Type D Retrovirus Capsid Assembly and Release Are Active Events Requiring ATP

Mason-Pfizer monkey virus (M-PMV), the prototype type D retrovirus, differs from most other retroviruses by assembling its Gag polyproteins into procapsids in the cytoplasm of infected cells. Once assembled, the procapsids migrate to the plasma membrane, where they acquire their envelope during budding. Because the processes of M-PMV protein transport, procapsid assembly, and budding are temporally and spatially unlinked, we have been able to determine whether cellular proteins play an active role during the different stages of procapsid morphogenesis. We report here that at least two stages of morphogenesis require ATP. Both procapsid assembly and procapsid transport to the plasma membrane were reversibly blocked by treating infected cells with sodium azide and 2-deoxy-d-glucose, which we show rapidly and reversibly depletes cellular ATP pools. Assembly of procapsids in vitro in a cell-free translation/assembly system was inhibited by the addition of nonhydrolyzable ATP analogs, suggesting that ATP hydrolysis and not just ATP binding is required. Since retrovirus Gag polyproteins do not bind or hydrolyze ATP, these results demonstrate that cellular components must play an active role during retrovirus morphogenesis.

Assembly and release of nascent retrovirus particles requires that the viral precursor polyproteins and genomic RNAs, and certain host cell tRNAs, migrate to the plasma membrane, where budding occurs. Two discrete intracellular transport pathways are utilized during the assembly of the infectious virion. The viral glycoproteins are synthesized on membrane-bound polysomes and are transported through the secretory pathway of the cell to the plasma membrane, where they colocalize with the immature capsid during the budding process (20). The major structural proteins of the viral capsid and the enzymatic proteins are synthesized in the cytoplasm on free polysomes and are transported to the underside of the plasma membrane (13, 36). While many of the details of the secretory pathway have been established, the mechanisms for intracytoplasmic protein transport are poorly understood.The major structural polyprotein (Gag) of a nascent retrovirus capsid is encoded by the gag gene. Unlike most enveloped RNA viruses in which the viral glycoproteins mediate assembly by stabilizing the interactions between the capsid proteins and the viral membrane, retroviral Gag proteins can drive capsid assembly and budding in the absence of all the other viral gene products (19, 55, 58). As such, they contain all cis-acting information necessary for intracytoplasmic transport, capsid assembly, membrane binding, envelopment, and release from the cell surface. Assembly of the immature retrovirus capsid begins shortly after the Gag polyproteins are synthesized and modified by myristylation (15, 17, 40, 47–49). The Gag proteins of most retroviruses (the type C avian and mammalian viruses, lentiviruses, and human T-cell leukemia virus/bovine leukemia virus-related viruses) migrate directly to the plasma membrane, where they coalesce into spherical, immature capsids and simultaneously bud through the lipid bilayer, thereby acquiring their envelope. During or shortly after release, the Gag protein is cleaved by the viral protease into the internal structural (NH₂-MA [matrix], CA [capsid], and NC [nucleocapsid]) proteins of the mature, infectious virion (22). In contrast, the Gag proteins of the mammalian and type B and D viruses (mouse mammary tumor virus [MMTV] and Mason-Pfizer monkey virus [M-PMV], respectively) accumulate in the cytoplasm, where they assemble into spherical structures in the absence of membranes. These nascent particles have been referred to as intracytoplasmic type A particles, but by analogy to other viruses and bacteriophages, we have redefined them as procapsids (55). Once assembled, procapsids are transported to the plasma membrane, from which they bud. Despite the different assembly strategies, the processes whereby Gag proteins assemble into procapsids are probably similar since a single amino acid change near the amino terminus of the Gag protein from M-PMV has been shown to convert it to the type C morphogenic pathway (41).Genetic analyses of the gag genes from different retroviruses have shown that Gag proteins contain specific domains which are required for capsid formation. A membrane binding (M) domain has been located at the amino-terminal end of Gag of several retroviruses (31, 43, 60, 61). A late (L) domain functions during the budding and release. In Rous sarcoma virus (RSV) and M-PMV, the L domain is located between the MA and CA domains (57, 59). An equivalent domain in the lentiviruses has been found near the carboxy terminus of the Gag precursor (34). A third domain (I), located near the CA-NC junction, appears to be a region of interaction between Gag proteins (3, 56). Despite the lack of any extensive sequence similarities between different Gag proteins, there is functional conservation between assembly domains. Chimeric Gag proteins containing the M, L, and I domains from different retroviruses can assemble into capsid-like structures and mediate budding at the plasma membrane (3, 9, 10, 34).The M-PMV Gag protein contains additional assembly elements which influence procapsid assembly, stability, and transport. This virus contains a region within Gag (known as p12) that is not found in either the type C viruses or lentiviruses. It has been suggested from biochemical data derived from studies with p12 deletion mutants that this domain assists in assembly by stabilizing intermolecular Gag associations (50). Protein stability and protein/procapsid transport depend on sequences in the MA domain which appear to be distinct from the M domain. As mentioned above, a single point mutation in MA at residue 55 results in a Gag protein that no longer assembles in the cytoplasm but rather assembles at the plasma membrane. This mutation lies within an 18-amino-acid region of the MA domain that has sequence similarity only to the type B retroviruses (41). The nuclear magnetic resonance-derived solution structure of a nonmyristylated M-PMV MA protein indicates that this region folds into a structured turn which is solvent accessible in the monomer and trimer models (8). Moreover, this structural feature is absent in human immunodeficiency virus (HIV), simian immunodeficiency virus, human T-cell leukemia virus, and bovine leukemia virus MA proteins (7, 18, 27–30, 37). It is reasonable, therefore, to suspect that this region contains a cytoplasmic protein transport signal which must interact with a cellular factor. In contrast, other mutations in either the myristic acid addition signal or at a variety of positions elsewhere in the MA coding region result in Gag proteins that fail to be released as virus-like particles despite assembling into procapsids in the cytoplasm (40, 43). Thus, the M-PMV Gag protein appears to contain a second cytoplasmic transport signal which normally directs assembled procapsids and not unassembled Gag proteins to the plasma membrane. It is implied in this model that the M-PMV Gag protein must utilize multiple cellular components during the different stages of assembly and release.The type D retroviruses provide a useful system for studying morphogenic events since procapsid assembly, protein transport, and budding are temporally and spatially unlinked. We report here that in infected cells and an in vitro translation/assembly system, procapsid assembly and transport to the plasma membrane require ATP. Thus, cellular proteins do play an active role during at least two stages of M-PMV morphogenesis.     

Endosomal Trafficking of HIV-1 Gag and Genomic RNAs Regulates Viral Egress

HIV-1 Gag can assemble and generate virions at the plasma membrane, but it is also present in endosomes where its role remains incompletely characterized. Here, we show that HIV-1 RNAs and Gag are transported on endosomal vesicles positive for TiVamp, a v-SNARE involved in fusion events with the plasma membrane. Inhibition of endosomal traffic did not prevent viral release. However, inhibiting lysosomal degradation induced an accumulation of Gag in endosomes and increased viral production 7-fold, indicating that transport of Gag to lysosomes negatively regulates budding. This also suggested that endosomal Gag-RNA complexes could access retrograde pathways to the cell surface and indeed, depleting cells of TiVamp-reduced viral production. Moreover, inhibition of endosomal transport prevented the accumulation of Gag at sites of cellular contact. HIV-1 Gag could thus generate virions using two pathways, either directly from the plasma membrane or through an endosome-dependent route. Endosomal Gag-RNA complexes may be delivered at specific sites to facilitate cell-to-cell viral transmission.The production of infectious retroviral particles is an ordered process that includes many steps (for review see Refs. 1–3). In particular, three major viral components, Gag, the envelope, and genomic RNAs have to traffic inside the cell to reach their assembly site. Viral biogenesis is driven by the polyprotein Gag, which is able to make viral-like particles when expressed alone (4). Upon release, HIV-1⁴ Gag is processed by the viral protease into matrix (MA(p17)), capsid (CA(p24)), nucleocapsid (NC(p7)), p6, and smaller peptides SP1 and SP2. Gag contains several domains that are essential for viral assembly: a membrane binding domain (M) in MA; a Gag-Gag interaction domain in CA; an assembly domain (I) in NC; and a late domain (L) in p6, which recruits the cellular budding machinery. Genomic RNAs are specifically recognized by NC, and they play fundamental roles in viral biogenesis by acting as a scaffold for Gag multimerization (5).It has been demonstrated that retroviruses bud by hijacking the endosomal machinery that sorts proteins into internal vesicles of multivesicular bodies (for review, see Refs. 6, 7). Indeed, these vesicles bud with the same topology as viral particles. Proteins sorted into this pathway are usually destined for degradation in lysosomes, but some can also recycle to the plasma membrane (for review see Refs. 8, 9). They are also frequently ubiquitinated on their cytoplasmic domain (10, 11), allowing their recognition by ESCRT complexes. ESCRT-0 and ESCRT-I recognize ubiquitinated cargo present at the surface of endosomes and recruit other ESCRT complexes (12–14). ESCRT-III is believed to function directly in the formation of multivesicular body intralumenal vesicles (12), even though its mechanism of action is currently not understood. Remarkably, Gag L domains interact directly with components of the multivesicular body-sorting machinery (for review see Ref. 15). HIV-1 Gag uses a PTAP motif to bind Tsg101, a component of ESCRT-I (16–19), and a YPLTSL motif to interact with Alix, a protein linked to ESCRT-I and -III (20–22). Finally, various ubiquitin ligases are also required directly or indirectly during HIV-1 biogenesis (23, 24; for review see Ref. 25).In many cell lines, Gag is found both at the plasma membrane and in endosomes. This has led to the hypothesis that there are several assembly sites for HIV-1 (1, 3). First, Gag can initiate and complete assembly at the plasma membrane. This is thought to occur predominantly in T lymphocytes, and this process is supported by several lines of evidences: (i) disruption of endosomal trafficking with drugs does not prevent viral production (26, 27); (ii) ESCRT complexes can be recruited at the plasma membrane, at sites where Gag accumulates (28–30); (iii) Gag can be seen multimerizing and budding from the plasma membrane in live cells (31). Second, Gag could initiate assembly in endosomes, and then traffic to the cell surface to be released. This is mainly supported by the presence of Gag in endosomes in several cell lines (32–34), including T cells and more strikingly macrophages (32, 35, 36–39). However, we are currently lacking functional experiments addressing the role of this endosomal pool of Gag, and it is still not clear to what extent it contributes to the production of viral particles. Nevertheless, the presence of Gag in endosomes might facilitate recruitment of ESCRT complexes (34, 40), packaging of viral genomic RNAs (32, 41), and incorporation of the envelope (42). It may also be important for polarized budding (43, 44) and to create a viral reservoir in infected cells (45, 46).Despite great progress, the traffic of HIV-1 components is still not fully elucidated. In particular, the transport of the genomic RNAs is poorly understood. In this study, we have used single molecule techniques to investigate the trafficking of HIV-1 RNAs in fixed and live cells, and we show that they are transported on endosomal vesicles. We also obtained functional evidence that Gag and viral RNAs can use at least two trafficking pathways to produce virions, one going directly from the plasma membrane and another one passing through endosomes.     

Analysis of Free Energy Signals Arising from Nucleotide Hybridization Between rRNA and mRNA Sequences during Translation in Eubacteria

A decoding algorithm is tested that mechanistically models the progressive alignments that arise as the mRNA moves past the rRNA tail during translation elongation. Each of these alignments provides an opportunity for hybridization between the single-stranded, -terminal nucleotides of the 16S rRNA and the spatially accessible window of mRNA sequence, from which a free energy value can be calculated. Using this algorithm we show that a periodic, energetic pattern of frequency 1/3 is revealed. This periodic signal exists in the majority of coding regions of eubacterial genes, but not in the non-coding regions encoding the 16S and 23S rRNAs. Signal analysis reveals that the population of coding regions of each bacterial species has a mean phase that is correlated in a statistically significant way with species () content. These results suggest that the periodic signal could function as a synchronization signal for the maintenance of reading frame and that codon usage provides a mechanism for manipulation of signal phase.[1,2,3,4,5,6,7,8,9,10,11,12,13,14,15,16,17,18,19,20,21,22,23,24,25,26,27,28,29,30,31,32]     

Sequence-Specific Binding of Human Immunodeficiency Virus Type 1 Nucleocapsid Protein to Short Oligonucleotides

We have analyzed the binding of recombinant human immunodeficiency virus type 1 nucleocapsid protein (NC) to very short oligonucleotides by using surface plasmon resonance (SPR) technology. Our experiments, which were conducted at a moderate salt concentration (0.15 M NaCl), showed that NC binds more stably to runs of d(G) than to other DNA homopolymers. However, it exhibits far more stable binding with the alternating base sequence d(TG)_n than with any homopolymeric oligodeoxyribonucleotide; thus, it shows a strong sequence preference under our experimental conditions. We found that the minimum length of an alternating d(TG) sequence required for stable binding was five nucleotides. Stable binding to the tetranucleotide d(TG)₂ was observed only under conditions where two tetranucleotide molecules were held in close spatial proximity. The stable, sequence-specific binding to d(TG)_n required that both zinc fingers be present, each in its proper position in the NC protein, and was quite salt resistant, indicating a large hydrophobic contribution to the binding. Limited tests with RNA oligonucleotides indicated that the preferential sequence-specific binding observed with DNA also occurs with RNA. Evidence was also obtained that NC can bind to nucleic acid molecules in at least two distinct modes. The biological significance of the specific binding we have detected is not known; it may reflect the specificity with which the parent Gag polyprotein packages genomic RNA or may relate to the functions of NC after cleavage of the polyprotein, including its role as a nucleic acid chaperone.A single protein species, the Gag polyprotein, is sufficient for assembly of retrovirus particles. Since this process includes the selective encapsidation of viral RNA, this protein is evidently capable of specific interactions with nucleic acids. The nature of these interactions is not well understood as yet. After the virion is released from the cell, the polyprotein is cleaved by the virus-encoded protease; one of the cleavage products, termed the nucleocapsid protein (NC), then binds to the genomic RNA, forming the ribonucleoprotein core of the mature particle (21, 35, 41).The interaction between Gag and the genomic RNA is known to involve the NC domain of the polyprotein, since mutants within this domain of Gag are defective in RNA packaging (e.g., references 2, 16, 17, 24–27, 31, 36, 37, and 39) and since the specificity of encapsidation tends to be determined by the NC domain in chimeric Gag molecules (9, 18, 49). However, NC is a basic protein and has frequently been described as binding to single-stranded DNA or RNA in a sequence-independent manner. Indeed, it is probably capable of binding to any single-stranded nucleic acid under appropriate conditions. This binding activity appears to be crucial at several stages of virus replication (13, 19, 28, 46).In the experiments described here, we have analyzed the binding of recombinant human immunodeficiency virus type 1 (HIV-1) NC to short oligonucleotides. These studies were performed at moderate ionic strengths, at which the nonspecific electrostatic interaction between NC and nucleic acids is minimized. We find that under these conditions, the protein exhibits profound sequence preferences. This sequence-specific binding is dependent upon the zinc fingers of the protein and has a strong hydrophobic component. The biological significance of this sequence specificity is not clear at present, but the results suggest that studies with very short oligonucleotides may provide important insights into NC function and perhaps functions of Gag as well.     

Algorithms for Finding Small Attractors in Boolean Networks

A Boolean network is a model used to study the interactions between different genes in genetic regulatory networks. In this paper, we present several algorithms using gene ordering and feedback vertex sets to identify singleton attractors and small attractors in Boolean networks. We analyze the average case time complexities of some of the proposed algorithms. For instance, it is shown that the outdegree-based ordering algorithm for finding singleton attractors works in time for , which is much faster than the naive time algorithm, where is the number of genes and is the maximum indegree. We performed extensive computational experiments on these algorithms, which resulted in good agreement with theoretical results. In contrast, we give a simple and complete proof for showing that finding an attractor with the shortest period is NP-hard.[1,2,3,4,5,6,7,8,9,10,11,12,13,14,15,16,17,18,19,20,21,22,23,24,25,26,27,28,29,30,31,32]     

A Putative α-Helical Structure Which Overlaps the Capsid-p2 Boundary in the Human Immunodeficiency Virus Type 1 Gag Precursor Is Crucial for Viral Particle Assembly

The capsid (CA) and nucleocapsid domains of the human immunodeficiency virus type 1 Gag polyprotein are separated by the p2 spacer peptide, which is essential for virus replication. Previous studies have revealed that p2 has an important role in virus morphogenesis. In this paper, we show that a crucial assembly determinant maps to the highly conserved N terminus of p2, which is predicted to form part of an α-helix that begins in CA. A mutational analysis indicates that the ability of the N terminus of p2 to adopt an α-helical structure is essential for its function during virus assembly. To prevent CA-p2 processing, it was necessary to mutate both the CA-p2 cleavage site and an internal cleavage site within p2. Virions produced by the double mutant lacked a conical core shell and instead contained a thin electron-dense shell about 10 nm underneath the virion membrane. These results suggest that p2 is transiently required for proper assembly, but needs to be removed from the C terminus of CA to weaken CA-CA interactions and allow the rearrangement of the virion core shell during virus maturation.The internal structural proteins of the human immunodeficiency virus type 1 (HIV-1) virion are synthesized in the form of a polyprotein (Pr55^gag) which can efficiently form enveloped virus-like particles even when expressed alone (17). Pr55^gag is modified by N-terminal myristylation, which is required for its stable association with the inner leaflet of the plasma membrane, where virus assembly occurs (4, 21). During or after the release of an immature particle from the plasma membrane, Pr55^gag is cleaved by the viral protease. The major Gag cleavage products are matrix (MA), capsid (CA), nucleocapsid (NC), and p6 (25, 34). MA, which has a crucial role in the incorporation of the viral surface glycoproteins (10, 52), remains associated with the host cell-derived lipid envelope of the virion (16). CA forms the shell of the characteristic cone-shaped core of the mature virion which encloses the viral genomic RNA (16, 27). NC is essential for the encapsidation of the viral genome and is believed to coat the viral RNA within the core of the virion (2, 19, 30). The C-terminal p6 domain of Pr55^gag facilitates the release of assembled viral particles from the cell surface (20) and is also needed for the incorporation of the regulatory viral protein Vpr (31, 39).Within the context of Pr55^gag, two spacer peptides, p2 and p1, are located between CA and NC and between NC and p6, respectively (24, 25). Cleavage between CA and p2 is much slower than that between p2 and NC or between MA and CA (41). As a consequence, a CA-p2 protein (p25) accumulates in virus-producing cells (34). However, CA-p2 is normally found only in trace amounts in virions. In addition to p2, which comprises 14 amino acids (Ala-363 through Met-376) of the HIV-1_HXB2 Gag precursor, a 10-amino-acid p2 fragment which extends from Ser-367 through Met-376 has been isolated from HIV-1 virions, indicating that the viral protease can also cleave within p2 (24, 25).Genetic analyses indicate that the region surrounding the CA-p2 boundary has an important role in particle assembly (21, 28, 50). Within CA, the N-terminal two-thirds forms a domain which appears dispensable for particle assembly but is required for the formation of the cone-shaped core of the mature virion (8, 44, 51). Recent structure determinations have revealed that the N-terminal HIV-1 CA domain is largely α-helical (18, 35). An exposed loop region between two α-helices interacts with the prolyl isomerase cyclophilin A (14), which leads to the incorporation of the cellular enzyme into virions (13, 48). The C-terminal third of CA forms a distinct domain which is essential for Gag oligomerization and particle assembly (8, 12, 44). While genetic and structural studies indicate that the N-terminal boundary of the CA assembly domain coincides with a uniquely conserved sequence, termed the major homology region (8, 15, 18, 32), its C-terminal boundary remains less well defined.The replacement of the scissile dipeptide Leu-Ala at the CA-p2 boundary with Ser-Arg in a mutant designated SVC-C2 led to the formation of grossly distorted capsid structures and caused a significant reduction in particle yield, indicating that the very C terminus of CA and/or p2 is crucial for HIV-1 morphogenesis (21). The possibility that the CA assembly domain extends into p2 is also suggested by the finding that the precise deletion of p2 from Pr55^gag markedly reduced particle production (28). Electron microscopy revealed an accumulation of large electron-dense plaques underneath the plasma membrane in the absence of p2 (28), a phenotype which is similar to that observed for the SVC-C2 cleavage site mutant (21). However, the role of p2 in virus assembly remains controversial, because its removal appeared to have no effect on particle release in another study (41).In the present study, we focused on the N-terminal portion of p2, since it is considerably more conserved than the C terminus and because it is predicted to be part of an α-helix which begins in CA. The analysis of a panel of single-amino-acid changes shows that the conserved N terminus of p2 is essential for virus replication and indicates that its predicted α-helical conformation is crucial for virus assembly. In contrast, a deletion which removed 5 out of 10 amino acids between a previously reported cleavage site within p2 and NC delayed but did not abolish virus replication, demonstrating that this relatively variable region of p2 has no essential function in the viral life cycle. We also show that processing of CA-p2 can be essentially prevented by disrupting both the CA-p2 cleavage site and the reported Met-Ser site (25) within p2. Interestingly, the mutant particles often contained a prominent circular structure underneath the viral membrane, indicating that the presence of p2 at the C terminus of CA prevented the rearrangement of the core into a conical tube.     

Analysis of the Assembly Function of the Human Immunodeficiency Virus Type 1 Gag Protein Nucleocapsid Domain

Previous studies have shown that in addition to its function in specific RNA encapsidation, the human immunodeficiency virus type 1 (HIV-1) nucleocapsid (NC) is required for efficient virus particle assembly. However, the mechanism by which NC facilitates the assembly process is not clearly established. Formally, NC could act by constraining the Pr55^gag polyprotein into an assembly-competent conformation or by masking residues which block the assembly process. Alternatively, the capacity of NC to bind RNA or make interprotein contacts might affect particle assembly. To examine its role in the assembly process, we replaced the NC domain in Pr55^gag with polypeptide domains of known function, and the chimeric proteins were analyzed for their abilities to direct the release of virus-like particles. Our results indicate that NC does not mask inhibitory domains and does not act passively, by simply providing a stable folded monomeric structure. However, replacement of NC by polypeptides which form interprotein contacts permitted efficient virus particle assembly and release, even when RNA was not detected in the particles. These results suggest that formation of interprotein contacts by NC is essential to the normal HIV-1 assembly process.Human immunodeficiency virus type 1 (HIV-1) encodes three major genes, gag, pol, and env, which are commonly found in all mammalian retroviruses. It also encodes accessory genes whose protein products are important for regulation of its life cycle (6, 30, 35). However, of all the genes encoded by HIV-1, only the protein product of the gag gene has been found to be necessary and sufficient for the assembly of virus-like particles (11, 13, 17, 22, 32, 33). The HIV-1 Gag protein initially is expressed as a 55-kDa polyprotein precursor (Pr55^gag), but during or shortly after particle release, Pr55^gag ordinarily is cleaved by the viral protease (PR). The products of the protease action are the four major viral proteins matrix (MA), capsid (CA), nucleocapsid (NC), and p6, and the two spacer polypeptides p2 and p1, which represent sequences between CA and NC and between NC and p6, respectively (15, 19, 23, 30).The HIV-1 nucleocapsid proteins have two Cys-X₂-Cys-X₄-His-X₄-Cys (Cys-His) motifs, reminiscent of the zinc finger motifs found in many DNA binding proteins, and NC has been shown to facilitate the specific encapsidation of HIV-1 genomic RNAs. In addition to its encapsidation function, NC influences virus particle assembly (7, 10, 17, 21, 40). In particular, Gag proteins lacking the NC domain fail to assemble virus particles efficiently. Nevertheless, some chimeric Gag proteins which carry foreign sequences in place of NC have been shown to assemble and release virus particles at wild-type (wt) levels (2, 37, 40). Thus, it appears that in some circumstances, the role that NC plays in virus particle assembly can be replaced. To date, it is not clear how NC affects particle assembly, although several possibilities might be envisioned. One possibility is that deletion of NC unmasks inhibitory sequences in p2 or the C terminus of CA. Alternatively, NC may simply provide a stable monomeric folded structure which locks CA or other Gag domains into an assembly-competent conformation. Another possibility is that NC facilitates assembly by forming essential protein-protein contacts between neighbor Pr^gag molecules, as suggested in cross-linking studies (21). Finally, the assembly role of NC may stem from its RNA binding capabilities, a hypothesis supported by studies of Campbell and Vogt (5), which have shown that RNA facilitates the in vitro assembly of retroviral Gag proteins into higher-order structures.To distinguish among possible mechanisms by which NC facilitates HIV-1 assembly, we replaced NC with polypeptides having known structural characteristics and examined particle assembly directed by these chimeric proteins. Using this approach, we have found that NC does not play a passive role in HIV-1 assembly as either a mask to assembly inhibitor domains or a nonspecific, stably folded structure. Rather, sequences known to form strong interprotein contacts were observed to enhance assembly, suggesting a similar role for the NC domain itself. With several assembly-competent chimeric proteins, we detected no particle-associated RNAs. These results suggest that while RNA may be essential to virus assembly in the context of the wt Pr55^gag protein, it is dispensable for formation of virus-like particles from chimeric proteins.     

Infection of Glial Cells by the Human Polyomavirus JC Is Mediated by an N-Linked Glycoprotein Containing Terminal α(2-6)-Linked Sialic Acids

The human JC polyomavirus (JCV) is the etiologic agent of the fatal central nervous system (CNS) demyelinating disease progressive multifocal leukoencephalopathy (PML). PML typically occurs in immunosuppressed patients and is the direct result of JCV infection of oligodendrocytes. The initial event in infection of cells by JCV is attachment of the virus to receptors present on the surface of a susceptible cell. Our laboratory has been studying this critical event in the life cycle of JCV, and we have found that JCV binds to a limited number of cell surface receptors on human glial cells that are not shared by the related polyomavirus simian virus 40 (C. K. Liu, A. P. Hope, and W. J. Atwood, J. Neurovirol. 4:49–58, 1998). To further characterize specific JCV receptors on human glial cells, we tested specific neuraminidases, proteases, and phospholipases for the ability to inhibit JCV binding to and infection of glial cells. Several of the enzymes tested were capable of inhibiting virus binding to cells, but only neuraminidase was capable of inhibiting infection. The ability of neuraminidase to inhibit infection correlated with its ability to remove both α(2-3)- and α(2-6)-linked sialic acids from glial cells. A recombinant neuraminidase that specifically removes the α(2-3) linkage of sialic acid had no effect on virus binding or infection. A competition assay between virus and sialic acid-specific lectins that recognize either the α(2-3) or the α(2-6) linkage revealed that JCV preferentially interacts with α(2-6)-linked sialic acids on glial cells. Treatment of glial cells with tunicamycin, but not with benzyl N-acetyl-α-d-galactosaminide, inhibited infection by JCV, indicating that the sialylated JCV receptor is an N-linked glycoprotein. As sialic acid containing glycoproteins play a fundamental role in mediating many virus-cell and cell-cell recognition processes, it will be of interest to determine what role these receptors play in the pathogenesis of PML.Approximately 70% of the human population worldwide is seropositive for JC virus (JCV). Like other polyomaviruses, JCV establishes a lifelong latent or persistent infection in its natural host (40, 49, 50, 68, 72). Reactivation of JCV in the setting of an underlying immunosuppressive illness, such as AIDS, is thought to lead to virus dissemination to the central nervous system (CNS) and subsequent infection of oligodendrocytes (37, 40, 66, 68). Reactivation of latent JCV genomes already present in the CNS has also been postulated to contribute to the development of progressive multifocal leukoencephalopathy (PML) following immunosuppression (19, 48, 55, 70, 75). Approximately 4 to 6% of AIDS patients will develop PML during the course of their illness (10). In the CNS, JCV specifically infects oligodendrocytes and astrocytes. Outside the CNS, JCV genomes have been identified in the urogenital system, in the lymphoid system, and in B lymphocytes (2, 17, 18, 30, 47, 59). In vitro, JCV infects human glial cells and, to a limited extent, human B lymphocytes (3, 4, 39, 41, 42). Recently, JCV infection of tonsillar stromal cells and CD34⁺ B-cell precursors has been described (47). These observations have led to the suggestion that JCV may persist in a lymphoid compartment and that B cells may play a role in trafficking of JCV to the CNS (4, 30, 47).Virus-receptor interactions play a major role in determining virus tropism and tissue-specific pathology associated with virus infection. Viruses that have a very narrow host range and tissue tropism, such as JCV, are often shown to interact with high affinity to a limited number of specific receptors present on susceptible cells (26, 44). In some instances, virus tropism is strictly determined by the presence of specific receptors that mediate binding and entry (7, 16, 27, 35, 46, 53, 56, 67, 73, 74, 76). In other instances, however, successful entry into a cell is necessary but not sufficient for virus growth (5, 8, 45, 57). In these cases, additional permissive factors that interact with viral regulatory elements are required.The receptor binding characteristics of several polyomaviruses have been described. The mouse polyomavirus (PyV) receptor is an N-linked glycoprotein containing terminal α(2-3)-linked sialic acid (12–14, 22, 28). Both the large and small plaque strains of PyV recognize α(2-3)-linked sialic acid. The small-plaque strain also recognizes a branched disialyl structure containing α(2-3)- and α(2-6)-linked sialic acids. Neither strain recognizes straight-chain α(2-6)-linked sialic acid. The ability of the large- and small-plaque strains of PyV to differentially recognize these sialic acid structures has been precisely mapped to a single amino acid in the major virus capsid protein VP1 (21). The large-plaque strains all contain a glycine at amino acid position 92 in VP1, and the small-plaque strains all contain a negatively charged glutamic acid at this position (21). In addition to forming small or large plaques, these strains also differ in the ability to induce tumors in mice (20). This finding suggests that receptor recognition plays an important role in the pathogenesis of PyV.The cell surface receptor for lymphotropic papovavirus (LPV) is an O-linked glycoprotein containing terminal α(2-6)-linked sialic acid (26, 33, 34). Infection with LPV is restricted to a subset of human B-cell lines, and recognition of specific receptors is a major determinant of the tropism of LPV for these cells (26).Unlike the other members of the polyomavirus family, infection of cells by simian virus 40 (SV40) is independent of cell surface sialic acids. Instead, SV40 infection is mediated by major histocompatibility complex (MHC)-encoded class I proteins (5, 11). MHC class I proteins also play a role in mediating the association of SV40 with caveolae, a prerequisite for successful targeting of the SV40 genome to the nucleus of a cell (1, 63). Not surprisingly, SV40 has been shown not to compete with the sialic acid-dependent polyomaviruses for binding to host cells (15, 26, 38, 58).Very little is known about the early steps of JCV binding to and infection of glial cells. Like other members of the polyomavirus family, JCV is known to interact with cell surface sialic acids (51, 52). A role for sialic acids in mediating infection of glial cells has not been described. It is also not known whether the sialic acid is linked to a glycoprotein or a glycolipid. In a previous report, we demonstrated that JCV bound to a limited number of cell surface receptors on SVG cells that were not shared by the related polyomavirus SV40 (38). In this report, we demonstrate that virus binding to and infection of SVG cells is dependent on an N-linked glycoprotein containing terminal α(2-3)- and α(2-6)-linked sialic acids. Competitive binding assays with sialic acid-specific lectins suggest that the virus preferentially interacts with α(2-6)-linked sialic acids. We are currently evaluating the role of this receptor in determining the tropism of JCV for glial cells and B cells.     

Enhanced Virus Clearance by Early Inducible Lymphocytic Choriomeningitis Virus-Neutralizing Antibodies in Immunoglobulin-Transgenic Mice

Following infection of mice with lymphocytic choriomeningitis virus (LCMV), virus-neutralizing antibodies appear late, after 30 to 60 days. Such neutralizing antibodies play an important role in protection against reinfection. To analyze whether a neutralizing antibody response which developed earlier could contribute to LCMV clearance during the acute phase of infection, we generated transgenic mice expressing LCMV-neutralizing antibodies. Transgenic mice expressing the immunoglobulin μ heavy chain of the LCMV-neutralizing monoclonal antibody KL25 (H25 transgenic mice) mounted LCMV-neutralizing immunoglobulin M (IgM) serum titers within 8 days after infection. This early inducible LCMV-neutralizing antibody response significantly improved the host’s capacity to clear the infection and did not cause an enhancement of disease after intracerebral (i.c.) LCMV infection. In contrast, mice which had been passively administered LCMV-neutralizing antibodies and transgenic mice exhibiting spontaneous LCMV-neutralizing IgM serum titers (HL25 transgenic mice expressing the immunoglobulin μ heavy and the κ light chain) showed an enhancement of disease after i.c. LCMV infection. Thus, early-inducible LCMV-neutralizing antibodies can contribute to viral clearance in the acute phase of the infection and do not cause antibody-dependent enhancement of disease.Against many cytopathic viruses such as poliovirus, influenza virus, rabies virus, and vesicular stomatitis virus, protective virus-neutralizing antibodies are generated early, within 1 week after infection (3, 31, 36, 44, 49). In contrast, several noncytopathic viruses (e.g., human immunodeficiency virus and hepatitis viruses B and C in humans or lymphocytic choriomeningitis virus [LCMV] in mice) elicit poor and delayed virus-neutralizing antibody responses (1, 7, 20, 24, 27, 35, 45, 48).In the mouse, the natural host of LCMV, the acute LCMV infection is predominantly controlled by cytotoxic T lymphocytes (CTLs) in an obligatory perforin-dependent manner (13, 18, 28, 50). In addition to the CTL response, LCMV-specific antibodies are generated. Early after infection (by day 8), a strong antibody response specific for the internal viral nucleoprotein (NP) is mounted (7, 19, 23, 28). These early LCMV NP-specific antibodies exhibit no virus-neutralizing capacity (7, 10). Results from studies of B-cell-depleted mice and B-cell-deficient mice implied that the early LCMV NP-specific antibodies are not involved in the clearance of LCMV (8, 11, 12, 40). Late after infection (between days 30 and day 60), LCMV-neutralizing antibodies develop (7, 19, 22, 28, 33); these antibodies are directed against the surface glycoprotein (GP) of LCMV (9, 10). LCMV-neutralizing antibodies have an important function in protection against reinfection (4, 6, 38, 41, 47).In some viral infections, subprotective virus-neutralizing antibody titers can enhance disease rather than promote host recovery (i.e., exhibit antibody-dependent enhancement of disease [ADE] [14, 15, 21, 46]). For example, neutralizing antibodies are involved in the resolution of a primary dengue virus infection and in the protection against reinfection. However, if subprotective neutralizing antibody titers are present at the time of reinfection, a severe form of the disease (dengue hemorrhagic fever/dengue shock syndrome [15, 21]), which might be caused by Fc receptor-mediated uptake of virus-antibody complexes leading to an enhanced infection of monocytes (15, 16, 25, 39), can develop. Similarly, an enhancement of disease after intracerebral (i.c.) LCMV infection was observed in mice which had been treated with virus-neutralizing antibodies before the virus challenge (6). ADE in LCMV-infected mice was either due to an enhanced infection of monocytes by Fc receptor-mediated uptake of antibody-virus complexes or due to CTL-mediated immunopathology caused by an imbalanced virus spread and CTL response.To analyze whether LCMV-neutralizing antibodies generated early after infection improve the host’s capacity to clear the virus or enhance immunopathological disease, immunoglobulin (Ig)-transgenic mice expressing LCMV-neutralizing IgM antibodies were generated. After LCMV infection of transgenic mice expressing the Ig heavy chain (H25 transgenic mice), LCMV-neutralizing serum antibodies were mounted within 8 days, which significantly improved the host’s capacity to eliminate LCMV. H25 transgenic mice did not show any signs of ADE after i.c. LCMV infection.Transgenic mice expressing the Ig heavy and light chains (HL25 transgenic mice) exhibited spontaneous LCMV-neutralizing serum antibodies and confirmed the protective role of preexisting LCMV-neutralizing antibodies, even though the neutralizing serum antibodies were of the IgM isotype. Similar to mice which had been treated with LCMV-neutralizing antibodies, HL25 transgenic mice developed an enhanced disease after i.c. LCMV infection, which indicated that ADE was due to an imbalance between virus spread and CTL response. Thus, the early-inducible LCMV-neutralizing antibody response significantly enhanced clearance of the acute infection without any risk of causing ADE.     

Multipattern Consensus Regions in Multiple Aligned Protein Sequences and Their Segmentation

Decomposing a biological sequence into its functional regions is an important prerequisite to understand the molecule. Using the multiple alignments of the sequences, we evaluate a segmentation based on the type of statistical variation pattern from each of the aligned sites. To describe such a more general pattern, we introduce multipattern consensus regions as segmented regions based on conserved as well as interdependent patterns. Thus the proposed consensus region considers patterns that are statistically significant and extends a local neighborhood. To show its relevance in protein sequence analysis, a cancer suppressor gene called p53 is examined. The results show significant associations between the detected regions and tendency of mutations, location on the 3D structure, and cancer hereditable factors that can be inferred from human twin studies.[1,2,3,4,5,6,7,8,9,10,11,12,13,14,15,16,17,18,19,20,21,22,23,24,25,26,27]