Three Arginine Residues within the RGG Box Are Crucial for ICP27 Binding to Herpes Simplex Virus 1 GC-Rich Sequences and for Efficient Viral RNA Export

ICP27 is a multifunctional protein that is required for herpes simplex virus 1 mRNA export. ICP27 interacts with the mRNA export receptor TAP/NXF1 and binds RNA through an RGG box motif. Unlike other RGG box proteins, ICP27 does not bind G-quartet structures but instead binds GC-rich sequences that are flexible in structure. To determine the contribution of arginines within the RGG box, we performed in vitro binding assays with N-terminal proteins encoding amino acids 1 to 160 of wild-type ICP27 or arginine-to-lysine substitution mutants. The R138,148,150K triple mutant bound weakly to sequences that were bound by the wild-type protein and single and double mutants. Furthermore, during infection with the R138,148,150K mutant, poly(A)⁺ RNA and newly transcribed RNA accumulated in the nucleus, indicating that viral RNA export was impaired. To determine if structural changes had occurred, nuclear magnetic resonance (NMR) analysis was performed on N-terminal proteins consisting of amino acids 1 to 160 from wild-type ICP27 and the R138,148,150K mutant. This region of ICP27 was found to be highly flexible, and there were no apparent differences in the spectra seen with wild-type ICP27 and the R138,148,150K mutant. Furthermore, NMR analysis with the wild-type protein bound to GC-rich sequences did not show any discernible folding. We conclude that arginines at positions 138, 148, and 150 within the RGG box of ICP27 are required for binding to GC-rich sequences and that the N-terminal portion of ICP27 is highly flexible in structure, which may account for its preference for binding flexible sequences.The herpes simplex virus 1 (HSV-1) protein ICP27 is a multifunctional regulatory protein that is required for productive viral infection. ICP27 interacts with a number of cellular proteins, and it binds RNA (35). One of the functions that ICP27 performs is to escort viral mRNAs from the nucleus to the cytoplasm for translation (2, 3, 5, 10, 13, 21, 34). ICP27 binds viral RNAs (5, 34) and interacts directly with the cellular mRNA export receptor TAP/NXF1 (2, 21), which is required for the export of HSV-1 mRNAs (20, 21). ICP27 also interacts with the export adaptor proteins Aly/REF (2, 3, 23) and UAP56 (L. A. Johnson, H. Swesey, and R. M. Sandri-Goldin, unpublished results), which form part of the TREX complex that binds to the 5′ end of mRNA through an interaction with CBP80 (26, 32, 41). Aly/REF does not appear to bind viral RNA directly (3), and it is not essential for HSV-1 RNA export based upon small interfering RNA (siRNA) knockdown studies (20), but it contributes to the efficiency of viral RNA export (3, 23). ICP27 also interacts with the SR splicing proteins SRp20 and 9G8 (11, 36), which have been shown to shuttle between the nucleus and the cytoplasm (1). SRp20 and 9G8 have also been shown to facilitate the export of some cellular RNAs (16, 17, 27) by binding RNA and interacting with TAP/NXF1 (14, 16, 18). The knockdown of SRp20 or 9G8 adversely affects HSV-1 replication and specifically results in a nuclear accumulation of newly transcribed RNA during infection (11). Thus, these SR proteins also contribute to the efficiency of viral RNA export. However, the overexpression of SRp20 was unable to rescue the defect in RNA export during infection with an ICP27 mutant that cannot bind RNA (11), suggesting that ICP27 is the major HSV-1 RNA export protein that links viral RNA to TAP/NXF1.ICP27 was shown previously to bind RNA through an RGG box motif located at amino acids 138 to 152 within the 512-amino-acid protein (28, 34). Using electrophoretic mobility shift assays (EMSAs), we showed that the N-terminal portion of ICP27 from amino acids 1 to 160 bound specifically to viral oligonucleotides that are GC rich and that are flexible and relatively unstructured (5). Here we report the importance of three arginine residues within the RGG box for ICP27 binding to GC-rich sequences in vitro and for viral RNA export during infection. We also performed nuclear magnetic resonance (NMR) structural analysis of the N-terminal portion of ICP27 for both the wild-type protein and an ICP27 mutant in which three arginines were replaced with lysines. The NMR data showed that the N-terminal portion of ICP27 is relatively unstructured but compact, and NMR analysis in the presence of oligonucleotide substrates to which the N-terminal portion of ICP27 binds did not show any discernible alterations in this highly flexible structure, nor did the arginine-to-lysine substitutions.     

Complementation of a Herpes Simplex Virus ICP0 Null Mutant by Varicella-Zoster Virus ORF61p

The herpes simplex virus (HSV) ICP0 protein acts to overcome intrinsic cellular defenses that repress viral α gene expression. In that vein, viruses that have mutations in ICP0''s RING finger or are deleted for the gene are sensitive to interferon, as they fail to direct degradation of promyelocytic leukemia protein (PML), a component of host nuclear domain 10s. While varicella-zoster virus is also insensitive to interferon, ORF61p, its ICP0 ortholog, failed to degrade PML. A recombinant virus with each coding region of the gene for ICP0 replaced with sequences encoding ORF61p was constructed. This virus was compared to an ICP0 deletion mutant and wild-type HSV. The recombinant degraded only Sp100 and not PML and grew to higher titers than its ICP0 null parental virus, but it was sensitive to interferon, like the virus from which it was derived. This analysis permitted us to compare the activities of ICP0 and ORF61p in identical backgrounds and revealed distinct biologic roles for these proteins.Alphaherpesviruses encode orthologs of the herpes simplex virus (HSV) α gene product ICP0. ICP0 is a nuclear phosphoprotein that behaves as a promiscuous activator of viral and cellular genes (7, 11, 28, 29). ICP0 also functions as an E3 ubiquitin ligase to target several host proteins for proteasomal degradation (4, 10, 11, 16, 26). Through this activity, ICP0 promotes degradation of components of nuclear domain 10 (ND10) bodies, including the promyelocytic leukemia protein (PML) and Sp100. These proteins are implicated in silencing of herpesvirus genomes (9, 10, 22, 34). Therefore, ICP0-mediated degradation of ND10 components may disrupt silencing of HSV genes to enable efficient gene expression. This hypothesis provides a plausible mechanistic explanation of how ICP0 induces gene activation.Introduction of DNA encoding the ICP0 orthologs from HSV, bovine herpesvirus, equine herpesvirus, and varicella-zoster virus (VZV) can also affect nuclear structures and proteins (27). In addition, and more specific to this report, ORF61p, the VZV ortholog, activates viral promoters and enhances infectivity of viral DNA like ICP0, the prototype for this gene family (24, 25). However, we have previously demonstrated two key biological differences between the HSV and VZV orthologs. We first showed that unlike ICP0, ORF61p is unable to complement depletion of BAG3, a host cochaperone protein. As a result, VZV is affected by silencing of BAG3 (15), whereas growth of HSV is altered only when ICP0 is not expressed (17). Furthermore, we have shown that while both proteins target components of ND10s, expression of ICP0 results in degradation of both PML and Sp100, whereas ORF61p specifically reduces Sp100 levels (16). These findings suggest that these proteins have evolved separately to provide different functions for virus replication.Virus mutants lacking the ICP0 gene have an increased particle-to-PFU ratio, a substantially lower yield, and decreased levels of α gene expression, in a multiplicity-of-infection (MOI)- and cell-type-dependent manner (2, 4, 8, 33). These mutants are also defective at degrading ND10 components (23). Depletion of PML and Sp100 accelerates virus gene expression and increases plaquing efficiency of HSV ICP0-defective viruses but has no effect on wild-type virus, suggesting that PML and Sp100 are components of an intrinsic anti-HSV defense mechanism that is counteracted by ICP0''s E3 ligase activity (9, 10). Interestingly, ICP0 null viruses are also hypersensitive to interferon (IFN) (26), a property that was suggested to be mediated via PML (3).To directly compare the activities of the two orthologs, we constructed an HSV mutant virus that expresses ORF61p in place of ICP0. The resulting chimeric virus only partially rescues the ICP0 null phenotype. Our studies emphasize the biological differences between ICP0 and ORF61p and shed light on the requirements for PML and Sp100 during infection.     

Analysis of the Functions of Herpes Simplex Virus Type 1 Regulatory Protein ICP0 That Are Critical for Lytic Infection and Derepression of Quiescent Viral Genomes

Herpes simplex virus type 1 (HSV-1) immediate-early regulatory protein ICP0 is important for stimulating the initiation of the lytic cycle and efficient reactivation of latent or quiescent infection. Extensive investigation has suggested several potential functions for ICP0, including interference in the interferon response, disruption of functions connected with PML nuclear bodies (ND10), and inhibition of cellular histone deacetylase (HDAC) activity through an interaction with the HDAC-1 binding partner CoREST. Analysis of the significance of these potential functions and whether they are direct or indirect effects of ICP0 is complicated because HSV-1 mutants expressing mutant forms of ICP0 infect cells with widely differing efficiencies. On the other hand, transfection approaches for ICP0 expression do not allow studies of whole cell populations because of their limited efficiency. To overcome these problems, we have established a cell line in which ICP0 expression can be induced at levels pertaining during the early stages of HSV-1 infection in virtually all cells in the culture. Such cells enable 100% complementation of ICP0-null mutant HSV-1. Using cells expressing the wild type and a variety of mutant forms of ICP0, we have used this system to analyze the role of defined domains of the protein in stimulating lytic infection and derepression from quiescence. Activity in these core functions correlated well the ability of ICP0 to disrupt ND10 and inhibit the recruitment of ND10 proteins to sites closely associated with viral genomes at the onset of infection, whereas the CoREST binding region was neither sufficient nor necessary for ICP0 function in lytic and reactivating infections.Herpes simplex virus type 1 (HSV-1) is an important human pathogen that infects the majority of the population at an early age and then establishes a life-long latent infection in sensory neurones. Periodic reactivation of latent virus causes episodes of active disease characterized by epithelial lesions at the site of the original primary infection. As with all herpesviruses, the ability of HSV-1 to establish and reactivate from latency is key to its clinical importance and evolutionary success. Therefore, the molecular mechanisms that regulate these processes have been the subject of intensive research (reviewed in reference 15). HSV-1 immediate-early (IE) protein ICP0 is required for efficient reactivation from latency in both mouse models and cultured cell systems of quiescent infection (15). ICP0 is also required to stimulate lytic infection by enhancing the probability that a cell receiving a viral genome will engage in productive infection (reviewed in references 19, 20 and 42). Therefore, a full understanding of the biology of HSV-1 infection requires a definition of the functions and mode of action of ICP0.The basic phenotype of ICP0-null mutant HSV-1 is a low probability of plaque formation, particularly in human diploid fibroblasts, that causes a high particle-to-PFU ratio (reference 20 and references therein). Biochemically, ICP0 is an E3 ubiquitin ligase of the RING finger class (4) that induces the degradation of several cellular proteins, including the promyelocytic leukemia (PML) protein (23), centromere proteins including CENP-C (54, 55), and the catalytic subunit of DNA-protein kinase (53, 72). Among the consequences of these activities are the disruption of PML nuclear bodies (herein termed nuclear domain 10 [ND10]) (24, 58) and centromeres (54). ICP0 has also been reported to interact with histone deacetylase enzymes (HDACs) (56) and the CoREST repressor protein, thereby disrupting the CoREST/HDAC-1 complex (37, 39). Evidence has also been presented that expression of ICP0 correlates with increased acetylation of histones on viral chromatin (12). ICP0-null mutant viruses replicate less efficiently than the wild type (wt) in cells pretreated with interferon (IFN) (44, 66), and there is evidence that ICP0 is able to impede an IFN-independent induction of IFN-stimulated genes that arises after infection with defective HSV-1 mutants (16, 59, 60, 65, 67, 76). As a further complication, ICP0-null mutant HSV-1 replicates more efficiently in cells that have been highly stressed by a variety of treatments (5, 6, 79).On the basis of this evidence, several not necessarily mutually exclusive hypotheses have been put forward to explain the biological effects of ICP0. These include (i) that ICP0 counteracts an intrinsic cellular resistance mechanism that involves PML and other components of ND10, (ii) that ICP0 overcomes the innate cellular antiviral defense based on the IFN pathway, and (iii) that ICP0 counteracts the establishment of a repressed chromatin structure on the viral genome by interfering with histone deacetylation. The aim of this paper is to investigate some of these issues using a novel inducible expression system. The question of the effects of ICP0 on IFN pathways is considered in the companion paper (28).The brief and by no means exhaustive summary of the functions and activities attributed to ICP0, presented above, illustrates that the understanding of ICP0 is a difficult issue. It is further complicated by the difficulty of working with ICP0-null mutant viruses under tightly controlled conditions. This arises because the defect varies greatly between different cell types, is highly dependent on the multiplicity of infection (MOI), and varies in a nonlinear manner with respect to virus dose (reference 20 and references therein). Furthermore, use of ICP0 mutant viruses in cultured cell models of reactivation of quiescent HSV-1 is complicated by competition between the resident quiescent viral genome targeted for reactivation and the genomes of the superinfecting virus used to induce the reactivation (75). Therefore, it is very difficult to establish infections with wt and ICP0 mutant viruses that are truly comparable in a way that allows clear distinctions between the direct effects of ICP0 and indirect effects that are due either to expression of other viral proteins that are expressed more efficiently in the presence of ICP0 or to less specific consequences of an active infection and subsequent effects on the cell. Here, we describe a system that enables expression of ICP0 in an inducible manner at levels similar to those at the early stages of infection in almost all cells in a population. We have used this system to study wt and mutant forms of ICP0 in assays of lytic infection and derepression of quiescent viral genomes in a cultured cell model of latency. We discuss the results in terms of the requirements of specific regions of the ICP0 protein for stimulating lytic infection and derepression of quiescent genomes, the potential biological significance of ND10 disruption, recruitment of ND10 components to the sites of HSV-1 genomes at the outset of virus infection, and the interaction of ICP0 with CoREST.     

African Swine Fever Virus Protein p17 Is Essential for the Progression of Viral Membrane Precursors toward Icosahedral Intermediates

The first morphological evidence of African swine fever virus (ASFV) assembly is the appearance of precursor viral membranes, thought to derive from the endoplasmic reticulum, within the assembly sites. We have shown previously that protein p54, a viral structural integral membrane protein, is essential for the generation of the viral precursor membranes. In this report, we study the role of protein p17, an abundant transmembrane protein localized at the viral internal envelope, in these processes. Using an inducible virus for this protein, we show that p17 is essential for virus viability and that its repression blocks the proteolytic processing of polyproteins pp220 and pp62. Electron microscopy analyses demonstrate that when the infection occurs under restrictive conditions, viral morphogenesis is blocked at an early stage, immediately posterior to the formation of the viral precursor membranes, indicating that protein p17 is required to allow their progression toward icosahedral particles. Thus, the absence of this protein leads to an accumulation of these precursors and to the delocalization of the major components of the capsid and core shell domains. The study of ultrathin serial sections from cells infected with BA71V or the inducible virus under permissive conditions revealed the presence of large helicoidal structures from which immature particles are produced, suggesting that these helicoidal structures represent a previously undetected viral intermediate.African swine fever virus (ASFV) (61, 72) is the only known DNA-containing arbovirus and the sole member of the Asfarviridae family (24). Infection by this virus of its natural hosts, the wild swine warthogs and bushpigs and the argasid ticks of the genus Ornithodoros, results in a mild disease, often asymptomatic, with low viremia titers, that in many cases develops into a persistent infection (3, 43, 71). In contrast, infection of domestic pigs leads to a lethal hemorrhagic fever for which the only available methods of disease control are the quarantine of the affected area and the elimination of the infected animals (51).The ASFV genome is a lineal molecule of double-stranded DNA of 170 to 190 kbp in length with convalently closed ends and terminal inverted repeats. The genome encodes more than 150 open reading frames, half of which lack any known or predictable function (16, 75).The virus particle, with an overall icosahedral shape and an average diameter of 200 nm (11), is organized in several concentric layers (6, 11, 15) containing more than 50 structural proteins (29). Intracellular particles are formed by an inner viral core, which contains the central nucleoid surrounded by a thick protein coat, referred to as core shell. This core is enwrapped by an inner lipid envelope (7, 34) on top of which the icosahedral capsid is assembled (26, 27, 31). Extracellular virions possess an additional membrane acquired during the budding from the plasma membrane (11). Both forms of the virus, intracellular and extracellular, are infective (8).The assembly of ASFV particles occurs in the cytoplasm of the infected cell, in viral factories located close to the cell nucleus (6, 13, 49). ASFV factories possess several characteristics similar to those of the cellular aggresomes (35), which are accumulations of aggregates of cellular proteins that form perinuclear inclusions (44).Current models propose that ASFV assembly begins with the modification of endoplasmic reticulum (ER) membranes, which are subsequently recruited to the viral factories and transformed into viral precursor membranes. These ER-derived viral membranes represent the precursors of the inner viral envelope and are the first morphological evidence of viral assembly (7, 60). ASFV viral membrane precursors evolve into icosahedral intermediates and icosahedral particles by the progressive assembly of the outer capsid layer at the convex face of the precursor membranes (5, 26, 27, 31) through an ATP- and calcium-dependent process (19). At the same time, the core shell is formed underneath the concave face of the viral envelope, and the viral DNA and nucleoproteins are packaged and condensed to form the innermost electron-dense nucleoid (6, 9, 12, 69). However, the assembly of the capsid and the internal envelope appears to be largely independent of the components of the core of the particle, since the absence of the viral polyprotein pp220 during assembly produces empty virus-like particles that do not contain the core (9).Comparative genome analysis suggests that ASFV shares a common origin with the members of the proposed nucleocytoplasmic large DNA viruses (NCLDVs) (40, 41). The reconstructed phylogeny of NCLDVs as well as the similitude in the structures and organizations of the genomes indicates that ASFV is more closely related to poxviruses than to other members of the NCLDVs. A consensus about the origin and nature of the envelope of the immature form of vaccinia virus (VV), the prototypical poxvirus, seems to be emerging (10, 17, 20, 54). VV assembly starts with the appearance of crescent-shaped structures within specialized regions of the cytoplasm also known as viral factories (21, 23). The crescent membranes originate from preexisting membranes derived from some specialized compartment of the ER (32, 37, 52, 53, 67), and an operative pathway from the ER to the crescent membrane has recently been described (38, 39). VV crescents apparently grow in length while maintaining the same curvature until they become closed circles, spheres in three dimensions, called immature virions (IV) (22). The uniform curvature is produced by a honeycomb lattice of protein D13L (36, 70), which attaches rapidly to the membranes so that nascent viral membranes always appear to be coated over their entirety. The D13L protein is evolutionarily related to the capsid proteins of the other members of the NCLDV group, including ASFV, but lacks the C-terminal jelly roll motif (40). This structural difference is probably related to the fact that poxviruses are the only member of this group without an icosahedral capsid; instead, the spherical D13L coat acts as a scaffold during the IV stage but is discarded in subsequent steps of morphogenesis (10, 28, 46, 66). Thus, although crescents in VV and precursors of the inner envelope in ASFV are the first morphogenetic stages discernible in the viral factories of these viruses, they seem to be different in nature. Crescents are covered by the D13L protein and are more akin to the icosahedral intermediates of ASFV assembly, whereas ASFV viral membrane precursors are more similar to the naked membranes seen when VV morphogenesis is arrested by rifampin treatment (33, 47, 48, 50) or when the expression of the D13L and A17L proteins are repressed during infection with lethal conditional VV viruses (45, 55, 56, 68, 74, 76).Although available evidence strongly supports the reticular origin of the ASFV inner envelope (7, 60), the mechanism of acquisition remains unknown, and the number of membranes present in the inner envelope is controversial. The traditional view of the inner envelope as formed by two tightly opposed membranes derived from ER collapsed cisternae (7, 59, 60) has recently been challenged by the careful examination of the width of the internal membrane of viral particles and the single outer mitochondrial membrane, carried out using chemical fixation, cryosectioning, and high-pressure freezing (34). The results suggest that the inner envelope of ASFV is a single lipid bilayer, which raises the question of how such a structure can be generated and stabilized in the precursors of the ASFV internal envelope. In the case of VV, the coat of the D13L protein has been suggested to play a key role in the stabilization of the single membrane structure of the crescent (10, 17, 36), but the ASFV capsid protein p72 is not a component of the viral membrane precursors. The identification and functional characterization of the proteins involved in the generation of these structures are essential for the understanding of the mechanisms involved in these early stages of viral assembly. For this reason, we are focusing our interest on the study of abundant structural membrane proteins that reside at the inner envelope of the viral particle. We have shown previously that one of these proteins, p54, is essential for the recruitment of ER membranes to the viral factory (59). Repression of protein p54 expression has a profound impact on virus production and leads to an early arrest in virion morphogenesis, resulting in the virtual absence of membranes in the viral factory.Protein p17, encoded by the late gene D117L in the BA71V strain, is an abundant structural protein (60, 65). Its sequence, which is highly conserved among ASFV isolates (16), does not show any significant similarity with the sequences present in the databases. Protein p17 is an integral membrane protein (18) that is predicted to insert in membranes with a Singer type I topology and has been localized in the envelope precursors as well as in both intracellular and extracellular mature particles (60), suggesting that it resides at the internal envelope, the only membranous structure of the intracellular particles.In this work, we analyze the role of protein p17 in viral assembly by means of an IPTG (isopropyl-β-d-thiogalactopyranoside)-dependent lethal conditional virus. The data presented indicate that protein p17 is essential for viral morphogenesis. The repression of this protein appears to block assembly at the level of viral precursor membranes, resulting in their accumulation at the viral factory.From the electron microscopy analysis of serial sections of viral factories at very early times during morphogenesis, we present experimental evidence that suggests that, during assembly, viral precursor membranes and core material organize into large helicoidal intermediates from which icosahedral particles emerge. The possible role of these structures during ASFV morphogenesis is discussed.     

Analysis of the Viral Elements Required in the Nuclear Import of HIV-1 DNA

HIV-1 possesses an exquisite ability to infect cells independently from their cycling status by undergoing an active phase of nuclear import through the nuclear pore. This property has been ascribed to the presence of karyophilic elements present in viral nucleoprotein complexes, such as the matrix protein (MA); Vpr; the integrase (IN); and a cis-acting structure present in the newly synthesized DNA, the DNA flap. However, their role in nuclear import remains controversial at best. In the present study, we carried out a comprehensive analysis of the role of these elements in nuclear import in a comparison between several primary cell types, including stimulated lymphocytes, macrophages, and dendritic cells. We show that despite the fact that none of these elements is absolutely required for nuclear import, disruption of the central polypurine tract-central termination sequence (cPPT-CTS) clearly affects the kinetics of viral DNA entry into the nucleus. This effect is independent of the cell cycle status of the target cells and is observed in cycling as well as in nondividing primary cells, suggesting that nuclear import of viral DNA may occur similarly under both conditions. Nonetheless, this study indicates that other components are utilized along with the cPPT-CTS for an efficient entry of viral DNA into the nucleus.Lentiviruses display an exquisite ability to infect dividing and nondividing cells alike that is unequalled among Retroviridae. This property is thought to be due to the particular behavior or composition of the viral nucleoprotein complexes (NPCs) that are liberated into the cytoplasm of target cells upon virus-to-cell membrane fusion and that allow lentiviruses to traverse an intact nuclear membrane (17, 28, 29, 39, 52, 55, 67, 79). In the case of the human immunodeficiency type I virus (HIV-1), several studies over the years identified viral components of such structures with intrinsic karyophilic properties and thus perfect candidates for mediation of the passage of viral DNA (vDNA) through the nuclear pore: the matrix protein (MA); Vpr; the integrase (IN); and a three-stranded DNA flap, a structure present in neo-synthesized viral DNA, specified by the central polypurine tract-central termination sequence (cPPT-CTS). It is clear that these elements may mediate nuclear import directly or via the recruitment of the host''s proteins, and indeed, several cellular proteins have been found to influence HIV-1 infection during nuclear import, like the karyopherin α2 Rch1 (38); importin 7 (3, 30, 93); the transportin SR-2 (13, 20); or the nucleoporins Nup98 (27), Nup358/RANBP2, and Nup153 (13, 56).More recently, the capsid protein (CA), the main structural component of viral nucleoprotein complexes at least upon their cytoplasmic entry, has also been suggested to be involved in nuclear import or in postnuclear entry steps (14, 25, 74, 90, 92). Whether this is due to a role for CA in the shaping of viral nucleoprotein complexes or to a direct interaction between CA and proteins involved in nuclear import remains at present unknown.Despite a large number of reports, no single viral or cellular element has been described as absolutely necessary or sufficient to mediate lentiviral nuclear import, and important controversies as to the experimental evidences linking these elements to this step exist. For example, MA was among the first viral protein of HIV-1 described to be involved in nuclear import, and 2 transferable nuclear localization signals (NLSs) have been described to occur at its N and C termini (40). However, despite the fact that early studies indicated that the mutation of these NLSs perturbed HIV-1 nuclear import and infection specifically in nondividing cells, such as macrophages (86), these findings failed to be confirmed in more-recent studies (23, 33, 34, 57, 65, 75).Similarly, Vpr has been implicated by several studies of the nuclear import of HIV-1 DNA (1, 10, 21, 43, 45, 47, 64, 69, 72, 73, 85). Vpr does not possess classical NLSs, yet it displays a transferable nucleophilic activity when fused to heterologous proteins (49-51, 53, 77, 81) and has been shown to line onto the nuclear envelope (32, 36, 47, 51, 58), where it can truly facilitate the passage of the viral genome into the nucleus. However, the role of Vpr in this step remains controversial, as in some instances Vpr is not even required for viral replication in nondividing cells (1, 59).Conflicting results concerning the role of IN during HIV-1 nuclear import also exist. Indeed, several transferable NLSs have been described to occur in the catalytic core and the C-terminal DNA binding domains of IN, but for some of these, initial reports of nuclear entry defects (2, 9, 22, 46, 71) were later shown to result from defects at steps other than nuclear import (60, 62, 70, 83). These reports do not exclude a role for the remaining NLSs in IN during nuclear import, and they do not exclude the possibility that IN may mediate this step by associating with components of the cellular nuclear import machinery, such as importin alpha and beta (41), importin 7 (3, 30, 93, 98), and, more recently, transportin-SR2 (20).The central DNA flap, a structure present in lentiviruses and in at least 1 yeast retroelement (44), but not in other orthoretroviruses, has also been involved in the nuclear import of viral DNA (4, 6, 7, 31, 78, 84, 95, 96), and more recently, it has been proposed to provide a signal for viral nucleoprotein complexes uncoating in the proximity of the nuclear pore, with the consequence of providing a signal for import (8). However, various studies showed an absence or weakness of nuclear entry defects in viruses devoid of the DNA flap (24, 26, 44, 61).Overall, the importance of viral factors in HIV-1 nuclear import is still unclear. The discrepancies concerning the role of MA, IN, Vpr, and cPPT-CTS in HIV-1 nuclear import could in part be explained by their possible redundancy. To date, only one comprehensive study analyzed the role of these four viral potentially karyophilic elements together (91). This study showed that an HIV-1 chimera where these elements were either deleted or replaced by their murine leukemia virus (MLV) counterparts was, in spite of an important infectivity defect, still able to infect cycling and cell cycle-arrested cell lines to similar efficiencies. If this result indicated that the examined viral elements of HIV-1 were dispensable for the cell cycle independence of HIV, as infections proceeded equally in cycling and arrested cells, they did not prove that they were not required in nuclear import, because chimeras displayed a severe infectivity defect that precluded their comparison with the wild type (WT).Nuclear import and cell cycle independence may not be as simply linked as previously thought. On the one hand, there has been no formal demonstration that the passage through the nuclear pore, and thus nuclear import, is restricted to nondividing cells, and for what we know, this passage may be an obligatory step in HIV infection in all cells, irrespective of their cycling status. In support of this possibility, certain mutations in viral elements of HIV affect nuclear import in dividing as well as in nondividing cells (4, 6, 7, 31, 84, 95). On the other hand, cell cycle-independent infection may be a complex phenomenon that is made possible not only by the ability of viral DNA to traverse the nuclear membrane but also by its ability to cope with pre- and postnuclear entry events, as suggested by the phenotypes of certain CA mutants (74, 92).Given that the cellular environment plays an important role during the early steps of viral infection, we chose to analyze the role of the four karyophilic viral elements of HIV-1 during infection either alone or combined in a wide comparison between cells highly susceptible to infection and more-restrictive primary cell targets of HIV-1 in vivo, such as primary blood lymphocytes (PBLs), monocyte-derived macrophages (MDM), and dendritic cells (DCs).In this study, we show that an HIV-1-derived virus in which the 2 NLSs of MA are mutated and the IN, Vpr, and cPPT-CTS elements are removed displays no detectable nuclear import defect in HeLa cells independently of their cycling status. However, this mutant virus is partially impaired for nuclear entry in primary cells and more specifically in DCs and PBLs. We found that this partial defect is specified by the cPPT-CTS, while the 3 remaining elements seem to play no role in nuclear import. Thus, our study indicates that the central DNA flap specifies the most important role among the viral elements involved thus far in nuclear import. However, it also clearly indicates that the role played by the central DNA flap is not absolute and that its importance varies depending on the cell type, independently from the dividing status of the cell.