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Nef: target for new potential HIV treatment - UMass Medical researchers uncover promising clue to battling HIV 'New treatment possibilities' - "New HIV Treatment Target: HIV: Antiviral action countered by Nef" - "SERINC3 and SERINC5 restrict HIV-1 infectivity and are counteracted by Nef"
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Two independent research teams at the University of Massachusetts Medical School have worked out a key step in HIV-1 infection - and their findings point to a possible way to fight the virus.
Further, new treatments for other infections caused by similar enveloped viruses could be speeded by the discovery.
Through different means, the two teams at the Worcester institution unraveled part of the mechanism of retroviral infection in host cells. The details could lead to the development of drugs targeting one of the few proteins the HIV-1 virus makes from its own DNA: called Nef. Drugs would aim to take that protein down.
Nef, the researchers showed, disables two cell membrane proteins -- SERINC5 and SERINC3 - that otherwise protect the host by somehow impeding the virus's ability to replicate and spread to other cells.
The two labs' papers will be published in the Oct. 8 issue of Nature, and are available now online. The study by the team of Jeremy Luban, M.D., on which scientists at two universities in Europe collaborated, is available here. The study by Heinrich Gottlinger, professor of molecular, cell and cancer biology at UMass Medical School, is available here.
"It's amazing, the magnitude of the effect that these proteins have on infectivity," Luban said in a statement Wednesday from the medical school. He is the David J. Freelander professor in AIDS Research and a professor of molecular medicine at the school. "The SERINC proteins reduce the infectivity of HIV-1 virions by more than 100-fold."
HIV-1 is the predominant strain of the AIDS-causing human immunodeficiency virus.
Luban's colleague Heinrich Gottlinger, professor of molecular, cell and cancer biology at UMass Medical School, said: "The ability of HIV to inhibit these SERINC proteins has a profound impact on its capacity to infect other cells. Disrupting this mechanism could be a very powerful strategy for treating HIV and similar viruses that express the Nef protein."
According to the statement from the medical school, the two studies used completely different, yet complementary, methods to probe the complex interaction between Nef and the cell surface membrane proteins SERINC5 and SERINC3, both of which are expressed in the immune system's T cells.
Dr. Luban - working with former members of his lab Massimo Pizzato, now of the University of Trento in Italy, and Federico Santoni of the University of Geneva in Switzerland - "performed massively parallel sequencing on 31 human cell lines that differed in terms of the magnitude of dependence on Nef for HIV-1 replication," the school said.
Gottlinger approached the problem biochemically. Analyzing proteins in purified virions, his lab was able to identify host cell proteins regulated by Nef. The statement from the medical school explains: "Viruses are relatively simple agents that lack the machinery needed to reproduce on their own. The HIV-1 virus, for example, consists of only nine genes. To replicate its genome, HIV-1 requires a host cell. Once the virus has infected a cell it takes over certain native cellular processes so that it can replicate. Ultimately, the infected host cell produces new virions carrying the HIV-1 genome. These virions, in turn, search for new cells to infect, continuing the cycle of infection." A virion is a complete, infective form of a virus outside a host cell.
When host proteins SERINC5 and SERINC3 have been allowed to do their jobs - that is, when the virus's Nef protein cannot counteract them - they enter the viral envelope as the virion leaves the host cell, and render the virion a dud, unable to keep infecting cells. The school said further research will aim to determine just what SERINC5 and SERINC3 do to block the virus from replicating. In some way they seem to lock the viral genome inside the envelope, preventing the genetic material from entering new host cells.
"It has been known for more than 20 years that Nef is needed to make HIV-1 such a deadly virus," Luban said in the statement. "Our new studies may finally give us an important glimpse into how Nef might do this."
And, how to stop it.
New HIV Treatment Target: HIV: Antiviral action countered by Nef.....http://www.natap.org/2015/HIV/100215_05.htm
The identification of SERINC3 and SERINC5 as antiviral proteins that are counteracted by diverse retroviruses suggests that these proteins may also target other enveloped viruses, which may in turn have different mechanisms for escaping their antiviral action. These two proteins can therefore be used as probes to examine the entry mechanisms of enveloped viruses. Although the available data suggest that these proteins target specific regions of viral glycoproteins, it is possible that they inhibit fusion indirectly by controlling the lipid composition or fluidity of the viral membrane. Regardless of the specific antiviral mechanism, the ability of Nef to counteract SERINC3 and SERINC5 adds to the impressive list of functions of this remarkable little viral protein.
SERINC3 and SERINC5 restrict HIV-1 infectivity and are counteracted by Nef
Nature Oct 2015
Yoshiko Usami1*, YuanfeiWu1* & Heinrich G. Gottlinger1
HIV-1 Nef and the unrelated mouse leukaemia virus glycosylated Gag (glycoGag) strongly enhance the infectivity of HIV-1 virions produced in certain cell types in a clathrin-dependent manner. Here we show that Nef and glycoGag prevent the incorporation of the multipass transmembrane proteins serine incorporator 3 (SERINC3) and SERINC5 into HIV-1 virions to an extent that correlates with infectivity enhancement. Silencing of both SERINC3 and SERINC5 precisely phenocopied the effects of Nef and glycoGag on HIV-1 infectivity. The infectivity of nef-deficient virions increased more than 100-fold when produced in double-knockout human CD4+ T cells that lack both SERINC3 and SERINC5, and re-expression experiments confirmed that the absence of SERINC3 and SERINC5 accounted for the infectivity enhancement. Furthermore, SERINC3 and SERINC5 together restricted HIV-1 replication, and this restriction was evaded by Nef. SERINC3 and SERINC5 are highly expressed in primary human HIV-1 target cells, and inhibiting their downregulation by Nef is a potential strategy to combat HIV/AIDS.
Nef is an accessory protein encoded by HIV-1 and other primate lentiviruses. In vivo, Nef is a major pathogenicity determinant that is required for high virus loads1, 2, 3. Although not essential for virus replication in cell culture, Nef enhances virus spreading in primary CD4+ T cells, particularly when such cells are infected before mitogenic stimulation4, 5, 6. Nef robustly downregulates the viral entry receptor CD4 from the surface of virus-producing cells by inducing its clathrin-dependent endocytosis and subsequent lysosomal degradation7, 8, 9, 10. A recent study suggests that an important physiological function of CD4 downregulation by Nef is to prevent the CD4-induced exposure of epitopes in HIV-1 envelope (Env) that make infected cells susceptible to antibody-dependent cell-mediated cytotoxicity11. Apart from CD4, Nef downregulates several other cell surface proteins12. The selective down-modulation of HLA-A and HLA-B but not of HLA-C by Nef serves to protect infected cells both from cytotoxic T cells and from natural killer cells13, 14, 15. The Nef proteins of most primate lentiviruses also down-modulate the T cell receptor complex, which is thought to protect infected T cells from activation-induced cell death in non-pathogenic natural SIV infections16. This function of Nef was lost in HIV-1 and closely related viruses, which may contribute to the pathogenicity of HIV-1 in humans16. One of the most conserved yet poorly understood functions of Nef is the enhancement of progeny virion infectivity17, 18. Although Nef exerts its effect on HIV-1 infectivity in virus producer cells, it does not detectably affect virus morphogenesis or maturation19, 20, 21, 22. Nevertheless, progeny virions produced in the absence of Nef do not efficiently reverse transcribe their genome in target cells19, 20. It has been reported that high levels of cell-surface CD4 inhibit the release or infectivity of HIV-1 progeny virions, and that Nef relieves these effects23, 24. However, the enhancement of HIV-1 infectivity depends on residues within Nef that are dispensable for its ability to downregulate CD4 (ref. 25). Furthermore, Nef enhances HIV-1 progeny virion infectivity even when CD4 is not expressed or cannot be downregulated17, 19, 20. Finally, the glycoGag protein of Moloney murine leukaemia virus (MLV) closely mimics the effect of Nef on HIV-1 infectivity, even though glycoGag does not downregulate CD4 (ref. 26). MLV glycoGag is an accessory protein whose translation begins at an inefficient CTG start codon upstream and in-frame with the gag gene27. The resulting product is a type II transmembrane protein with an amino-terminal cytosolic non-Gag portion and an extracellular Gag domain28. The potent Nef-like activity of glycoGag on HIV-1 infectivity resides entirely in its cytosolic domain, which is unrelated to Nef29. Nevertheless, the effects of Nef and glycoGag on HIV-1 infectivity appear mechanistically related. Both are similarly dependent on the producer cell type26, are similarly determined by variable regions of HIV-1 Env30, and exhibit a similar reliance on clathrin-mediated endocytosis29, 31, 32. However, the molecular basis for these similarities remains unknown.
Our findings reveal that HIV-1 Nef and MLV glycoGag efficiently downregulate SERINC3 and SERINC5 from the cell surface, which prevents their incorporation into HIV-1 virions and consequently counteracts their inhibitory effect on HIV-1 infectivity. Importantly, these findings offer an explanation for why the enhancement of HIV-1 infectivity by Nef and glycoGag is highly dependent on dynamin 2, clathrin and the AP-2 clathrin adaptor complex29, 31. SERINC family members are present in all eukaryotes, but their functions remain largely unknown. SERINC proteins reportedly enhance the incorporation of serine into phosphatidylserine and sphingolipids33. In principle, this activity could affect the lipid composition of the viral envelope, which is considered crucial for virion infectivity40. Our data demonstrate that SERINC3 and SERINC5 together account for most if not all of the effects of Nef on HIV-1 infectivity and on HIV-1 replication in JTAg cells. Notably, Nef enhances HIV-1 infectivity and stimulates HIV-1 replication in human PBMC4, 5, 6, 18, 41, whose SERINC3 and SERINC5 mRNA levels exceed those of Jurkat cells (Extended Data Fig. 4). The ability of virions produced in the absence of Nef to reverse transcribe their genome in target cells is impaired17, 19, 20. Consistent with these observations, we find that SERINC5 in virus producer cells strongly inhibits the ability of Nef- HIV-1 virions to complete reverse transcription. We also find that SERINC5 can in principle abolish the ability of progeny HIV-1 virions to fuse with target cells. However, lower levels of SERINC5 inhibited the fusion step to a lesser extent than the ability of progeny virions to productively infect target cells. Although there is controversy about the effect of Nef on HIV-1 entry35, 42, 43, a twofold reduction in the ability of Nef- HIV-1 virions to fuse with target cells was noted in one study35. In all of these studies, virus was produced in 293T cells, whose endogenous SERINC5 mRNA levels are low (Extended Data Fig. 4). It is conceivable that relatively low levels of virion-associated SERINC5 impair primarily fusion pore enlargement, which poses a higher energy barrier to overcome than pore formation44. This would be expected to impair passage of the viral core but not necessarily of the much smaller BlaM-Vpr fusion indicator into target cells. Consistent with a role in entry, Nef enhances the cytoplasmic delivery of viral cores45. Further, the requirement for Nef is determined by HIV-1 Env30.
Interestingly, the Env proteins of HIV-1NL43 and HIV-1SF162, which are highly responsive to Nef and glycoGag, require a higher number of Env trimers to complete entry than the poorly Nef/glycoGag-responsive EnvJRFL30, 46. Furthermore, the naturally occurring Asn160Lys mutation in the V2 loop of HIV-1 Env, which results in the loss of a glycosylation site, can increase both the responsiveness to Nef and glycoGag and the stoichiometry of entry30, 46. Mechanistically, a link between Nef/glycoGag responsiveness and the stoichiometry of entry could be due to an inhibitory effect of virion-associated SERINCs on the clustering of Env trimers. Notably, such clusters have been visualized on the surface of mature HIV-1 virions and, most prominently, at virus-cell contact zones47, 48. Alternatively, SERINCs embedded in the virion membrane could increase the energy barrier for fusion pore expansion. In both scenarios, differences in Nef/glycoGag-responsiveness among HIV-1 Envs, as well as differences in SERINC-sensitivity, may ultimately be due to differences in the amount of energy that these Envs provide towards fusion. Regardless of the mechanism, our observation that viruses as distant as HIV-1 and MLV have evolved to counteract SERINC3 and SERINC5 raises the possibility that these proteins have a broader role in innate antiviral immunity.

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