy (Figure 5). hT5Cyp is the most potent HIV-1 inhibitor of the TRIM5 proteins we compared, including rhTRIM5α, in both single-cycle and spreading infections of human T cells with HIV-1 (Figure 5). This difference in activity might be due to the apparently higher CA affinity of the Cyp domain of hT5Cyp (46). In addition to its superior potency, hT5Cyp is composed of human components with no amino acids derived from non-human orthologs. Despite having what we believe is a novel fusion junction, hT5Cyp is likely to have the lowest antigenic potential among these TRIM5-based gene therapies.

HIV-1 rev, tat, and gag (4, 47, 51), as well as the HIV-1 co-receptor CCR5 (48–50), have been targeted by various means including siRNAs (4, 50, 51) and zinc finger endonucleases (49). With either approach there are concerns regarding toxicity (51), off-target effects (48), and viral resistance (4, 51). Transdominant revM10 proteins have been tested in patients without reducing viremia (47). Furthermore, revM10-resistant HIV-1 strains were observed in vitro (52). Similarly, for siRNA approaches, escape mutants arise readily, since single-nucleotide changes are sufficient to escape the siRNA-mediated blocks to HIV-1 replication (51). CCR5 disruption, one of the most promising approaches to date, could select for CCR5-independent viruses (53) and is not without consequence, as the CCR5Δ32 allele is a risk factor for fatal West Nile Virus infection (54).

hT5Cyp is a broadly acting, antilentiviral agent that blocks CCR5- and CXCR4-tropic HIV-1 clones and primary isolates as well as some HIV-2, SIV, and FIV clones (Figures 4 and 6). TRIM5 isoforms form hetero-multimers, and hT5Cyp might interfere with endogenous hTRIM5α function (55). However, no significant decrease in hTRIM5α-mediated restriction of N-MLV, and no alteration in cell surface markers or cytokine secretion, was observed in hT5Cyp-transduced cells (Supplemental Figure 4). Despite extensive efforts to identify hT5Cyp-resistant viruses, none were detected, perhaps due to the potency of the antiviral activity and the fact that hT5Cyp blocks HIV-1 prior to RT, thus precluding the generation of the genetic diversity required for emergence of resistance. hT5Cyp additionally blocked a natural HIV-1 CA variant reported to be resistant to AoT5Cyp (28) (Figure 3D). These results suggest that, if hT5Cyp were utilized as anti–HIV-1 gene therapy in people, viral resistance would not emerge readily and host cell function would remain intact.

The evaluation of HIV-1 therapies is limited by a lack of adequate animal models for HIV-1 replication and pathogenesis. The hCD4+-Rag2–/–γc–/– mouse developed here is a straightforward alternative to the HIV-1 mouse models used to date (56–59). It permitted high-level HIV-1 viremia and assessment of the effect of hT5Cyp transduction on HIV-1 infection and CD4+ T cell protection (Figure #8). Autotransfusion of ex vivo expanded CD4+ T cells is of clinical benefit to HIV-1–infected people (60). Human CD4+ T cells can be transduced at high efficiency and expanded to more than 109 cells ex vivo. Given that hT5Cyp-transduced CD4+ cells can be expanded in vitro and in vivo (Figures 6 and 7), and that these cells exhibit a selective advantage during HIV-1 infection (Figure 6G and Figure 8A), there might be an impressive effect of hT5Cyp transduction on autologous CD4+ T cell gene therapy in the clinical setting.

CD34+ hematopoietic stem cells transduced with the state-of-the-art lentiviral vectors described here and elsewhere (61) achieve long-term engraftment of Rag2–/–γc–/– mice, but transgene expression in the mature CD4+ T cells that develop within these animals has not been consistently detected. Nonetheless, hT5Cyp modestly reduced HIV-1 viremia in humanized Rag2–/–γc–/– mice (Supplemental Figure #8). With improved transduction methods, it will be important to examine the effect of stem cell transduction with hT5Cyp on immune cell development and HIV-1 infection within one of the humanized mouse models currently under development aimed at enhancing de novo T cell development and in vivo immune function (62, 63).

"I have tried hard--but life is difficult, and I am a very useless person. I can hardly be said to have an independent existence. I was just a screw or a cog in the great machine I called life, and when I dropped out of it I found I was of no use anywhere else."

Offline Inchlingblue

  • Member
  • Posts: 3,119
  • Chad Ochocinco PETA Ad
Re: Engineered Human Fusion Protein Inhibits HIV-1 Replication
« Reply #1 on: September 09, 2009, 02:04:50 PM »
This is great news, it always made logical sense to me to try this.

There was a previous thread discussing a scientist, Dr. Hatziioannou, whose work is the opposite of this: she is trying to get monkeys not to recognize TRIM5 so they can get sick from HIV (!), in order to make them into better models for testing purposes. This makes no sense to me on so many levels, I even emailed her (not that she gives a shit, she probably thinks her approach is brilliant).

Duh (!) wouldn't it make more sense to make a human TRIM5 which might inhibit HIV in humans?

Thankfully, other researchers appear to be more enlightened than Hatziioannou.
 
LINK:

http://forums.poz.com/index.php?topic=27059.msg335239#msg335239

Offline Rev. Moon

  • Member
  • Posts: 3,782
  • Smart ass faggot ©
Re: Engineered Human Fusion Protein Inhibits HIV-1 Replication
« Reply #2 on: September 09, 2009, 02:21:50 PM »
There was a previous thread discussing a scientist, Dr. Hatziioannou, whose work is the opposite of this: she is trying to get monkeys not to recognize TRIM5 so they can get sick from HIV (!), in order to make them into better models for testing purposes. This makes no sense to me on so many levels, I even emailed her (not that she gives a shit, she probably thinks her approach is brilliant).

Duh (!) wouldn't it make more sense to make a human TRIM5 which might inhibit HIV in humans?

That is simply appalling.  Working on infecting a different species as an approach to the understanding of this virus?  I guess she's taking an "if it ain't broke, break it" type of approach.  I should send her an email as well (animal lover that I am  ;D)

Let's wait and see what else comes out of these findings/experiments from the Swiss.
"I have tried hard--but life is difficult, and I am a very useless person. I can hardly be said to have an independent existence. I was just a screw or a cog in the great machine I called life, and when I dropped out of it I found I was of no use anywhere else."

Offline Inchlingblue

  • Member
  • Posts: 3,119
  • Chad Ochocinco PETA Ad
Re: Engineered Human Fusion Protein Inhibits HIV-1 Replication
« Reply #3 on: September 09, 2009, 02:28:11 PM »
  I guess she's taking an "if it ain't broke, break it" type of approach.  

LOL that's funny, I've never heard that expression, did you just make it up?

I just noticed in the article that, in the lab at least, hT5Cyp also seems to work against FIV (good for the kitties!):

CXCR4- and CCR5-tropic HIV-1 clones and primary isolates were inhibited from infecting multiple human macrophage and T cell lines and primary cells by hT5Cyp, as were HIV-2ROD, SIVAGMtan, FIVPET, and a circulating HIV-1 isolate previously reported to be AoT5Cyp resistant.
« Last Edit: September 09, 2009, 02:34:32 PM by Inchlingblue »

Offline Rev. Moon

  • Member
  • Posts: 3,782
  • Smart ass faggot ©
Re: Engineered Human Fusion Protein Inhibits HIV-1 Replication
« Reply #4 on: September 09, 2009, 02:31:21 PM »
Nah, it was the title to some dumb corporate book that I had to read a few years ago  :)
"I have tried hard--but life is difficult, and I am a very useless person. I can hardly be said to have an independent existence. I was just a screw or a cog in the great machine I called life, and when I dropped out of it I found I was of no use anywhere else."

Offline freewillie99

  • Member
  • Posts: 304
Re: Engineered Human Fusion Protein Inhibits HIV-1 Replication
« Reply #5 on: September 09, 2009, 04:11:20 PM »
I should send her an email as well (animal lover that I am  ;D)

So you're rooting for Michael Vick this NFL season then?
Beware Romanians bearing strange gifts

Offline Rev. Moon

  • Member
  • Posts: 3,782
  • Smart ass faggot ©
Re: Engineered Human Fusion Protein Inhibits HIV-1 Replication
« Reply #6 on: September 09, 2009, 07:36:42 PM »
So you're rooting for Michael Vick this NFL season then?

LOL, heck no... Jets fan here.
"I have tried hard--but life is difficult, and I am a very useless person. I can hardly be said to have an independent existence. I was just a screw or a cog in the great machine I called life, and when I dropped out of it I found I was of no use anywhere else."

Offline brazilianman

  • Member
  • Posts: 92
Re: Engineered Human Fusion Protein Inhibits HIV-1 Replication
« Reply #7 on: September 10, 2009, 02:16:19 PM »
a little more of the same

Preintegration HIV-1 Inhibition by a Combination Lentiviral Vector Containing a Chimeric TRIM5 Protein, a CCR5 shRNA, and a TAR Decoy

Human immunodeficiency virus (HIV) gene therapy offers a promising alternative approach to current antiretroviral treatments to inhibit HIV-1 infection. Various stages of the HIV life cycle including pre-entry, preintegration, and postintegration can be targeted by gene therapy to block viral infection and replication. By combining multiple highly potent anti-HIV transgenes in a single gene therapy vector, HIV-1 resistance can be achieved in transduced cells while prohibiting the generation of escape mutants. Here, we describe a combination lentiviral vector that encodes three highly effective anti-HIV genes functioning at separate stages of the viral life cycle including a CCR5 short hairpin RNA (shRNA) (pre-entry), a human/rhesus macaque chimeric TRIM5 (postentry/preintegration), and a transactivation response element (TAR) decoy (postintegration). The major focus on designing this anti-HIV vector was to block productive infection of HIV-1 and to inhibit any formation of provirus that would maintain the viral reservoir. Upon viral challenge, potent preintegration inhibition of HIV-1 infection was achieved in combination vector–transduced cells in both cultured and primary CD34+ hematopoietic progenitor cell (HPC)–derived macrophages. The generation of escape mutants was also blocked as evaluated by long-term culture of challenged cells. The ability of this combination anti-HIV lentiviral vector to prevent HIV-1 infection, in vitro, warrants further evaluation of its in vivo efficacy.


http://www.nature.com/mt/journal/vaop/ncurrent/abs/mt2009187a.html
« Last Edit: September 10, 2009, 02:18:59 PM by brazilianman »

Offline Inchlingblue

  • Member
  • Posts: 3,119
  • Chad Ochocinco PETA Ad
Re: Engineered Human Fusion Protein Inhibits HIV-1 Replication
« Reply #8 on: September 10, 2009, 05:28:22 PM »
wow, that's a genetic ARV cocktail....very cool

Offline georgep77

  • Member
  • Posts: 145
Re: Engineered Human Fusion Protein Inhibits HIV-1 Replication
« Reply #9 on: September 11, 2009, 09:07:33 AM »
wow, that's a genetic ARV cocktail....very cool
Can't wait for my genetic cocktail !!   woo hoo
Come on Sangamo,  Geovax,  Bionor immuno, ...Make us happy !!!
+ 2008

Offline brazilianman

  • Member
  • Posts: 92
Re: Engineered Human Fusion Protein Inhibits HIV-1 Replication
« Reply #10 on: September 11, 2009, 10:53:08 AM »
study phase zero poit zero comma zero


study phase zero.
will still take another 10 years.
my fear and find a preventive vaccine.
after that we'll be left behind.

 


Terms of Membership for these forums
 

© 2014 Smart + Strong. All Rights Reserved.   terms of use and your privacy
Smart + Strong® is a registered trademark of CDM Publishing, LLC.