Protein and DNA Effectors Control the TraI Conjugative Helicase of Plasmid R1

The mechanisms controlling progression of conjugative DNA processing from a preinitiation stage of specific plasmid strand cleavage at the transfer origin to a stage competent for unwinding the DNA strand destined for transfer remain obscure. Linear heteroduplex substrates containing double-stranded DNA binding sites for plasmid R1 relaxosome proteins and various regions of open duplex for TraI helicase loading were constructed to model putative intermediate structures in the initiation pathway. The activity of TraI was compared in steady-state multiple turnover experiments that measured the net production of unwound DNA as well as transesterase-catalyzed cleavage at nic. Helicase efficiency was enhanced by the relaxosome components TraM and integration host factor. The magnitude of stimulation depended on the proximity of the specific protein binding sites to the position of open DNA. The cytoplasmic domain of the R1 coupling protein, TraDΔN130, stimulated helicase efficiency on all substrates in a manner consistent with cooperative interaction and sequence-independent DNA binding. Variation in the position of duplex opening also revealed an unsuspected autoinhibition of the unwinding reaction catalyzed by full-length TraI. The activity reduction was sequence dependent and was not observed with a truncated helicase, TraIΔN308, lacking the site-specific DNA binding transesterase domain. Given that transesterase and helicase domains are physically tethered in the wild-type protein, this observation suggests that an intramolecular switch controls helicase activation. The data support a model where protein-protein and DNA ligand interactions at the coupling protein interface coordinate the transition initiating production and uptake of the nucleoprotein secretion substrate.Controlled duplex DNA unwinding is a crucial prerequisite for the expression and maintenance of genomes. Genome-manipulating and -regulating proteins are central to that biological function in recognizing appropriate DNA targets at initiation sequences and unwinding the complementary strands to provide single-stranded DNA (ssDNA) templates for nucleic acid synthesis and other processing reactions. The protein machineries involved include nucleic acid helicases. DNA helicases are powerful enzymes that convert the energy of nucleoside triphosphate hydrolysis to directional DNA strand translocation and separation of the double helix into its constituent single strands (for reviews, see references 13, 14, 16, 38, 55, and 64). By necessity, these enzymes interact with DNA strands via mechanisms independent of sequence recognition. At replication initiation helicases gain controlled access to the double-stranded genome at positions determined by the DNA binding properties of initiator proteins that comprise an origin recognition complex (1, 9, 17, 31, 45, 66). The mechanisms supporting localized unwinding within the complex include initiator-induced DNA looping, wrapping, and bending and feature regions of low thermodynamic stability. The exposed ssDNA mediates helicase binding followed by directional translocation along that strand until the enzyme engages the duplex for unwinding.In the MOB_F family of conjugation systems, the plasmid DNA strand destined for transfer (T strand) is unwound from its complement by a dedicated conjugative helicase, TraI of F-like plasmids or TrwC of the IncW paradigm. These enzymes are remarkable in that the same polypeptides additionally harbor in a distinct domain a DNA transesterase activity. That function is required to recognize and cleave the precise phosphodiester bond, nic, in the T strand where unwinding of the secretion substrate begins. In current models the conjugative helicases are thus targeted to the transfer origin (oriT) of their cognate plasmid by the high-affinity DNA sequence interactions of their N-terminal DNA transesterase domains. In the bacterial cell, recruitment and activation of the conjugative helicase occur not on naked DNA but within an initiator complex called the relaxosome (67). For the F-like plasmid R1, sequence-specific DNA binding properties of the plasmid proteins TraI, TraY, TraM, and the host integration factor (IHF) direct assembly of the relaxosome at oriT (10, 12, 29, 33, 51, 52). Integration of protein TraM confers recognition features to the relaxosome, which permit its selective docking to TraD, the coupling protein associated with the conjugative type IV secretion system (T4CP) (2, 15, 49). In current models, the T4CP forms a hexameric translocation pore at the cytoplasmic membrane that not only governs substrate entry to the envelope spanning type IV secretion machinery but also provides energy for macromolecular transport via ATP hydrolysis (36, 50). These models propose that T4CPs provide not only a physical bridge between the plasmid and the type IV transporter but also a unique control function in distinguishing one plasmid (relaxosome) from another (7, 8). Before the current study (see accompanying report [41]), evidence indicating that regulation of the initiation of conjugative DNA processing also takes place at this interface had not been reported.F plasmid TraI protein, originally named Escherichia coli DNA helicase I, was initially characterized in the Hoffman-Berling laboratory (19). The purified enzyme exhibits properties in vitro consistent with its function in conjugative DNA strand transfer including a very high 1,100-bp/s rate of duplex unwinding, high processivity, and a 5′-to-3′ directional bias (relative to the strand to which it is bound) (34, 54). Together these features should readily support the observed rate of conjugative DNA translocation as well as concomitant replacement synthesis of the mobilized T strand from the 3′ OH product of nic cleavage.Comparatively little is known about the mechanisms of initiating TraI helicase activity. The enzyme requires ssDNA 5′ to the duplex junction (32), and a minimum length of 30 nucleotides (nt) is necessary to promote efficient duplex unwinding on substrates lacking oriT (11, 54). To our knowledge, oriT is the only sequence where the helicase activity is naturally initiated, however. Moreover, the unique fusion of a helicase to the site- and strand-specific DNA transesterase domains within MOB_F enzymes is expected to pose intriguing regulatory challenges during initiation. The combination within a single polypeptide of a site-specific DNA binding capacity with a helical motor activity would seem counterproductive. The extraordinary efficiency of these proteins in intercellular DNA strand transfer belies this prediction and instead hints strongly at a coordinated progression of the initiation pathway. Since relaxosome assembly is thus far insufficient to initiate helicase activity on supercoiled oriT substrates in vitro, we have developed a series of heteroduplex DNA substrates which support the unwinding reaction and model possible intermediate structures of R1 plasmid strand transfer initiation (10). In this system linear double-stranded DNA (dsDNA) substrates with a central region of sequence heterogeneity trap defined lengths of R1 oriT sequence in unwound conformation. Unexpectedly, efficient helicase activity initiated from a melted oriT duplex required ssDNA twice as long (60 nt) as that previously observed on substrates lacking this sequence (11).In the current report, we describe an application of these models where variation in the position of duplex opening in the vicinity of nic, as well as the additional presence of auxiliary relaxosome proteins, has revealed novel insights into control of a conjugative helicase involving both DNA and protein interactions. Moreover, we observe a sequence-independent stimulation of the unwinding reaction in the presence of T4CP TraD. These results support a model where docking of the preinitiation relaxosome assembly to the T4CP alters the composition and architecture of the complex in a manner essential to the subsequent initiation of T-strand unwinding.     

Plasmid R1 Conjugative DNA Processing Is Regulated at the Coupling Protein Interface

Selective substrate uptake controls initiation of macromolecular secretion by type IV secretion systems in gram-negative bacteria. Type IV coupling proteins (T4CPs) are essential, but the molecular mechanisms governing substrate entry to the translocation pathway remain obscure. We report a biochemical approach to reconstitute a regulatory interface between the plasmid R1 T4CP and the nucleoprotein relaxosome dedicated to the initiation stage of plasmid DNA processing and substrate presentation. The predicted cytosolic domain of T4CP TraD was purified in a predominantly monomeric form, and potential regulatory effects of this protein on catalytic activities exhibited by the relaxosome during transfer initiation were analyzed in vitro. TraDΔN130 stimulated the TraI DNA transesterase activity apparently via interactions on both the protein and the DNA levels. TraM, a protein interaction partner of TraD, also increased DNA transesterase activity in vitro. The mechanism may involve altered DNA conformation as TraM induced underwinding of oriT plasmid DNA in vivo (ΔL_k = −4). Permanganate mapping of the positions of duplex melting due to relaxosome assembly with TraDΔN130 on supercoiled DNA in vitro confirmed localized unwinding at nic but ruled out formation of an open complex compatible with initiation of the TraI helicase activity. These data link relaxosome regulation to the T4CP and support the model that a committed step in the initiation of DNA export requires activation of TraI helicase loading or catalysis.Type IV secretion systems (T4SS) in gram-negative bacteria mediate translocation of macromolecules out of the bacterial cell (14). The transmission of effector proteins and DNA into plant cells or other bacteria via cell-cell contact is one example of their function, and conjugation systems as well as the transferred DNA (T-DNA) delivery system of the phytopathogen Agrobacterium tumefaciens are prototypical of the T4SS family. Macromolecular translocation is achieved by a membrane-spanning protein machinery comprised of 12 gene products, VirB1 to VirB11 and an associated factor known as the coupling protein (VirD4) (66). The T4SS-associated coupling protein (T4CP) performs a crucial function in recognition of appropriate secretion substrates and governing entry of those molecules to the translocation pathway (7, 8, 10, 30, 41). In conjugation systems substrate recognition is applied to the relaxosome, a nucleoprotein complex of DNA transfer initiator proteins assembled specifically at the plasmid origin of transfer (oriT). In current models, initiation of the reactions that provide the single strand of plasmid (T-strand) DNA for secretion to recipient bacteria is expected to resemble the initiation of chromosomal replication (for reviews, see references 18, 54, and 81). Controlled opening of the DNA duplex is required to permit entry of the DNA processing machinery. The task of remodeling the conjugative oriT is generally ascribed to two or three relaxosome auxiliary factors, of host and plasmid origin, which occupy specific DNA binding sites at this locus. Intrinsic to the relaxosome is also a site- and strand-specific DNA transesterase activity that breaks the phosphodiester backbone at nic (5). Upon cleavage, the transesterase enzyme (also called relaxase) forms a reversible phosphotyrosyl linkage to the 5′ end of the DNA. Duplex unwinding initiating from this site produces the single-stranded T strand to be exported. A wealth of information is available supporting the importance of DNA sequence recognition and binding by relaxosome components at oriT to the transesterase reaction in vitro and for effective conjugative transfer (for reviews, see references 18, 54, and 81). On the other hand, the mechanisms controlling release of the 3′-OH generated at nic and the subsequent DNA unwinding stage remain obscure.Equally little is known about the process of nucleoprotein uptake by the transport channel. DNA-independent translocation of the relaxases TrwC (R388), MobA (RSF1010), and VirD2 (Ti plasmid) has been demonstrated; thus, current models propose that the relaxase component of the protein-DNA adduct is the substrate actively secreted by the transport system after interaction with the T4CP (42, 66). Cotransport of the covalently linked single-stranded T strand occurs concurrently (42). The mechanisms underlying relaxosome recognition by T4CPs are not understood. Direct interactions have been observed biochemically between the RP4 TraG protein and relaxase proteins of the cognate plasmid (65) and heterologous relaxosomes that it mobilizes (73, 76). TrwB of R388 interacts in vitro with relaxase TrwC and an auxiliary component, TrwA (44). TraD proteins of plasmid R1 and F are known to interact with the auxiliary relaxosome protein TraM (20) via a cluster of C-terminal amino acids (3, 62). Extensive mutagenic analyses (45) plus recent three-dimensional structural data for a complex of the TraM tetramerization domain and the C-terminal tail of TraD (46) have provided more detailed models for the intermolecular contacts involved in recognition.Application of the Cre recombinase assay for translocation of conjugative relaxases as well as effector proteins to eukaryotic cells is currently the most promising approach to elucidate protein motifs recognized by T4CPs (56, 68, 78, 79). Despite that progress, the nature of the interactions between a T4CP and its target protein that initiate secretion and the mechanisms controlling this step remain obscure. In contrast to systems dedicated specifically to effector protein translocation, conjugation systems mobilize nucleoprotein complexes that additionally exhibit catalytic activities, which can be readily monitored. These models are therefore particularly well suited to investigate aspects of regulation occurring at the physical interface of a T4CP and its secretion substrate. For this purpose the MOB_F family of DNA-mobilizing systems is additionally advantageous, since DNA processing within this family features the fusion of a dedicated conjugative helicase to the DNA transesterase enzyme within a single bifunctional protein. The TraI protein of F-like plasmids, originally described as Escherichia coli DNA helicase I (1, 2, 23), and the related TrwC protein of plasmid R388 (25) are well characterized (reviewed in reference 18). Early work by Llosa et al. revealed a complex domain arrangement for TrwC (43). Similar analyses with TraI identified nonoverlapping transesterase and helicase domains (6, 77), while the remaining intermediate and C-terminal regions of the protein additionally provide functions essential to effective conjugative transfer (49, 71). The ability to physically separate the catalytic domains of TraI and TrwC has facilitated a detailed biochemical characterization of their DNA transesterase, ATPase, and DNA-unwinding reactions. Nonetheless, failure of the physically disjointed polypeptides to complement efficient conjugative transfer when coexpressed indicates a role(s) for these proteins in the strand transfer process that goes beyond the need for their dual catalytic activities (43, 50). The assignment of additional functional properties to regions within TraI is a focus of current investigation (16, 29, 49).In all systems studied thus far, conditions used to reconstitute relaxosomes on a supercoiled oriT plasmid have not supported the initiation steps necessary to enable duplex unwinding by a conjugative helicase. The question remains open whether additional protein components are required and/or whether the pathway of initiation is subject to specific repression. In the present study, we applied the IncFII plasmid R1 paradigm to investigate the potential for interaction between purified components of the relaxosome and its cognate T4CP, TraD, to exert regulatory effects on relaxosome activities in vitro. In this and in the accompanying report (72), we present evidence for wide-ranging stimulatory effects of the cytoplasmic domain of TraD protein and its interaction partner TraM on multiple aspects of relaxosome function.     

Alternative Mechanisms for Coordinating Polymerase α and MCM Helicase

Functional coordination between DNA replication helicases and DNA polymerases at replication forks, achieved through physical linkages, has been demonstrated in prokaryotes but not in eukaryotes. In Saccharomyces cerevisiae, we showed that mutations that compromise the activity of the MCM helicase enhance the physical stability of DNA polymerase α in the absence of their presumed linker, Mcm10. Mcm10 is an essential DNA replication protein implicated in the stable assembly of the replisome by virtue of its interaction with the MCM2-7 helicase and Polα. Dominant mcm2 suppressors of mcm10 mutants restore viability by restoring the stability of Polα without restoring the stability of Mcm10, in a Mec1-dependent manner. In this process, the single-stranded DNA accumulation observed in the mcm10 mutant is suppressed. The activities of key checkpoint regulators known to be important for replication fork stabilization contribute to the efficiency of suppression. These results suggest that Mcm10 plays two important roles as a linker of the MCM helicase and Polα at the elongating replication fork—first, to coordinate the activities of these two molecular motors, and second, to ensure their physical stability and the integrity of the replication fork.The key players of the replication machinery are the DNA polymerases that synthesize the leading and lagging daughter strands and the replicative helicase that unwinds the parental strands ahead of the polymerases. Coordination between the helicase and the polymerases is critical during replication. Uncoupling of these two molecular machines, especially during lagging strand synthesis, may result in an unrestrained helicase and the exposure of extensive single-stranded DNA (ssDNA), as observed in checkpoint mutants treated with hydroxyurea (HU) (37). Although there is no direct evidence, the implication is that the replicative helicase would be moving at a faster pace than would the DNA polymerase if synchrony were destroyed. In Escherichia coli, the replicative helicase (DnaB) and the primase (DnaG) are coupled by direct contact to form a tight complex (3). In T7, processivity of the gp5 polymerase in lagging strand synthesis requires coupling to the gp4 helicase (16). Recent studies of the budding yeast Saccharomyces cerevisiae suggest that Mrc1 may couple DNA polymerase ɛ and the MCM helicase on the leading strand as well as activate the checkpoint response under replication stress (1, 22, 28). A candidate for coupling DNA polymerase α primase and the MCM helicase on the lagging strand is Mcm10, because Mcm10 interacts with subunits of the Mcm2-7 helicase (26, 29) as well as Polα (14, 33) and the stability of Polα requires Mcm10 in both budding yeast and human cells (8, 33). Mcm10 is an essential protein known to be involved in various aspects of the replication process. It is required during both initiation and elongation steps of DNA replication and interacts with a wide range of replication factors, such as ORC (17, 23, 29), MCM helicase, DNA polymerases ɛ and δ (23), Cdc45 (34), and Polα (33). Therefore, Mcm10 is important for the overall stability of the elongation complex, but its essential function remains unknown.Accumulating evidence suggests that the major function of many checkpoint proteins is the stabilization of the replication machinery at the fork (9, 22, 39), in addition to regulation of the temporal and spatial firing of origins and prevention of premature mitosis (31, 35, 39). The main signal that leads to checkpoint activation is believed to be the exposure of RPA-coated ssDNA (42). In Xenopus, ssDNA exposure has been shown to be mediated by a functional uncoupling between the polymerase and the helicase (7), and it has been shown that the level of checkpoint activation depended on the extent of ssDNA accumulation. This observation suggests that uncoupling of the polymerase and the helicase activity would result in ssDNA accumulation that in turn would activate the checkpoint pathway to stabilize the fork.In our study, we carried out a random and a gene-targeted mutagenesis screen to identify mutations that suppress the conditional lethality of mcm10 caused by the lability of Mcm10 in budding yeast (27). We found suppressor mutations in MCM2, which encodes one of the six distinct subunits of the MCM helicase. These mcm2 mutations correct the fork defects of mcm10, particularly that which leads to Polα instability. The altered helicase activity and activation of the checkpoint pathway of the mcm2 mutants appeared to be required for viability of mcm10 mcm2. We showed that uncoupling the MCM helicase and DNA polymerase α by destabilizing Mcm10 leads to accumulation of ssDNA, which is suppressed by reducing the MCM helicase activity. Our findings suggest that the physical coupling of Polα and the helicase by Mcm10 may be replaced by an alternative stabilization mechanism that involves slowing down the helicase and activating the checkpoint proteins.     

Widely Conserved Recombination Patterns among Single-Stranded DNA Viruses

The combinatorial nature of genetic recombination can potentially provide organisms with immediate access to many more positions in sequence space than can be reached by mutation alone. Recombination features particularly prominently in the evolution of a diverse range of viruses. Despite rapid progress having been made in the characterization of discrete recombination events for many species, little is currently known about either gross patterns of recombination across related virus families or the underlying processes that determine genome-wide recombination breakpoint distributions observable in nature. It has been hypothesized that the networks of coevolved molecular interactions that define the epistatic architectures of virus genomes might be damaged by recombination and therefore that selection strongly influences observable recombination patterns. For recombinants to thrive in nature, it is probably important that the portions of their genomes that they have inherited from different parents work well together. Here we describe a comparative analysis of recombination breakpoint distributions within the genomes of diverse single-stranded DNA (ssDNA) virus families. We show that whereas nonrandom breakpoint distributions in ssDNA virus genomes are partially attributable to mechanistic aspects of the recombination process, there is also a significant tendency for recombination breakpoints to fall either outside or on the peripheries of genes. In particular, we found significantly fewer recombination breakpoints within structural protein genes than within other gene types. Collectively, these results imply that natural selection acting against viruses expressing recombinant proteins is a major determinant of nonrandom recombination breakpoint distributions observable in most ssDNA virus families.Genetic recombination is a ubiquitous biological process that is both central to DNA repair pathways (10, 57) and an important evolutionary mechanism. By generating novel combinations of preexisting nucleotide polymorphisms, recombination can potentially accelerate evolution by increasing the population-wide genetic diversity upon which adaptive selection relies. Recombination can paradoxically also prevent the progressive accumulation of harmful mutations within individual genomes (18, 35, 53). Whereas its ability to defend high-fitness genomes from mutational decay possibly underlies the evolutionary value of sexuality in higher organisms, in many microbial species where pseudosexual genetic exchange is permissible among even highly divergent genomes, recombination can enable access to evolutionary innovations that would otherwise be inaccessible by mutation alone.Such interspecies recombination is fairly common in many virus families (8, 17, 27, 44, 82). It is becoming clear, however, that as with mutation events, most recombination events between distantly related genomes are maladaptive (5, 13, 38, 50, 63, 80). As genetic distances between parental genomes increase, so too does the probability of fitness defects in their recombinant offspring (16, 51). The viability of recombinants is apparently largely dependent on how severely recombination disrupts coevolved intragenome interaction networks (16, 32, 51). These networks include interacting nucleotide sequences that form secondary structures, sequence-specific protein-DNA interactions, interprotein interactions, and amino acid-amino acid interactions within protein three-dimensional folds.One virus family where such interaction networks appear to have a large impact on patterns of natural interspecies recombination are the single-stranded DNA (ssDNA) geminiviruses. As with other ssDNA viruses, recombination is very common among the species of this family (62, 84). Partially conserved recombination hot and cold spots have been detected in different genera (39, 81) and are apparently caused by both differential mechanistic predispositions of genome regions to recombination and natural selection disfavoring the survival of recombinants with disrupted intragenome interaction networks (38, 51).Genome organization and rolling circle replication (RCR)—the mechanism by which geminiviruses and many other ssDNA viruses replicate (9, 67, 79; see reference 24 for a review)—seem to have a large influence on basal recombination rates in different parts of geminivirus genomes (20, 33, 39, 61, 81). To initiate RCR, virion-strand ssDNA molecules are converted by host-mediated pathways into double-stranded “replicative-form” (RF) DNAs (34, 67). Initiated by a virus-encoded replication-associated protein (Rep) at a well-defined virion-strand replication origin (v-ori), new virion strands are synthesized on the complementary strand of RF DNAs (28, 73, 74) by host DNA polymerases. Virion-strand replication is concomitant with the displacement of old virion strands, which, once complete, yields covalently closed ssDNA molecules which are either encapsidated or converted into additional RF DNAs. Genome-wide basal recombination rates in ssDNA viruses are probably strongly influenced by the specific characteristics of host DNA polymerases that enable RCR. Interruption of RCR has been implicated directly in geminivirus recombination (40) and is most likely responsible for increased basal recombination rates both within genes transcribed in the opposite direction from that of virion-strand replication (40, 71) and at the v-ori (1, 9, 20, 69, 74).Whereas most ssDNA virus families replicate via either a rolling circle mechanism (the Nanoviridae, Microviridae, and Geminiviridae) (3, 23, 24, 31, 59, 67, 74) or a related rolling hairpin mechanism (the Parvoviridae) (25, 76), among the Circoviridae only the Circovirus genus is known to use RCR (45). Although the Gyrovirus genus (the other member of the Circoviridae) and the anelloviruses (a currently unclassified ssDNA virus group) might also use RCR, it is currently unknown whether they do or not (78). Additionally, some members of the Begomovirus genus of the Geminiviridae either have a second genome component, called DNA-B, or are associated with satellite ssDNA molecules called DNA-1 and DNA-Beta, all of which also replicate by RCR (1, 47, 68).Recombination is known to occur in the parvoviruses (19, 43, 70), microviruses (66), anelloviruses (40, 46), circoviruses (11, 26, 60), nanoviruses (30), geminivirus DNA-B components, and geminivirus satellite molecules (2, 62). Given that most, if not all, of these ssDNA replicons are evolutionarily related to and share many biological features with the geminiviruses (22, 31, 36), it is of interest to determine whether conserved recombination patterns observed in the geminiviruses (61, 81) are evident in these other groups. To date, no comparative analyses have ever been performed with different ssDNA virus families to identify, for example, possible influences of genome organization on recombination breakpoint distributions found in these viruses.Here we compare recombination frequencies and recombination breakpoint distributions in most currently described ssDNA viruses and satellite molecules and identify a number of sequence exchange patterns that are broadly conserved across this entire group.     

Functional Dissection of the Conjugative Coupling Protein TrwB

The conjugative coupling protein TrwB is responsible for connecting the relaxosome to the type IV secretion system during conjugative DNA transfer of plasmid R388. It is directly involved in transport of the relaxase TrwC, and it displays an ATPase activity probably involved in DNA pumping. We designed a conjugation assay in which the frequency of DNA transfer is directly proportional to the amount of TrwB. A collection of point mutants was constructed in the TrwB cytoplasmic domain on the basis of the crystal structure of TrwBΔN70, targeting the nucleotide triphosphate (NTP)-binding region, the cytoplasmic surface, or the internal channel in the hexamer. An additional set of transfer-deficient mutants was obtained by random mutagenesis. Most mutants were impaired in both DNA and protein transport. We found that the integrity of the nucleotide binding domain is absolutely required for TrwB function, which is also involved in monomer-monomer interactions. Polar residues surrounding the entrance and inside the internal channel were important for TrwB function and may be involved in interactions with the relaxosomal components. Finally, the N-terminal transmembrane domain of TrwB was subjected to random mutagenesis followed by a two-hybrid screen for mutants showing enhanced protein-protein interactions with the related TrwE protein of Bartonella tribocorum. Several point mutants were obtained with mutations in the transmembranal helices: specifically, one proline from each protein may be the key residue involved in the interaction of the coupling protein with the type IV secretion apparatus.Bacterial conjugation can be viewed mechanistically as a rolling-circle replication system linked to a type IV secretion process. The two processes come into contact through the activity of a protein that couples the plasmid replication machinery to the export system in the membrane, allowing horizontal dissemination of the replicating DNA molecule (35). This key protein is called “coupling protein” (here “T4CP” for “type IV CP”). It is present in all conjugative systems as well as in many type IV secretion systems (T4SS) involved in bacterial virulence (16). The secreted substrate in bacterial conjugation is the relaxase or pilot protein, attached to the DNA strand. The shoot-and-pump model for bacterial conjugation proposes that, after secretion of the protein through the T4SS, the T4CP works as a motor for export of the rest of the DNA molecule (36). In addition to its presumed role as a DNA transporter, TrwB is also required for transport of relaxase TrwC in the absence of DNA transfer (15).In accordance with its proposed coupling activity, early genetic experiments made patent that the function of conjugative T4CPs depended on interactions with both the cytoplasmic substrate complex (the relaxosome) and the T4SS (6, 7). Thus, T4CP interactions with other conjugation proteins are a key aspect of their function. There have been several reports of interactions between T4CPs from conjugative plasmids and either relaxosomal components—such as F-TraD with TraM (14, 38), RP4-TraG with TraI (49), and pCF10-PcfC with PcfF and PcfG (11)—or T4SS components such as R27-TraG with TrhB (17). T4CP-T4SS interactions have also been reported for the VirB/D4 T4SS involved in DNA transfer from Agrobacterium tumefaciens to plant cells (1, 9). Both sets of interactions have only been concurrently shown for TrwB, the T4CP of plasmid R388. TrwB interacts with proteins TrwA and TrwC, which form the R388 relaxosome, and with the R388 T4SS component TrwE (37). While the interaction with the relaxosome is highly specific for its cognate system (24, 37, 48), the interaction between the T4CP and the T4SS is less specific: a single T4CP can interact functionally with several conjugative T4SS. Interestingly, a correlation was observed between the strength of the T4CP-TrwE-like interaction and the efficiency of DNA transfer (37). T4CPs also interact with TrwE-like components of T4SS involved in virulence (13). In the case of the highly related Trw T4SS systems of plasmid R388 and the human pathogen Bartonella, it was further demonstrated that R388 TrwE could be functionally replaced by the Bartonella tribocorum TrwE homolog, TrwE_Bt (13).T4CPs are integral membrane proteins anchored to the inner membrane by an N-terminal transmembrane domain (TMD). The soluble cytoplasmic domain of TrwB (TrwBΔN70), lacking this TMD, has been biochemically and structurally analyzed in detail. It retains the ability to bind NTPs and to unspecifically bind DNA (42). The characterization of its DNA-dependent ATPase activity (53) strengthened the possibility that T4CPs work as DNA motors. This activity is also stimulated by the oriT-binding protein TrwA (52).The determination of the three-dimensional (3D) structure of TrwBΔN70 indicated a quaternary structure consisting of hexamers that form an almost spherical, orange-shaped structure with a 20-Å inner channel (ICH) (18, 19). Each monomer is composed of two main structural domains: the nucleotide-binding domain (NBD) and the all-alpha domain (AAD). The NBD has α/β topology and is reminiscent of RecA and DNA ring helicases. The AAD is facing the cytoplasmic side and bears significant structural similarity to the N-terminal domain of site-specific recombinase XerD and also to a 40-residue segment of the DNA binding domain of protein TraM, the component of the relaxosome of F-like plasmids that interacts with its cognate T4CP, TraD. The structure of the hexamer as a whole resembles that of the F₁-ATPase, raising interesting perspectives into the possible way of action of coupling proteins as molecular motors in conjugation (5).There have been several attempts to functionally dissect T4CPs. In F-TraD, it was determined that its C terminus is essential for relaxosomal specificity, probably through an interaction with TraM (4, 39, 48). The cytoplasmic domain of the related TraD protein of plasmid R1 stimulates both transesterase and helicase activities of its cognate relaxase, TraI (41, 51). A series of random mutations were shown to affect TraD oligomerization (23). In VirD4, the T4CP of the VirB T4SS of A. tumefaciens, both the periplasmic domain plus key residues of the NBD are required for its location at the cell poles (31); its interaction with the T4SS protein substrate VirE2 does not require the N-terminal TMD (2). Mutational analysis of R27 TraG showed that the periplasmic residues are essential for interaction with the T4SS (22). An N-terminal deletion variant of PcfC, the T4CP of the Enterococcus plasmid pCF10, loses its membrane localization but retains its ability to bind relaxosomal components (11). Biochemical analysis of full-length R388 TrwB showed that the N-terminal TMD stabilizes the protein, aids oligomerization, and affects nucleotide selection (25-27). This region is essential for T4SS interaction, but TrwBΔN70 retains the ability to interact with the relaxosomal components TrwA and TrwC (37). Taken together, these analyses suggested that the N-terminal TMD of the T4CPs is necessary for T4SS interaction, oligomerization, and cellular location and that the C-terminal cytoplasmic domain is necessary for relaxosomal interactions and ATPase activity associated with DNA transport.In this study, we set up different assays to search for mutants affecting TrwB function in DNA and protein transfer. We constructed a series of TrwB point mutants based on the 3D structure of TrwBΔN70. Most selected residues were essential for TrwB function in conjugation, especially under conditions where TrwB was in limiting quantities. We analyzed the in vivo properties of selected mutants with a battery of in vivo assays to map functional domains. Also, random mutants in the TMD were screened for improved interactions with the T4SS, which allowed mapping of the TrwB-TrwE interaction domain.     

Quantitative Fluorescence Resonance Energy Transfer Microscopy Analysis of the Human Immunodeficiency Virus Type 1 Gag-Gag Interaction: Relative Contributions of the CA and NC Domains and Membrane Binding

The human immunodeficiency virus type 1 structural polyprotein Pr55^Gag is necessary and sufficient for the assembly of virus-like particles on cellular membranes. Previous studies demonstrated the importance of the capsid C-terminal domain (CA-CTD), nucleocapsid (NC), and membrane association in Gag-Gag interactions, but the relationships between these factors remain unclear. In this study, we systematically altered the CA-CTD, NC, and the ability to bind membrane to determine the relative contributions of, and interplay between, these factors. To directly measure Gag-Gag interactions, we utilized chimeric Gag-fluorescent protein fusion constructs and a fluorescence resonance energy transfer (FRET) stoichiometry method. We found that the CA-CTD is essential for Gag-Gag interactions at the plasma membrane, as the disruption of the CA-CTD has severe impacts on FRET. Data from experiments in which wild-type (WT) and CA-CTD mutant Gag molecules are coexpressed support the idea that the CA-CTD dimerization interface consists of two reciprocal interactions. Mutations in NC have less-severe impacts on FRET between normally myristoylated Gag proteins than do CA-CTD mutations. Notably, when nonmyristoylated Gag interacts with WT Gag, NC is essential for FRET despite the presence of the CA-CTD. In contrast, constitutively enhanced membrane binding eliminates the need for NC to produce a WT level of FRET. These results from cell-based experiments suggest a model in which both membrane binding and NC-RNA interactions serve similar scaffolding functions so that one can functionally compensate for a defect in the other.The human immunodeficiency virus type 1 (HIV-1) structural precursor polyprotein Pr55^Gag is necessary and sufficient for the assembly of virus-like particles (VLPs). Gag is composed of four major structural domains, matrix (MA), capsid (CA), nucleocapsid (NC), and p6, as well as two spacer peptides, SP1 and SP2 (3, 30, 94). Following particle assembly and release, cleavage by HIV-1 protease separates these domains. However, these domains must work together in the context of the full-length Gag polyprotein to drive particle assembly.Previous studies have mapped two major functional domains involved in the early steps of assembly: first, Gag associates with cellular membranes via basic residues and N-terminal myristoylation of the MA domain (10, 17, 20, 35, 39, 87, 91, 106); second, the Gag-Gag interaction domains that span the CA C-terminal domain (CA-CTD) and NC domain promote Gag multimerization (3, 11, 14, 16, 18, 23, 27, 29, 30, 33, 36, 46, 64, 88, 94, 102, 103). Structural and genetic studies have identified two residues (W184 and M185) within a dimerization interface in the CA-CTD that are critical to CA-CA interactions (33, 51, 74, 96). Analytical ultracentrifugation of heterodimers formed between wild-type (WT) Gag and Gag mutants with changes at these residues suggests that the dimerization interface consists of two reciprocal interactions, one of which can be disrupted to form a “half-interface” (22).In addition to the CA-CTD, NC contributes to assembly via 15 basic residues (8, 9, 11, 14, 18, 23, 25, 28, 34, 40, 43, 54, 57, 58, 74, 79, 88, 97, 104, 105), although some researchers have suggested that NC instead contributes to the stability of mature virions after assembly (75, 98, 99). It is thought that the contribution of NC to assembly is due to its ability to bind RNA, since the addition of RNA promotes the formation of particles in vitro (14-16, 37, 46), and RNase treatment disrupts Gag-Gag interactions (11) and immature viral cores (67). However, RNA is not necessary per se, since dimerization motifs can substitute for NC (1, 4, 19, 49, 105). This suggests a model in which RNA serves a structural role, such as a scaffold, to promote Gag-Gag interactions through NC. Based on in vitro studies, it has been suggested that this RNA scaffolding interaction facilitates the low-order Gag multimerization mediated by CA-CTD dimerization (4, 37, 49, 62, 63, 85). Despite a wealth of biochemical data, the relative contributions of the CA-CTD and NC to Gag multimerization leading to assembly are yet to be determined in cells.Mutations in Gag interaction domains alter membrane binding in addition to affecting Gag multimerization. In particular, mutations or truncations of CA reduce membrane binding (21, 74, 82), and others previously reported that mutations or truncations of NC affect membrane binding (13, 78, 89, 107). These findings are consistent with a myristoyl switch model of membrane binding in which Gag can switch between high- and low-membrane-affinity states (38, 71, 76, 83, 86, 87, 92, 95, 107). Many have proposed, and some have provided direct evidence (95), that Gag multimerization mediated by CA or NC interactions promotes the exposure of the myristoyl moiety to facilitate membrane associations.Gag membrane binding and multimerization appear to be interrelated steps of virus assembly, since membrane binding also facilitates Gag multimerization. Unlike betaretroviruses that fully assemble prior to membrane targeting and envelopment (type B/D), lentiviruses, such as HIV, assemble only on cellular membranes at normal Gag expression levels (type C), although non-membrane-bound Gag complexes exist (45, 58, 60, 61, 65). Consistent with this finding, mutations that reduce Gag membrane associations cause a defect in Gag multimerization (59, 74). Therefore, in addition to their primary effects on Gag-Gag interactions, mutations in Gag interaction domains cause a defect in membrane binding, which, in turn, causes a secondary multimerization defect. To determine the relative contributions of the CA-CTD and the NC domain to Gag-Gag interactions at the plasma membrane, it is essential to eliminate secondary effects due to a modulation of membrane binding.Except for studies using a His-tag-mediated membrane binding system (5, 46), biochemical studies of C-type Gag multimerization typically lack membranes. Therefore, these studies do not fully represent particle assembly, which occurs on biological membranes in cells. Furthermore, many biochemical and structural approaches are limited to isolated domains or truncated Gag constructs. Thus, some of these studies are perhaps more relevant to the behavior of protease-cleaved Gag in mature virions. With few exceptions (47, 74), cell-based studies of Gag multimerization have typically been limited to measuring how well mutant Gag is incorporated into VLPs when coexpressed or not with WT Gag. Since VLP production is a complex multistep process, effects of mutations on other steps in the process can confound this indirect measure. For example, NC contributes to VLP production by both promoting multimerization and interacting with the host factor ALIX to promote VLP release (26, 80). To directly assay Gag multimerization in cells, several groups (24, 45, 52, 56) developed microscopy assays based on fluorescence resonance energy transfer (FRET). These assays measure the transfer of energy between donor and acceptor fluorescent molecules that are brought within ∼5 nm by the association of the proteins to which they are attached (41, 48, 90). However, these microscopy-based Gag FRET assays have not been used to fully elucidate several fundamental aspects of HIV-1 Gag multimerization at the plasma membrane of cells, such as the relative contributions of the CA-CTD and NC and the effect of membrane binding on Gag-Gag interactions. In this study, we used a FRET stoichiometry method based on calibrated spectral analysis of fluorescence microscopy images (41). This algorithm determines the fractions of both donor and acceptor fluorescent protein-tagged Gag molecules participating in FRET. For cells expressing Gag molecules tagged with donor (cyan fluorescent protein [CFP]) and acceptor (yellow fluorescent protein [YFP]) molecules, this method measures the apparent FRET efficiency, which is proportional to the mole fraction of Gag constructs in complex. By measuring apparent FRET efficiencies, quantitative estimates of the mole fractions of interacting proteins can be obtained.Using this FRET-based assay, we aim to answer two questions: (i) what are the relative contributions of CA-CTD and NC domains to Gag multimerization when secondary effects via membrane binding are held constant, and (ii) what is the effect of modulating membrane binding on the ability of Gag mutants to interact with WT Gag?Our data demonstrate that the CA-CTD dimerization interface is essential for Gag multimerization at the plasma membrane, as fully disrupting the CA-CTD interaction abolishes FRET, whereas a modest level of FRET is still detected in the absence of NC. We also present evidence that the CA-CTD dimerization interface consists of two reciprocal interactions, allowing the formation of a half-interface that can still contribute to Gag multimerization. Notably, when Gag derivatives with an intact CA-CTD were coexpressed with WT Gag, either membrane binding ability or NC was required for the Gag mutants to interact with WT Gag, suggesting functional compensation between these factors.     

Analysis of Human Immunodeficiency Virus Type 1 Matrix Binding to Membranes and Nucleic Acids

The human immunodeficiency virus type 1 (HIV-1) matrix (MA) protein targets HIV-1 precursor Gag (PrGag) proteins to assembly sites at plasma membrane (PM) sites that are enriched in cholesterol and phosphatidylinositol-(4,5)-bisphosphate [PI(4,5)P₂]. MA is myristoylated, which enhances membrane binding, and specifically binds PI(4,5)P₂ through headgroup and 2′ acyl chain contacts. MA also binds nucleic acids, although the significance of this association with regard to the viral life cycle is unclear. We have devised a novel MA binding assay and used it to examine MA interactions with membranes and nucleic acids. Our results indicate that cholesterol increases the selectivity of MA for PI(4,5)P₂-containing membranes, that PI(4,5)P₂ binding tolerates 2′ acyl chain variation, and that the MA myristate enhances membrane binding efficiency but not selectivity. We also observed that soluble PI(4,5)P₂ analogues do not compete effectively with PI(4,5)P₂-containing liposomes for MA binding but surprisingly do increase nonspecific binding to liposomes. Finally, we have demonstrated that PI(4,5)P₂-containing liposomes successfully outcompete nucleic acids for MA binding, whereas other liposomes do not. These results support a model in which RNA binding protects MA from associating with inappropriate cellular membranes prior to PrGag delivery to PM assembly sites.The matrix (MA) domain of the human immunodeficiency virus type 1 (HIV-1) precursor Gag (PrGag) protein serves several functions in the viral replication cycle. One essential function is to target PrGag proteins to their assembly sites at the plasma membranes (PMs) of infected cells (4, 5, 11, 16, 25, 29, 30, 33, 35, 39, 43-45, 47, 50, 54, 56, 57). A second function is the recruitment of the viral surface/transmembrane (SU/TM; also referred to as gp120/gp41) envelope (Env) protein complex into virions (14, 15, 18, 19, 27, 51-53). In addition to these activities, numerous reports have attributed nucleic acid binding properties to retroviral MAs (24, 38, 47), and with some viruses MA appears to serve in an encapsidation capacity (24). While no encapsidation role has been assigned for HIV-1 MA, experiments have shown that MA can substitute for the HIV-1 nucleocapsid (NC) protein assembly function (38) under some circumstances, presumably by virtue of its facility to concentrate PrGag proteins by binding them to RNAs (38).A number of structural studies have been conducted on HIV-1 MA (1, 22, 41, 42, 49). The protein is N terminally myristoylated and composed of six α helices, capped by a three-strand β sheet (7, 22, 41, 42, 49). The protein trimerizes in solution and in crystals (22, 28, 49) and recently has been shown to organize as hexamers of trimers on lipid membranes (1). The membrane binding face of HIV-1 MA is basic, fostering its ability to associate with negatively charged phospholipid headgroups (1, 22, 30, 41, 42, 49). The importance of such an interaction has been underscored in molecular genetic experiments which demonstrated that depletion of PM phosphatidylinositol-(4,5)-bisphosphate [PI(4,5)P₂] reduced the assembly efficiency of HIV-1 (9, 36). Consistent with these observations, HIV-1 MA preferentially binds to soluble PI(4,5)P₂ mimics through contacts with the headgroup and 2′ acyl chain, and binding promotes exposure of the MA myristate group and protein oligomerization (17, 21, 40-43, 46). However, PI(4,5)P₂ is not the only lipid to demonstrate an association with HIV-1. In particular, HIV-1 appears to assemble at cholesterol-rich PM sites, cholesterol is highly enriched in HIV-1 virions, and cholesterol depletion reduces viral infectivity (2, 6, 8, 20, 23, 26, 31, 34, 37). The HIV-1 lipidome shows additional differences from the PM lipids of infected cells (2, 5, 8), suggesting that other lipids could affect PrGag-membrane binding or virus assembly site selection.To gain a better understanding of the functions and interactions of HIV-1 MA, we have examined the liposome and nucleic acid binding properties of purified myristoylated MA. Using liposome flotation assays and a novel liposome bead binding assay, we have demonstrated that the PI(4,5)P₂ binding specificity of MA is enhanced by cholesterol, that protein myristoylation increases membrane binding efficiency but not specificity, and that 2′ acyl chain variation is compatible with PI(4,5)P₂ binding. We also examined whether soluble PI(4,5)P₂ mimics could compete with liposomes for MA binding. Surprisingly, we found that soluble mimics not only failed to compete with PI(4,5)P₂ liposomes but also increased MA binding to membranes that do not contain acidic phospholipids. Finally, we have observed that while MA does bind nucleic acids, nucleic acid binding is outcompeted by PI(4,5)P₂-containing liposomes. Our results suggest models for PrGag-membrane and RNA association and the HIV-1 assembly pathway.     

Lagging-Strand DNA Replication Origins Are Required for Conjugal Transfer of the Promiscuous Plasmid pMV158

The promiscuous streptococcal plasmid pMV158 is mobilizable by auxiliary plasmids and replicates by the rolling-circle mechanism in a variety of bacterial hosts. The plasmid has two lagging-strand origins, ssoA and ssoU, involved in the conversion of single-stranded DNA intermediates into double-stranded plasmid DNA during vegetative replication. Transfer of the plasmid also would involve conversion of single-stranded DNA molecules into double-stranded plasmid forms in the recipient cells by conjugative replication. To test whether lagging-strand origins played a role in horizontal transfer, pMV158 derivatives defective in one or in both sso''s were constructed and tested for their ability to colonize new hosts by means of intra- and interspecies mobilization. Whereas either sso supported transfer between strains of Streptococcus pneumoniae, only plasmids that had an intact ssoU could be efficiently mobilized from S. pneumoniae to Enterococcus faecalis. Thus, it appears that ssoU is a critical factor for pMV158 promiscuity and that the presence of a functional sso plays an essential role in plasmid transfer.Conjugation of bacterial plasmids is, together with transposition, the most important source of horizontal gene transfer among bacteria of the same or of different species (42). Conjugation implies the unidirectional transfer of one plasmid DNA strand from a donor to a recipient cell. This is initiated by the activity of a plasmid-encoded protein generically termed relaxase, in a process that resembles replication by the rolling-circle mechanism (13, 29). In the case of numerous, small plasmids (<10 kb) isolated primarily from gram-positive bacteria, two pioneer findings led to the discovery of the rolling-circle mechanism of replication (RCR) plasmids (reviewed in references 20 and 21). First, the strand-specific single-stranded DNA (ssDNA) molecules which act as replication intermediates were identified (41) and, second, the relaxing activity on the supercoiled DNA via the recognition of a specific sequence (the double-strand origin) of the Rep initiator proteins were described (22). Most RCR plasmids are not self-transmissible; instead, they encode not only the Rep topoisomerase-like initiator but also a Mob protein with relaxase activity involved in mobilization mediated by auxiliary plasmids. Such is the case of the promiscuous plasmid pMV158, which can be mobilized between various bacterial species by the pMV158-encoded MobM protein and by helper conjugative plasmids belonging to the Inc18 plasmid family, such as pAMβ1 (15), or even by IncP plasmids, such as RP4 (11). The relaxing activity of MobM on supercoiled DNA of pMV158 and the site of cleavage were first demonstrated in vitro (5, 17), and later the same activity of the MobA protein of the Staphylococcus aureus RCR plasmid pC221 was demonstrated (3, 39).Initiation of transfer, like initiation of RCR, involves cleavage of the phosphodiester bond of a specific dinucleotide on one of the plasmid strands. Cleavage is mediated either by the plasmid-encoded Mob protein at the origin of transfer (oriT) during conjugation or by the plasmid-encoded Rep protein at the dso during replication. In both processes, this initial stage is followed by displacement of the cleaved strand in a unidirectional manner (8, 21, 29, 36). Thus, RCR and conjugal transfer are equivalent processes in the sense that they generate strand-specific ssDNA plasmid intermediates that correspond only to the cleaved strand (9, 16, 41). The ssDNA intermediates are generated in the plasmid host by the activity of the Rep initiator protein (replication) or generated and transferred to the recipient cell (T-DNA) and closed by the Mob relaxase (conjugation), where they are converted into double-stranded plasmid DNA (dsDNA) molecules by lagging-strand synthesis. Replication of the lagging strand is initiated at the single-strand origins (sso) by the host RNA polymerase (RNAP), upon recognition of a specific site on ssDNA and synthesis of a short RNA primer (pRNA). The pRNA is used by DNA polymerase I for limited extension synthesis, followed by replication of the lagging strand by DNA polymerase III (27). Features of the sso include the potential to generate stem-loop structures on ssDNA (9, 16, 41) that can conform a ssDNA promoter, which is inactive in the dsDNA configuration. This kind of promoter was described in the coliphage N4 (18), as recognized by the virion RNAP (4, 14). A different kind of ssDNA promoter, F_rpo, was reported for the Escherichia coli plasmid F and was demonstrated to be used for gene expression and appeared to play a role during plasmid conjugation (34). The presence of ssDNA promoters has also been demonstrated in plasmids pMV158 (27) and ColI-P9 (1, 35). The organization of this kind of promoters showed that they are placed on the DNA strand that is partially complementary to the template strand.The first sso was described in the staphylococcal RCR plasmid pT181, in which a deletion located out of the replicon led to instability, reduction in copy number, and accumulation of ssDNA intermediates (16). Plasmid pMV158 exhibits two sso''s, ssoA and ssoU (23). Two conserved regions were found in the ssoA of pLS1 plasmid (a nonmobilizable pMV158 derivative lacking ssoU): a short region termed recombination site B, RS_B, supposedly involved in plasmid cointegration (16, 38), and a 6-nucleotide (nt) consensus sequence (5′-TAGCGT-3′, termed CS-6). Determination of the roles of these two conserved sites showed that, whereas RS_B was the primary site of RNAP binding (located at the stem of the hairpin), CS-6 was the termination site for the synthesis of a 20-nt pRNA in the loop of the hairpin (27). The predicted intrastrand pairings in the pMV158-ssoA showed the presence of an ssDNA promoter in the vicinity of the RS_B, which would have a consensus −35 region (5′-TTGACA-3′) but a weak −10 region (5′-TAcgcT-3′). With this situation, RNA synthesis should start and proceed in the direction toward the binding site of RNAP, being thus opposite to RNA synthesis from classic promoters (27) (see Fig. ​Fig.1A).1A). Sites homologous to RS_B and CS-6 were later observed in pMV158-ssoU (24).Open in a separate windowFIG. 1.Features of pMV158 and its two lagging-strand origins. (A) Schematic map of the plasmid transfer module indicating relevant restriction sites and the relative positions (shadowed) of the two lagging-strand origins of replication (ssoA and ssoU). Plasmid-encoded MobM protein (arrow below the map) and the position of the oriT are depicted. Direction of DNA transfer is indicated. The EcoRI fragment deleted to construct the pLS1 derivative (28) and the positions of primers used are also shown. A representation of the secondary structures of ssoA (left) and of ssoU (right) indicates the positions of the CS-6 and the RS_B regions (boxed). The locations of the G3 and G7 mutations in the ssoA and of the restriction sites used to generate deletions in the ssoU are shown. The start point and direction for the RNA primer (pRNA) synthesis, downstream to CS-6 sequence, is indicated by a wavy arrow. (B) Relevant sequence features of the two pMV158 sso. The RS_B and CS-6 sequences are shown in boxes. The restriction sites of BsaI and DraI used to generate ssoU-ΔBD mutant are also indicated, as well as the nucleotides changed (boldface) to construct the ssoA-G3G7 mutant (sequence indicated beneath).In the present study we have addressed the question of whether and, eventually, which of the two pMV158-ssos plays a role in conjugal transfer. With this objective, we constructed pMV158-derivatives defective in one or both sso''s and tested their role on intra- and interspecies mobilization. Whereas either sso supported transfer between strains of Streptococcus pneumoniae with the same efficiency as the parental pMV158, only the ssoU could do so when conjugal transfer was assayed between S. pneumoniae and Enterococcus faecalis. Our findings show that the functionality of ssoU is a critical factor in the colonization of a broad range of gram-positive bacteria for the pMV158 promiscuous plasmid and demonstrate that efficient transfer and replication in enterococci depend upon a functional ssoU. We suggest that sso''s lacking functionality for vegetative replication in a specific host should not be efficient in conjugative transfer and vice versa, since both events are mechanistically identical. As far as we know, this is the first report that shows the effect of sso functionality on horizontal gene transfer by plasmid conjugation, as well as the efficiency of the ssoA and ssoU in E. faecalis.     

Virus Entry via the Alternative Coreceptors CCR3 and FPRL1 Differs by Human Immunodeficiency Virus Type 1 Subtype

Human immunodeficiency virus type 1 (HIV-1) infects target cells by binding to CD4 and a chemokine receptor, most commonly CCR5. CXCR4 is a frequent alternative coreceptor (CoR) in subtype B and D HIV-1 infection, but the importance of many other alternative CoRs remains elusive. We have analyzed HIV-1 envelope (Env) proteins from 66 individuals infected with the major subtypes of HIV-1 to determine if virus entry into highly permissive NP-2 cell lines expressing most known alternative CoRs differed by HIV-1 subtype. We also performed linear regression analysis to determine if virus entry via the major CoR CCR5 correlated with use of any alternative CoR and if this correlation differed by subtype. Virus pseudotyped with subtype B Env showed robust entry via CCR3 that was highly correlated with CCR5 entry efficiency. By contrast, viruses pseudotyped with subtype A and C Env proteins were able to use the recently described alternative CoR FPRL1 more efficiently than CCR3, and use of FPRL1 was correlated with CCR5 entry. Subtype D Env was unable to use either CCR3 or FPRL1 efficiently, a unique pattern of alternative CoR use. These results suggest that each subtype of circulating HIV-1 may be subject to somewhat different selective pressures for Env-mediated entry into target cells and suggest that CCR3 may be used as a surrogate CoR by subtype B while FPRL1 may be used as a surrogate CoR by subtypes A and C. These data may provide insight into development of resistance to CCR5-targeted entry inhibitors and alternative entry pathways for each HIV-1 subtype.Human immunodeficiency virus type 1 (HIV-1) infects target cells by binding first to CD4 and then to a coreceptor (CoR), of which C-C chemokine receptor 5 (CCR5) is the most common (6, 53). CXCR4 is an additional CoR for up to 50% of subtype B and D HIV-1 isolates at very late stages of disease (4, 7, 28, 35). Many other seven-membrane-spanning G-protein-coupled receptors (GPCRs) have been identified as alternative CoRs when expressed on various target cell lines in vitro, including CCR1 (76, 79), CCR2b (24), CCR3 (3, 5, 17, 32, 60), CCR8 (18, 34, 38), GPR1 (27, 65), GPR15/BOB (22), CXCR5 (39), CXCR6/Bonzo/STRL33/TYMSTR (9, 22, 25, 45, 46), APJ (26), CMKLR1/ChemR23 (49, 62), FPLR1 (67, 68), RDC1 (66), and D6 (55). HIV-2 and simian immunodeficiency virus SIVmac isolates more frequently show expanded use of these alternative CoRs than HIV-1 isolates (12, 30, 51, 74), and evidence that alternative CoRs other than CXCR4 mediate infection of primary target cells by HIV-1 isolates is sparse (18, 30, 53, 81). Genetic deficiency in CCR5 expression is highly protective against HIV-1 transmission (21, 36), establishing CCR5 as the primary CoR. The importance of alternative CoRs other than CXCR4 has remained elusive despite many studies (1, 30, 70, 81). Expansion of CoR use from CCR5 to include CXCR4 is frequently associated with the ability to use additional alternative CoRs for viral entry (8, 16, 20, 63, 79) in most but not all studies (29, 33, 40, 77, 78). This finding suggests that the sequence changes in HIV-1 env required for use of CXCR4 as an additional or alternative CoR (14, 15, 31, 37, 41, 57) are likely to increase the potential to use other alternative CoRs.We have used the highly permissive NP-2/CD4 human glioma cell line developed by Soda et al. (69) to classify virus entry via the alternative CoRs CCR1, CCR3, CCR8, GPR1, CXCR6, APJ, CMKLR1/ChemR23, FPRL1, and CXCR4. Full-length molecular clones of 66 env genes from most prevalent HIV-1 subtypes were used to generate infectious virus pseudotypes expressing a luciferase reporter construct (19, 57). Two types of analysis were performed: the level of virus entry mediated by each alternative CoR and linear regression of entry mediated by CCR5 versus all other alternative CoRs. We thus were able to identify patterns of alternative CoR use that were subtype specific and to determine if use of any alternative CoR was correlated or independent of CCR5-mediated entry. The results obtained have implications for the evolution of env function, and the analyses revealed important differences between subtype B Env function and all other HIV-1 subtypes.