HIV-1 Vpr-chloramphenicol acetyltransferase fusion proteins: sequence requirement for virion incorporation and analysis of antiviral effect

Yao, Xiaojian; Kobinger, Gary P.; Dandache, Serge; Rougeau, Nicole; Cohen, Éric A.

doi:10.1038/sj.gt.3300988

Cited by 31 publications

(27 citation statements)

References 38 publications

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“…The VprW54R-UNG fusion is efficiently incorporated into virions, and enzymatic assays performed from purified virions show that the UNG fused to VprW54R was still catalytically active. These results confirm that Vpr can efficiently target proteins within HIV-1 particles without affecting the catalytic properties of the cargo (20,(37)(38)(39). Moreover, the observation that the Vpr-UNG fusions can restore the mutant frequency phenotype indicates that Vpr and the virion-associated UNG are directly responsible for the modulation of the virus mutant frequency in vivo.…”

Section: Discussionsupporting

confidence: 68%

Vpr-mediated Incorporation of UNG2 into HIV-1 Particles Is Required to Modulate the Virus Mutation Rate and for Replication in Macrophages

Chen

Rouzic

Kearney

et al. 2004

Journal of Biological Chemistry

111

131

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Human immunodeficiency virus type 1 is able to infect nondividing cells, such as macrophages, and the viral Vpr protein has been shown to participate in this process. Here, we investigated the impact of the recruitment into virus particles of the nuclear form of uracil DNA glycosylase (UNG2), a cellular DNA repair enzyme, on the virus mutation rate and on replication in macrophages. We demonstrate that the interaction of Vpr with UNG2 led to virion incorporation of a catalytically active enzyme that is directly involved with Vpr in modulating the virus mutation rate. The lack of UNG in virions during virus replication in primary monocyte-derived macrophages further exacerbated virus mutant frequencies to an 18-fold increase compared with the 4-fold increase measured in actively dividing cells. Because the presence of UNG is also critical for efficient infection of macrophages, these observations extend the role of Vpr to another early step of the virus life cycle, e.g. viral DNA synthesis, that is essential for replication of human immunodeficiency virus type 1 in nondividing cells.

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Section: Discussionsupporting

confidence: 68%

Vpr-mediated Incorporation of UNG2 into HIV-1 Particles Is Required to Modulate the Virus Mutation Rate and for Replication in Macrophages

Chen

Rouzic

Kearney

et al. 2004

Journal of Biological Chemistry

111

131

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“…In addition, various proteins fused to Vpr were shown to be efficiently incorporated into HIV-1 particles while retaining their original properties (17,32,57,93,94). Since the myristoylation of Nef is important for most of the functions of Nef investigated so far (8,82,91), we decided to fuse Vpr to the C-terminal end of Nef as depicted in Fig.…”

Section: Viral Infectivity Correlates With Cellular and Viral Levels Ofmentioning

confidence: 99%

Human Immunodeficiency Virus Type 1 Nef Incorporation into Virions Does Not Increase Infectivity

Laguette

Bénichou

Basmaciogullari

2009

J Virol

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The viral protein Nef contributes to the optimal infectivity of human and simian immunodeficiency viruses. The requirement for Nef during viral biogenesis particles suggests that Nef might play a role in this process. Alternatively, because Nef is incorporated into viruses, it might play a role when progeny virions reach target cells. We challenged these hypotheses by manipulating the amounts of Nef incorporated in viruses while keeping its expression level constant in producer cells. This was achieved by forcing the incorporation of Nef into viral particles by fusing a Vpr sequence to the C-terminal end of Nef. A cleavage site for the viral protease was introduced between Nef and Vpr to allow the release of Nef fragments from the fusion protein during virus maturation. We show that the resulting Nef-CS-Vpr fusion partially retains the ability of Nef to downregulate cell surface CD4 and that high amounts of Nef-CS-Vpr are incorporated into viral particles compared with what is seen for wild-type Nef. The fusion protein is processed during virion maturation and releases Nef fragments similar to those found in viruses produced in the presence of wild-type Nef. Unlike viruses produced in the presence of wild-type Nef, viruses produced in the presence of Nef-CS-Vpr do not have an increase in infectivity and are as poorly infectious as viruses produced in the absence of Nef. These findings demonstrate that the presence of Nef in viral particles is not sufficient to increase human immunodeficiency virus type 1 infectivity and suggest that Nef plays a role during the biogenesis of viral particles.

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“…To construct different CMV-YFP-IN deletion mutants, cDNA fragments encoding amino acids 1-212 and 1-240 of IN were generated using PCR with 5Ј-BglII and 3Ј primers (5-CAATTCCCGGGTTTGTAT-GTCTGTTTGC-3; 5-CCAGACCCGGGTTGCTGGTCCTT-TCCA-3), and was inserted into the pEYFP-C1 vector at BglII and XmaI sites. Different IN substitution mutants were generated by a two-step PCR-based method (46), using a 5Ј-BglII primer, 3Ј-XhoI primer, and complementary primers containing the desired mutations. The amplified IN cDNAs harboring specific mutations were then cloned into pEYFP-C1 vector.…”

Section: Construction Of Different Viral and Cellular Proteinmentioning

confidence: 99%

Interaction of Human Immunodeficiency Virus Type 1 Integrase with Cellular Nuclear Import Receptor Importin 7 and Its Impact on Viral Replication

Huang

Yao

et al. 2007

Journal of Biological Chemistry

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Similar to all other viruses, human immunodeficiency virus type 1 (HIV-1) depends heavily on cellular factors for its successful replication. In this study we have investigated the interaction of HIV-1 integrase (IN) with several host nuclear import factors using co-immunoprecipitation assays. Our results indicate that IN interacts specifically with host importin 7 (Imp7) in vivo, but does not interact with importin 8 (Imp8) or importin ␣ (Rch1). To carry out a successful infection, human immunodeficiency virus type 1 (HIV-1) 4 takes advantage of various host cellular proteins and cellular pathways. The interaction between cellular proteins and viral components takes place during various steps of the HIV-1 life cycle, including viral DNA nuclear import. The most striking feature of HIV-1 is its ability to replicate in non-dividing cells. This feature depends on the ability of the virus to transport its cDNA, as part of a large preintegration complex (PIC), from the cytoplasm to the nucleus by an active and energy-dependent process (1-3). However, the mechanism by which the PIC translocates across the nuclear membrane into the nucleus of non-dividing cells is still not fully understood. It has been shown that three HIV-1 PIC-associated proteins including MAp17, IN, and Vpr possess karyophilic properties, and contribute to nuclear translocation of viral PICs. This action is accomplished through their interactions with karyophilic cellular proteins, thereby directing the PIC through the nuclear pore complex (4 -10). In addition, a cis-acting element named the central DNA flap, which is located in the 3Ј region of the pol gene sequence, was also shown to contribute to HIV-1 nuclear import in both dividing and non-dividing cells (11)(12)(13)(14). Nuclear import of proteins in mammalian cells can be mediated by several distinct pathways. The importin ␣/␤ heterodimer meditates nuclear import of proteins harboring a classical nuclear localization signal, which either contains a cluster of basic amino acids or two basic clusters separated by 10 -20 amino acids (bipartite nuclear localization signal) (for reviews see Refs. 15 and 16). Also, importin ␤ (Imp␤) was shown to bind to and import HIV-1 proteins, such as HIV-1 Tat, Rev, and HTLV Rex, independently of importin ␣ (Imp␣) (17-21). Similarly, transportin, an Imp␤-related receptor, imports its substrates (heterogeneous nuclear ribonucleoproteins) by directly binding to the glycine-rich M9 domain of the protein (22,23). Based on the similarity to Imp␤, several other nuclear import factors, including Imp7 and Imp8, have also been identified (24). Imp7 is one of several cellular importins that bind to and mediate nuclear import of ribosomal proteins in mammalian cells, and it was also found to translocate other proteins, such as glucocorticoid receptor and histone H1 into the nucleus (18,(25)(26)(27). In the case of histone H1, Jakel et al. (27) have demonstrated that two receptors, Imp␤ and Imp7,

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HIV-1 Vpr-chloramphenicol acetyltransferase fusion proteins: sequence requirement for virion incorporation and analysis of antiviral effect

Cited by 31 publications

References 38 publications

Vpr-mediated Incorporation of UNG2 into HIV-1 Particles Is Required to Modulate the Virus Mutation Rate and for Replication in Macrophages

Vpr-mediated Incorporation of UNG2 into HIV-1 Particles Is Required to Modulate the Virus Mutation Rate and for Replication in Macrophages

Human Immunodeficiency Virus Type 1 Nef Incorporation into Virions Does Not Increase Infectivity

Interaction of Human Immunodeficiency Virus Type 1 Integrase with Cellular Nuclear Import Receptor Importin 7 and Its Impact on Viral Replication

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