A peptide derived from the N‐terminal region of HIV‐1 Vpr promotes nuclear import in permeabilized cells: elucidation of the NLS region of the Vpr

To identify the domains of Vpr that are involved nuclear localization, we transfected HeLa cells with a panel of expression vectors that encode mutant Vpr protein with deletions or substitutions within putative domains. Immunofluorescence staining of transfected cells revealed that wild-type Vpr was localized predominantly in the nucleus and the nuclear envelope and certainly in the cytoplasm. Introduction of substitutions or deletions within ␣H1 or ␣H2 resulted, by contrast, in diffuse expression over the entire cell. In addition, double mutations within both of these ␣-helical domains led to the complete absence of Vpr from nuclei. Next, we prepared HeLa cells that express chimeric proteins which consist of the ␣H1 and ␣H2 domains fused individually with green fluorescent protein (GFP) and a Flag tag and extracted them with digitonin and Triton X-100 prior to fixation. Flag-␣H1-GFP was detected in the nucleus but not in the cytoplasm, while Flag-␣H2-GFP was retained predominantly in the nucleus and in a small amount in the cytoplasm. The immunostaining patterns were almost eliminated by substitutions in each chimeric protein. Thus, it appeared that the two ␣-helical domains might be involved in nuclear import by binding to certain cellular factors. Taken together, our data suggest that the two putative ␣-helical domains mediate the nuclear localization of Vpr by at least two mechanisms.

mentioning

confidence: 67%

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confidence: 99%

Two Putative α-Helical Domains of Human Immunodeficiency Virus Type 1 Vpr Mediate Nuclear Localization by at Least Two Mechanisms

Kamata¹,

Aida²

2000

“…It has been suggested that Vpr acts like an importin-␤ homologue through its direct binding to nucleoporins within the NPC, although this appears to be context dependent (18,58,74). While MA and integrase utilize the importin-␣/importin-␤-dependent pathway of nuclear import, Vpr possesses noncanonical NLSs and is not imported exclusively through these classical mechanisms (21,22,35,38,64). Thus HIV-1 may have adapted a novel strategy to bypass cellular defense mechanisms targeted at excluding viruses from the nucleus.…”

Section: Discussionmentioning

confidence: 99%

Nuclear Export of Vpr Is Required for Efficient Replication of Human Immunodeficiency Virus Type 1 in Tissue Macrophages

Sherman

Noronha

Eckstein

et al. 2003

Retroviruses must gain access to the host cell nucleus for subsequent replication and viral propagation. Human immunodeficiency virus type 1 (HIV-1) and other primate lentiviruses are distinguished from the gammaretroviruses by their ability to infect nondividing cells such as macrophages, an important viral reservoir in vivo. Rather than requiring nuclear membrane breakdown during cell division, the HIV-1 preintegration complex (PIC) enters the nucleus by traversing the central aqueous channel of the limiting nuclear pore complex. The HIV-1 PIC contains three nucleophilic proteins, matrix, integrase, and Vpr, all of which have been implicated in nuclear targeting. The mechanism by which Vpr can display such nucleophilic properties and yet also be available for incorporation into virions assembling at the plasma membrane is unresolved. We recently characterized Vpr as a nucleocytoplasmic shuttling protein that contains two novel nuclear import signals and an exportin-1-dependent nuclear export signal (NES). We now demonstrate that mutation of this NES impairs the incorporation of Vpr into newly formed virions. Furthermore, we find that the Vpr NES is required for efficient HIV replication in tissue macrophages present in human spleens and tonsils. These findings underscore how the nucleocytoplasmic shuttling of Vpr not only contributes to nuclear import of the HIV-1 PIC but also enables Vpr to be present in the cytoplasm for incorporation into virions, leading to enhancement of viral spread within nondividing tissue macrophages.Human immunodeficiency virus type 1 (HIV-1) and other primate lentiviruses are able to infect nondividing cells, notably terminally differentiated macrophages (41), an important viral reservoir within the infected host (31,34,53). This biological feature distinguishes the lentiviruses from the oncoretroviruses (or gammaretroviruses), in which cell division associated with nuclear membrane dissolution is required for infection (33,42,62). HIV-1 is also able to infect resting, nondividing T cells in lymphoid tissues (13). These nondividing T cells may contribute to the establishment of protected reservoirs in the host, undermining attempts to eradicate virusproducing cells in the long term (5,6,15,56,77).After entry by fusion and uncoating, the viral reverse transcriptase complex traverses the cytoplasm while reverse transcribing the two strands of RNA into DNA (20), forming the viral preintegration complex (PIC). The nuclear envelope forms a barrier that the PIC must negotiate. The nuclear envelope is studded with nuclear pore complexes (NPCs) that form a conduit with a central aqueous channel mediating bidirectional transport of many macromolecules. The NPC corresponds to a 125-MDa structure comprising 50 to 100 polypeptides. Many of these proteins are members of the nucleoporin family characterized by FG repeats (46). During active transport, the central aqueous channel accommodates protein complexes as large as 25 nm in diameter. However, the HIV-1 PIC exhibits a Stokes diameter of 56 n...

“…HIV-1 encodes three karyophilic proteins, Vpr, matrix, and integrase, that function in a redundant or possibly cooperative manner to promote translocation of the relatively immense viral PIC across the limiting nuclear pore. While matrix and integrase utilize the classical importin ␣/importin ␤-dependent pathway of nuclear import, Vpr lacks identifiable canonical NLSs and, moreover, Vpr import is not blocked by inhibitors of the importin ␣/importin ␤ or M9 pathways (14,15,26,30). Instead, Vpr contains at least two novel import signals, one in the helical, aminoterminal portion of Vpr between amino acids 1 and 71 (26,30) FIG.…”

Section: Discussionmentioning

confidence: 99%

Nucleocytoplasmic Shuttling by Human Immunodeficiency Virus Type 1 Vpr

Sherman

Noronha

Heusch

et al. 2001

109

106

Human immunodeficiency virus type 1 (HIV-1) is capable of infecting nondividing cells such as macrophages because the viral preintegration complex is able to actively traverse the limiting nuclear pore due to the redundant and possibly overlapping nuclear import signals present in Vpr, matrix, and integrase. We have previously recognized the presence of at least two distinct and novel nuclear import signals residing within Vpr that, unlike matrix and integrase, bypass the classical importin ␣/␤-dependent signals and do not require energy or a RanGTP gradient. We now report that the carboxy-terminal region of Vpr (amino acids 73 to 96) contains a bipartite nuclear localization signal (NLS) composed of multiple arginine residues. Surprisingly, when the leucine-rich Vpr(1-71) fragment, previously shown to harbor an NLS, or full-length Vpr is fused to the C terminus of a green fluorescent protein-pyruvate kinase (GFP-PK) chimera, the resultant protein is almost exclusively detected in the cytoplasm. However, the addition of leptomycin B (LMB), a potent inhibitor of CRM1-dependent nuclear export, produces a shift from a cytoplasmic localization to a nuclear pattern, suggesting that these Vpr fusion proteins shuttle into and out of the nucleus. Studies of nuclear import with GFP-PK-Vpr fusion proteins in the presence of LMB reveals that both of the leucine-rich ␣-helices are required for effective nuclear uptake and thus define a unique NLS. Using a modified heterokaryon analysis, we have localized the Vpr nuclear export signal to the second leucine-rich helix, overlapping a portion of the amino-terminal nuclear import signal. These studies thus define HIV-1 Vpr as a nucleocytoplasmic shuttling protein.