Genetic and functional analysis of the human immunodeficiency virus (HIV) type 1-inhibiting F12-HIVnef allele

D'Aloja, Paola; Santarcangelo, Anna Claudia; Arold, Stefan T.; Baur, Andreas S.; Federico, Maurizio

doi:10.1099/0022-1317-82-11-2735

Cited by 31 publications

(23 citation statements)

References 46 publications

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“…A second well known property of Nef, the down-regulation of surface molecules such as CD4 and MHC I, has also been shown to be defective in Nef7 (31). Our results show that in HeLa cells WT Nef down-regulates both CD4 and MHC I, with MHC I down-regulation being much more efficient.…”

Section: Discussionsupporting

confidence: 63%

“…Nef7 is a derivative of F12-HIV, a non-producing HIV-1 strain (30,31); it is defective for a number of the typical Nef functions, including CD4 and major histocompatibility complex (MHC) class I down-regulation, p21-activated kinase 2 (PAK-2) activation, and binding to the V1H regulatory subunit of the vacuolar ATPase (31). Importantly, although WT Nef is incorporated into the HIV-1 core, Nef7 has the ability to be incorporated into virions at a much higher level.…”

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confidence: 99%

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Inhibition of HIV-1 Infection and Replication by Enhancing Viral Incorporation of Innate Anti-HIV-1 Protein A3G

Green

Liu

2009

Journal of Biological Chemistry

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APOBEC3G (A3G) is a cellular protein that has been identified as an innate anti-human immunodeficiency virus type 1 (HIV-1) factor. One of the major functions of HIV-1 virion infectivity protein (Vif) protein is to target A3G for ubiquitination/proteasome-mediated degradation and, as a result, evade the host innate defense mechanism. Thus, we wished to devise a strategy to restore the anti-HIV activity of A3G by actively targeting it into HIV-1 virions and countering HIV-1 Vif-targeted degradation. In the current study we performed a series of proof-of-concept experiments for this strategy using as a delivery vehicle of A3G, a derivate of non-pathogenic Nef mutant Nef7 that is capable of being efficiently incorporated into HIV-1 virions. We demonstrate that the Nef7.A3G fusion protein retains several important properties of Nef7; that is, the higher virion incorporation efficiency, no PAK-2 (p21-activated kinase 2) activation, and no CD4 and major histocompatibility complex I down-regulation. Meanwhile, we show that virion incorporated Nef7.A3G possesses the anti-HIV infectivity function of A3G. Moreover, we show that virus-like particle-mediated inverse fusion delivery of Nef7.A3G into HIV-infected CD4؉ T lymphocytes leads to potent inhibition of HIV-1 replication in these cells. Taken together, these results indicate that Nef7.A3G can effectively restrict HIV infection and replication by restoring the virion incorporation of A3G, even in the presence of Vif.Apolipoprotein mRNA editing enzyme catalytic peptide 3G (APOBEC3G, or A3G) is an innate antiviral cellular protein that dramatically reduces human immunodeficiency virus type 1 (HIV-1) 2 infectivity when incorporated into virions (1, 2). It is a single-stranded DNA deaminase that functions immediately after viral infection, during the first round of reverse transcription, to deaminate cytidine to uracil on the minus strand of the proviral DNA. This results in hypermutations that lead to loss of genetic stability and protein function (3, 4). The key to the A3G anti-HIV function is virion incorporation. When HIV-1 infects a cell, A3G is incorporated into the progeny virions from that cell through interactions with HIV-1 nucleocapsid protein and RNA. When those virions then infect a second cell, A3G renders them non-replicative. However, lentiviruses such as HIV-1 have evolved a mechanism to prevent the antiviral effects of A3G and other members of APOBEC3 family. The HIV-1 virion infectivity protein (Vif) binds to A3G and targets it for degradation by recruiting the E3 ubiquitin ligase cullin 5-elongin B/C (5), thus preventing A3G encapsidation (6 -9).HIV-1 Nef protein is an accessory protein of ϳ27 kDa. It is post-translationally modified by phosphorylation and by the addition of a myristoyl moiety to its second amino acid (glycine), which aids in its membrane targeting and is required for many Nef functions (10 -12). Although Nef is dispensable for in vitro HIV-1 replication, it is essential for efficient HIV-1 replication and pathogenesis in vivo (13-16). ...

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Section: Discussionsupporting

confidence: 63%

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confidence: 99%

Inhibition of HIV-1 Infection and Replication by Enhancing Viral Incorporation of Innate Anti-HIV-1 Protein A3G

Green

Liu

2009

Journal of Biological Chemistry

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“…This suggested that receptor-independent HIV-1 endocytosis (23,27,49) is not involved in the transfer of viral products. To test this hypothesis, U937 cells previously infected with VSV-G-pseudotyped NL4-3/Nef F12 , i.e., an HIV-1 variant unable to release viral particles in the supernatant (14,39), were cocultured with 293/CD8T cells in the presence of 20 M azidothymidine. We noticed that the transfer of HIV-1 Gag products to target cells occurred at levels similar to those detected using U937 cells infected with wild-type (wt) HIV-1 as donor cells (Fig.…”

Section: Resultsmentioning

confidence: 99%

Macrophages Transmit Human Immunodeficiency Virus Type 1 Products to CD4-Negative Cells: Involvement of Matrix Metalloproteinase 9

Muratori¹,

Sistigu²,

Ruggiero³

et al. 2007

J Virol

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It was previously reported that human immunodeficiency virus type 1 (HIV-1) spreads in CD4 lymphocytes through cell-to-cell transmission. Here we report that HIV-1-infected macrophages, but not lymphocytes, transmit HIV-1 products to CD4-negative cells of either epithelial, neuronal, or endothelial origin in the absence of overt HIV-1 infection. This phenomenon was detectable as early as 1 h after the start of cocultivation and depended on cell-to-cell contact but not on the release of viral particles from donor cells. Transfer of HIV-1 products occurred upon their polarization and colocalization within zones of cell-to-cell contact similar to virological synapses. Neither HIV-1 Env nor Nef expression was required but, interestingly, we found that an HIV-1-dependent increase in matrix metalloproteinase 9 production from donor cells significantly contributed to the cell-to-cell transmission of the viral products. The macrophage-driven transfer of HIV-1 products to diverse CD4-negative cell types may have a significant role in AIDS pathogenesis.It is widely documented that viruses can spread through mechanisms alternative to receptor-mediated cell internalization. These include transcytosis (31), transinfection (57), and cell-to-cell infection. This latter way of infection has been documented for human herpesviruses (13), human cytomegalovirus (17), measles virus (22), and human hepatitis C virus (58). The human T-lymphotropic virus type 1 retrovirus was found to propagate exclusively through the formation of zones of tight cell-to-cell adhesion (virological synapses) very similar to the contact between antigen-presenting cells and lymphocytes (immunological synapses) (32). Human immunodeficiency virus type 1 (HIV-1) was found to propagate very efficiently by cell-to-cell transmission (33, 48, 53) through a mechanism requiring expression of HIV Env receptors in donor cells and of CD4 and CXCR4 or CCR5 cell receptors in target cells, although coreceptor-independent HIV-1 transfer to peripheral blood mononuclear cells was also described (5).HIV infection can lead either to cell death, mostly in activated CD4 lymphocytes, or to persistent infection in cells controlling HIV gene expression and/or resisting its cytopathic effects, as is the case with macrophages (55). These cells play a critical role in AIDS pathogenesis, both as viral reservoirs during highly active anti-retroviral therapy (36) and by affecting the pattern of released soluble factors involved in both innate and adaptive immunity. In addition, the nature of macrophages as migratory blood cells strongly favors their interaction with cells of different types, e.g., epithelial, stromal, or endothelial cells. This is also the case with the central nervous system counterpart of macrophagic cells, i.e., microglia cells (41).Macrophages are good producers of matrix metalloproteinases (MMPs), i.e., zinc-dependent extracellular proteases that function at a neutral pH to cleave a wide variety of substrates (61). These include basement membrane and extracellular ...

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“…F12Nef is defective for a number of functions described for wild-type Nef. In particular, it does not downregulate either CD4 (D'Aloja et al, 1998) or class I major histocompatibility complex (MHC) membrane expression (M. Doria, personal communication), it does not activate Nefassociated kinase/p21-activated kinase (NAK/PAK) Nunn and Marsh, 1996;Renkema et al, 1999;D'Aloja et al, 2001), and it fails to interact with the V1H subunit of vacuolar ATPase (V-ATPase) (Lu et al, 1998;D'Aloja et al, 2001). On the other hand, F12Nef maintains its ability to dispose at the cell margin, as well as to interact with the CD4 intracytoplasmic tail, both being functions required for its antiviral phenotype .…”

Section: Introductionmentioning

confidence: 96%

Inducible Expression of the ΔNGFr/F12Nef Fusion Protein as a New Tool for Anti-Human Immunodeficiency Virus Type 1 Gene Therapy

et al. 2002

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Expression of the human immunodeficiency virus type 1 (HIV-1) Nef triple mutant F12Nef strongly inhibits HIV-1 replication. We exploited such a unique feature in a novel anti-HIV-1 gene therapy design by constructing an HIV-1 Tat-defective lentivirus vector expressing the product of fusion between the low-affinity human nerve growth factor receptor truncated in its intracytoplasmic domain (deltaNGFr, NH(2) moiety), and F12Nef (COOH moiety), under the control of the HIV-1 long terminal repeats. In this manner, both the selection marker (deltaNGFr) and the anti-HIV-1 effector are comprised in the same fusion protein, the expression of which is targetable by HIV-1 infection. Such a vector was proved to transduce human cells efficiently and, on HIV-1 infection, it expressed high levels of the fusion protein. In addition, strong antiviral activity of the deltaNGFr/F12Nef-expressing vector was demonstrated in cell lines as well as in primary cell cultures challenged with T- or M-tropic HIV-1 isolates. Thus, the HIV-1-targetable expression of the deltaNGFr/F12Nef fusion protein represents a novel and powerful tool for an effective anti-HIV-1 gene therapy strategy.

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Genetic and functional analysis of the human immunodeficiency virus (HIV) type 1-inhibiting F12-HIVnef allele

Cited by 31 publications

References 46 publications

Inhibition of HIV-1 Infection and Replication by Enhancing Viral Incorporation of Innate Anti-HIV-1 Protein A3G

Inhibition of HIV-1 Infection and Replication by Enhancing Viral Incorporation of Innate Anti-HIV-1 Protein A3G

Macrophages Transmit Human Immunodeficiency Virus Type 1 Products to CD4-Negative Cells: Involvement of Matrix Metalloproteinase 9

Inducible Expression of the ΔNGFr/F12Nef Fusion Protein as a New Tool for Anti-Human Immunodeficiency Virus Type 1 Gene Therapy

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