Human immunodeficiency virus type 1 viral protein R localization in infected cells and virions

The subcellular localization of human immunodeficiency virus type 1 (HIV-1) viral protein R (Vpr) was examined by subcellular fractionation. In HIV-1-infected peripheral blood mononuclear cells, Vpr was found in the nuclear and membrane fractions as well as the conditioned medium. Expression of Vpr without other HIV-1 proteins, in two different eukaryotic expression systems, demonstrated a predominant localization of Vpr in the nuclear matrix and chromatin extract fractions. Deletion of the carboxyl-terminal 19-amino-acid arginine-rich sequence impaired Vpr nuclear localization. Indirect immunofluorescence confirmed the nuclear localization of Vpr and also indicated a perinuclear location. Expression of Vpr alone did not result in export of the protein from the cell, but when coexpressed with the Gag protein, Vpr was exported and found in virus-like particles. A truncated Gag protein, missing the p6 sequence and a portion of the p9 sequence, was incapable of exporting Vpr from the cell. Regulation of Vpr localization may be important in the influence of this protein on virus replication.     

Mapping of functional domains for HIV-2 gag assembly into virus-like particles

The human immunodeficiency virus type 2 gag precursor protein, pr41, self assembles as virus-like particles (VLP) when the gag gene is expressed in insect cells. To map the functional domains for HIV-2 gag VLP formation, a series of deletion mutants was constructed by removing sequentially the C-terminal region of HIV-2 gag precursor protein and expressing the truncated gag genes in SF9 insect cells by means of recombinant baculoviruses. We found that deletion of up to 143 amino acids at the C-terminus of HIV-2 gag, leaving 376 amino acids at the N-terminus of the protein, did not prevent VLP formation. However, an additional four amino acids deletion from the C-terminus, which represents 372 amino acids at the N-terminus, made gag protein fail to form VLP. There is a proline-rich region at amino acid positions 372 and 377 of HIV-2 gag. To analyze the role of these proline residues, we generated five mutants in which proline was changed sequentially into leucine. Our results showed that replacement of one or two prolines did not stop gag VLP formation, whereas replacement of all three prolines by leucine residues completely abolished VLP assembly. Our data demonstrate that the C-terminal p12 region of HIV-2 gag precursor protein and the zinc finger domain are dispensable for gag VLP assembly, but the presence of at least one of the three proline residues located between amino acid positions 372 and 377 of HIV-2NIH-Z is required.     

Mutations in murine Mx1: effects on localization and antiviral activity

Murine Mx1 is a nuclear localized protein of 631 amino acids with antiviral activity against influenza virus. Fourteen mutations in murine Mx1 were constructed in vitro, expressed in chicken embryo fibroblasts via replication-competent retroviruses, and the effects of the mutations on Mx localization and antiviral activity were assayed. The results suggest that a nuclear location is not sufficient for antiviral activity, that there are intricate structural constrains on the Mx protein for antiviral activity and that multiple domains of the Mx protein are required for the characteristic punctate nuclear Mx distribution. These conclusions are based on the findings showing that: (i) none of the mutants retained antiviral activity; (ii) only a mutant with a Leu to Lys substitution at residue 612 within the nuclear targeting signal retained the characteristic punctate nuclear localization of wildtype Mx1; (iii) diffuse nuclear localization was observed for mutants with substitutions of Pro for Leu at residue 619, 626, or both 619 and 626, and deletions of residues 23 to 95, 159 to 185, 369 to 409, 387 to 440, 522 to 560, or 541 to 596; and (iv) cytoplasmic localization was observed for mutants with carboxy-terminal truncations of 15, 30, or 61 amino acids, or a deletion of residues 610 to 624.     

Nuclear import, virion incorporation, and cell cycle arrest/differentiation are mediated by distinct functional domains of human immunodeficiency virus type 1 Vpr

The vpr gene product of human immunodeficiency virus type 1 (HIV-1) is a virion-associated protein that is essential for efficient viral replication in monocytes/macrophages. Vpr is primarily localized in the nucleus when expressed in the absence of other viral proteins. Vpr is packaged efficiently into viral particles through interactions with the p6 domain of the Gag precursor polyprotein p55gag. We developed a panel of expression vectors encoding Vpr molecules mutated in the amino-terminal helical domain, leucine-isoleucine (LR) domain, and carboxy-terminal domain to map the different functional domains and to define the interrelationships between virion incorporation, nuclear localization, cell cycle arrest, and differentiation functions of Vpr. We observed that substitution mutations in the N-terminal domain of Vpr impaired both nuclear localization and virion packaging, suggesting that the helical structure may play a vital role in modulating both of these biological properties. The LR domain was found to be involved in the nuclear localization of Vpr. In contrast, cell cycle arrest appears to be largely controlled by the C-terminal domain of Vpr. The LR and C-terminal domains do not appear to be essential for virion incorporation of Vpr. Interestingly, we found that two Vpr mutants harboring single amino acid substitutions (A30L and G75A) retained the ability to translocate to the nucleus but were impaired in the cell cycle arrest function. In contrast, mutation of Leu68 to Ser resulted in a protein that localizes in the cytoplasm while retaining the ability to arrest host cell proliferation. We speculate that the nuclear localization and cell cycle arrest functions of Vpr are not interrelated and that these functions are mediated by separable putative functional domains of Vpr.     

NMR structure of the (52-96) C-terminal domain of the HIV-1 regulatory protein Vpr: molecular insights into its biological functions

The HIV-1 regulatory protein Vpr (96 amino acid residues) is incorporated into the virus particle through a mechanism involving its interaction with the C-terminal portion of Gag. Vpr potentiates virus replication by interrupting cell division in the G2 phase and participates in the nuclear transport of proviral DNA. The domain encompassing the 40 C-terminal residues of Vpr was shown to be involved in cell cycle arrest and binding of nucleocapsid protein NCp7, and suggested to promote nuclear provirus transfer. Accordingly, we show here that the synthetic 52-96 but not 1-51 sequences of Vpr interact with HIV-1 RNA. Based on these results, the structure of (52-96)Vpr was analysed by two-dimensional 1H-NMR in aqueous TFE (30%) solution and refined by restrained molecular dynamics. The structure is characterized by a long (53-78) amphipathic alpha-helix, followed by a less defined (79-96) C-terminal domain. The Leu60 and Leu67 side-chains are located on the hydrophobic side of the helix, suggesting their involvement in Vpr dimerization through a leucine zipper-type mechanism. Accordingly, their replacement by Ala eliminates Vpr dimerization in the two hybrid systems, while mutations of Ile74 and Ile81 have no effect. This was confirmed by gel filtration measurements and circular dichroism, which also showed that the alpha-helix still exists in (52-96)Vpr and its Ala60, Ala67 mutant in the presence and absence of TFE. Based on these results, a model of the coiled-coil Vpr dimer has been described, and its biological relevance as well as that of the structural characteristics of the 52-96 domain for the different functions of Vpr, including HIV-1 RNA binding, are discussed.     

Viral protein R regulates docking of the HIV-1 preintegration complex to the nuclear pore complex

Replication of human immunodeficiency virus type 1 (HIV-1) in non-dividing cells depends critically on import of the viral preintegration complex into the nucleus. Recent evidence suggests that viral protein R (Vpr) plays a key regulatory role in this process by binding to karyopherin alpha, a cellular receptor for nuclear localization signals, and increasing its affinity for the nuclear localization signals. An in vitro binding assay was used to investigate the role of Vpr in docking of the HIV-1 preintegration complex (PIC) to the nuclear pore complex. Mutant HIV-1 PICs that lack Vpr were impaired in the ability to dock to isolated nuclei and recombinant nucleoporins. Although Vpr by itself associated with nucleoporins, the docking of Vpr+ PICs was dependent on karyopherin beta and was blocked by antibodies to beta. Vpr stabilized docking by preventing nucleoporin-stimulated dissociation of the import complex. These results suggest a biochemical mechanism for Vpr function in transport of the HIV-1 genome across the nuclear pore complex.     

Nuclear import and export of influenza virus nucleoprotein

Influenza virus nucleoprotein (NP) shuttles between the nucleus and the cytoplasm. A nuclear localization signal (NLS) has been identified in NP at amino acids 327 to 345 (J. Davey et al., Cell 40:667-675, 1985). However, some NP mutants that lack this region still localize to the nucleus, suggesting an additional NLS in NP. We therefore investigated the nucleocytoplasmic transport of NP from influenza virus A/WSN/33 (H1N1). NP deletion constructs lacking the 38 N-terminal amino acids, as well as those lacking the 38 N-terminal amino acids and the previously identified NLS, localized to both the cytoplasm and the nucleus. Nuclear localization of a protein containing amino acids 1 to 38 of NP fused to LacZ proved that these 38 amino acids function as an NLS. Within this region, we identified two basic amino acids, Lys7 and Arg8, that are crucial for NP nuclear import. After being imported into the nucleus, the wild-type NP and the NP-LacZ fusion construct containing amino acids 1 to 38 of NP were both transported back to the cytoplasm, where they accumulated. These data indicate that NP has intrinsic structural features that allow nuclear import, nuclear export, and cytoplasmic accumulation in the absence of any other viral proteins. Further, the information required for nuclear import and export is located in the 38 N-terminal amino acids of NP, although other NP nuclear export signals may exist. Treatment of cells with a protein kinase C inhibitor increased the amounts of nuclear NP, whereas treatment of cells with a phosphorylation stimulator increased the amounts of cytoplasmic NP. These findings suggest a role of phosphorylation in nucleocytoplasmic transport of NP.     

The long amino-terminal tail domain of annexin XI is necessary for its nuclear localization

Annexin XI is a newly identified annexin which localizes mainly in the nucleus of rat embryonic fibroblasts. There are no typical nuclear localization signals (NLS) in the molecule. To define the region responsible for its nuclear localization, a series of mutants and chimeric cDNA were constructed. These were transiently expressed in COS-7 cells, and the subcellular distributions of the mutants and chimeric proteins were determined by indirect immunofluorescence microscopy. Wild-type annexin XI was located predominantly within the nucleus. Deletion of the N-terminal tail domain (residues 3-196) changed the distribution of the protein from the nucleus to the cytoplasm whereas deletion of the C-terminal core domain (residues 208-504) still kept the protein sorting to the nucleus. Three other mutants lacking 60-80 amino acids in the N-terminal region (residues 3-61, 61-115, and 115-197, respectively) no longer efficiently imported into the nucleus. Furthermore, Escherichia coli beta-galactosidase polypeptide was efficiently localized to the nucleus only when fused with the whole N-terminal region of annexin XI (residues 1-207), not with part of the N-terminal region. In primary cultured rat hepatocytes, annexin XI was distributed in the cytoplasm but not in the nucleus. These results suggest that the whole N-terminal tail domain of annexin XI is necessary and sufficient for its nuclear localization, and may function as NLS in a cell-type specific manner.     

The putative alpha helix 2 of human immunodeficiency virus type 1 Vpr contains a determinant which is responsible for the nuclear translocation of proviral DNA in growth-arrested cells

Several viral determinants were shown to play a role in the ability of human immunodeficiency virus type 1 (HIV-1) to infect nondividing cells. In particular, Vpr and Gag matrix (MA) were recognized to be involved in the nuclear transport of the viral preintegration complex. The goal of the present study was to evaluate the ability of isogenic HIV-1 viruses harboring different vpr and gag genes to infect nondividing cells. Surprisingly, our results reveal that the introduction of mutations in the MA nuclear localization signal marginally affected the ability of proviruses to establish infection in growth-arrested HeLa or MT4 cells. In contrast, we show that in our experimental system, the absence of Vpr expression leads to a reduction in viral infectivity and production which correlates with a decrease in the synthesis and nuclear transport of proviral DNA as determined by PCR analysis. Moreover, our data demonstrate that this reduction of viral replication is also observed with proviruses containing different mutated Vpr alleles. In particular, the Vpr Q65E mutant, which contains a substitution in the second predicted amphipathic alpha-helical structure located in the central region of the protein, is associated with an impairment of the protein nuclear localization and a concomitant reduction of the nuclear transport of proviral DNA. The results of this study provide evidence that a putative amphipathic alpha-helical structure in the central region of Vpr contains a determinant involved in the nuclear translocation of the preintegration complex in nondividing cells.     

Changes in high K+-evoked serotonin release and serotonin 2A/2C receptor binding in the frontal cortex of rats with thioacetamide-induced hepatic encephalopathy

Protein import into the nucleus is generally considered to involve specific nuclear localization signals (NLS) though it is becoming increasingly clear that efficient and well controlled import of proteins which lack a canonical NLS also occurs in cells. Human immunodeficiency virus type 1 (HIV-1) Vpr is one such protein which does not have an identifiable canonical NLS and yet efficiently localizes to the nuclear compartment. Here, we use confocal microscopy to demonstrate that mutations in the putative central hydrophobic helix of Vpr result in the retention of the protein in highly localized ring-like structures around the nuclear periphery with striking impairment in their ability to enter the nuclear interior. By characterizing other biological activities associated with this protein, such as its ability to incorporate into budding virions and its ability to arrest cells in G2, we show that this helical domain is specific for the nuclear translocation of the protein with very little effect on these other functions. Interestingly, however, perturbation of this helical motif also perturbs the protein's ability to augment viral replication in primary human macrophages indicating that the integrity of this secondary structure is essential for optimal infection in these non-dividing cells.     

Role of the charged tail in localization of a surface protein antigen of Streptococcus mutans

To make clear the role of the C terminus of a surface protein antigen (PAc) of Streptococcus mutans, stepwise truncations beginning at the C terminus of PAc were performed by utilizing site-directed mutagenesis. A remarkable increase in the amount of cell-free PAc was observed upon deletion of four or more amino acid residues at the C terminus. On the other hand, the amount of cell surface PAc gradually decreased when increasing numbers (four or more) of amino acid residues were deleted at the C terminus, and deletion of six amino acids involving both the total charged tail and Leu, an amino acid residue immediately upstream of the charged tail, resulted in a drastic reduction in the amount of cell surface PAc. These results indicate that the cytoplasmic charged tail and Leu residue are required for cell surface localization of PAc in S. mutans.     

Deletion analysis of MotA and MotB, components of the force-generating unit in the flagellar motor of Salmonella

MotA and MotB are cytoplasmic membrane proteins that form the force-generating unit of the flagellar motor in Salmonella typhimurium and many other bacteria. Many missense mutations in both proteins are known to cause slow motor rotation (slow-motile phenotype) or no rotation at all (non-motile or paralysed phenotype). However, large stretches of sequence in the cytoplasmic regions of MotA and in the periplasmic region of MotB have failed to yield these types of mutations. In this study, we have investigated the effect of a series of 10-amino-acid deletions in these phenotypically silent regions. In the case of MotA, we found that only the C-terminal 5 amino acids were completely dispensable; an adjacent 10 amino acids were partially dispensable. In the cytoplasmic loop region of MotA, deletions made the protein unstable. For MotB, we found that two large segments of the periplasmic region were dispensable: the results with individual deletions showed that the first consisted of six deletions between the sole transmembrane span and the peptidoglycan binding motif, whereas the second consisted of four deletions at the C-terminus. We also found that deletions in the MotB cytoplasmic region at the N-terminus impaired motility but did not abolish it. Further investigations in MotB were carried out by combining dispensable deletion segments. The most extreme version of MotB that still retained some degree of function lacked a total of 99 amino acids in the periplasmic region, beginning immediately after the transmembrane span. These results indicate that the deleted regions in the MotA cytoplasmic loop region are essential for stability; they may or may not be directly involved in torque generation. Part of the MotA C-terminal cytoplasmic region is not essential for torque generation. MotB can be divided into three regions: an N-terminal region of about 30 amino acids in the cytoplasm, a transmembrane span and about 260 amino acids in the periplasm, including a peptidoglycan binding motif. In the periplasmic region, we suggest that the first of the two dispensable stretches in MotB may comprise part of a linker between the transmembrane span of MotB and its attachment point to the peptidoglycan layer, and that the length or specific sequence of much of that linker sequence is not critical. About 40 residues at the C-terminus are also unimportant.     

Nuclear localization of cyclin B1 controls mitotic entry after DNA damage

While the Vpr protein of HIV-1 has been implicated in import of the viral preintegration complex across the nuclear pore complex (NPC) of nondividing cellular hosts, the mechanism by which Vpr enters the nucleus remains unknown. We now demonstrate that Vpr contains two discrete nuclear targeting signals that use two different import pathways, both of which are distinct from the classical nuclear localization signal (NLS)- and the M9-dependent pathways. Vpr import does not appear to require Ran-mediated GTP hydrolysis and persists under conditions of low energy. Competition experiments further suggest that Vpr directly engages the NPC at two discrete sites. These sites appear to form distal components of a common import pathway used by NLS- and M9-containing proteins. Together, our data suggest that Vpr bypasses many of the soluble receptors involved in import of cellular cargoes. Rather, this viral protein appears to directly access the NPC, a property that may help to ensure the capacity of HIV to replicate in nondividing cellular hosts.     

Virion-targeted viral inactivation of human immunodeficiency virus type 1 by using Vpr fusion proteins

Inactivation of progeny virions with chimeric virion-associated proteins represents a novel therapeutic approach against human immunodeficiency virus (HIV) replication. The HIV type 1 (HIV-1) Vpr gene product, which is packaged into virions, is an attractive candidate for such a strategy. In this study, we developed Vpr-based fusion proteins that could be specifically targeted into mature HIV-1 virions to affect their structural organization and/or functional integrity. Two Vpr fusion proteins were constructed by fusing to the first 88 amino acids of HIV-1 Vpr the chloramphenicol acetyltransferase enzyme (VprCAT) or the last 18 C-terminal amino acids of the HIV-1 Vpu protein (VprIE). These Vpr fusion proteins were initially designed to quantify their efficiency of incorporation into HIV-1 virions when produced in cis from the provirus. Subsequently, CD4+ Jurkat T-cell lines constitutively expressing the VprCAT or the VprIE fusion protein were generated with retroviral vectors. Expression of the VprCAT or the VprIE fusion protein in CD4+ Jurkat T cells did not interfere with cellular viability or growth but conferred substantial resistance to HIV replication. The resistance to HIV replication was more pronounced in Jurkat-VprIE cells than in Jurkat-VprCAT cells. Moreover, the antiviral effect mediated by VprIE was dependent on an intact p6(gag) domain, indicating that the impairment of HIV-1 replication required the specific incorporation of Vpr fusion protein into virions. Gene expression, assembly, or release was not affected upon expression of these Vpr fusion proteins. Indeed, the VprIE and VprCAT fusion proteins were shown to affect the infectivity of progeny virus, since HIV virions containing the VprCAT or the VprIE fusion proteins were, respectively, 2 to 3 times and 10 to 30 times less infectious than the wild-type virus. Overall, this study demonstrated the successful transfer of resistance to HIV replication in tissue cultures by use of Vpr-based antiviral genes.