Host-Specific Replication of BK Virus DNA in Mouse Cell Extracts Is Independently Controlled by DNA Polymerase α-Primase and Inhibitory Activities

The activation of the human polyomavirus BK causes polyomavirus-associated nephropathy in immunocompromised humans. Studies of the virus have been restricted since the virus DNA replication is species specific. Cell-based and cell-free DNA replication systems, including the BK virus (BKV) monopolymerase DNA replication system using purified proteins, reproduce the species specificity (28). Therefore, the major host proteins comprising this assay, DNA polymerase α-primase (Pol-prim) and replication protein A (RPA), were intensively studied here. We demonstrate that Pol-prim plays a major role in the species specificity of BKV DNA replication. Both large subunits p180 and p68 of the enzyme complex have central functions in modulating the host specificity. Recently, an inhibitory activity of BKV DNA replication was described (C. Mahon, B. Liang, I. Tikhanovich, J. R. Abend, M. J. Imperiale, H. P. Nasheuer, and W. R. Folk, J. Virol. 83:5708-5717, 2009), but neither mouse Pol-prim nor mouse RPA diminishes cell-free BKV DNA replication. However, the inhibition of BKV DNA replication in mouse extracts depends on sequences flanking the core origin. In the presence of human Pol-prim, the inhibitory effect of mouse cell factors is abolished with plasmid DNAs containing the murine polyomavirus early promoter region, whereas the late enhancer region and the core origin are supplied from BKV. Thus, BKV replication is regulated by both Pol-prim, as a core origin species-specific factor, and inhibitory activities, as origin-flanking sequence-dependent factor(s).BK virus (BKV) is a human polyomavirus that was first isolated in the 1970s (15). Up to 90% of adults have serologic evidence of exposure to BKV, but in most humans the virus remains latent (25, 26). Almost all disease accompanied by BKV reactivation has been found in immunocompromised patients (22). In recent years, BKV has been associated with nephropathy (polyomavirus-associated nephropathy, or PVAN) in up to 10% of renal transplant patients. Once established, the disease results in allograft loss in 45 to 70% of the patients (18). Importantly, BKV preferentially replicates in human cells and less well in cells of other primates, and the virus is highly tumorigenic in rodents (21, 41, 44). This fact and the lack of sustainable viral replication in rodents or other convenient, experimental animal models have been an enormous setback to the study of PVAN.As with other members of the Polyomaviridae family, BKV virions are nonenveloped icosahedral particles with a diameter of 45 nm that contain a circular double-stranded DNA genome of 5.3 kb (1). In BKV and in other polyomaviruses, three genomic areas have been distinguished: (i) a noncoding control region including the origin of viral DNA replication, (ii) the early genes encoding large and small T antigens (TAgs), and (iii) the late genes which code for the capsid proteins VP-1, VP-2, and VP-3 and the agnoprotein (22).BKV DNA replication is similar to that of all other members of the Polyomaviridae family and requires only one viral protein, the multifunctional large TAg, whereas all other replication factors are supplied by the host (13, 14, 28, 39, 47). As the first step, TAg binds to the core origin, which contains the early palindrome, an AT-rich sequence, and the TAg binding site II, which consists of two pairs of G(G/A)GGC pentanucleotides. In the presence of ATP, TAg forms a double hexamer and partially melts the early palindrome (EP) and untwists the AT-rich sequence of the BKV core origin (5, 6, 14). Then the TAg double hexamers bidirectionally unwind the viral replication origin, which requires ATP hydrolysis. In the following process the two hexamers remain associated with each other, with the separated single-stranded DNA (ssDNA) threading through the hexameric channels (14). The viral core origin is sufficient to constitute a functional replication origin, but the presence of auxiliary domains increases its activity 5- to 100-fold in vivo (16, 30). After the viral TAg unwinds the core origin and its flanking sequences, replication protein A (RPA), the main eukaryotic ssDNA-binding protein, covers the resulting stretches of ssDNA, whereas topoisomerase I releases the resulting torsional stress and enhances initiation of DNA replication (5, 7, 43). Then, DNA polymerase α-primase (Pol-prim) is loaded onto this TAg-RPA-topoisomerase 1-DNA complex, yielding a functional initiation complex. In the following step, Pol-prim synthesizes short RNA primers at the origin, and these RNA primers are elongated by the DNA polymerase function of the enzyme complex (9, 35, 47). After a polymerase switch from Pol-prim to DNA polymerase δ (Pol δ) with the help of RPA, replication factor C (RFC), and proliferating cell nuclear antigen (PCNA), processive DNA synthesis is completed by Pol δ in association with PCNA, the sliding clamp, on the leading strand (38, 51, 54, 59). Lagging-strand synthesis is discontinuous, and multiple initiation events catalyzed by Pol-prim must take place. Again, after the elongation of the RNA primers by Pol-prim, DNA synthesis is switched to Pol δ, which then synthesizes the complete Okazaki fragments. The maturation of these Okazaki fragments requires the collaboration of RNase H, PCNA, flap endonuclease 1 (Fen-1), Pol δ, and DNA ligase I to establish a continuous strand also on the lagging strand (9, 19, 20, 51, 55).TAg functions in infected cells rely heavily on specific associations with host proteins; for example, TAg interacts with RPA, Pol-prim, and topoisomerase I to replicate viral DNA. Selective interactions with the host p180 and p48 subunits of Pol-prim were shown to be responsible for species-specific replication of simian virus 40 (SV40) and murine polyomavirus (mPyV) DNAs, respectively (8, 47, 50). The subunits of Pol-prim are highly conserved since 88, 80, 89, and 90% of the amino acids are identical between human and murine p180, p68, p58, and p48, respectively. Biochemical studies have shown that TAg interacts independently with all four subunits of Pol-prim (8, 12, 57). Moreover, the p180, p58, and p48 subunits of Pol-prim also physically bind to RPA (7, 11, 57). RPA and TAg binding sites in the Pol-prim complex are essential for SV40 DNA replication in vitro since the presence of an excess of these purified binding peptides diminishes viral DNA replication in vitro (52, 53). Interestingly, species specificity requires the viral origin of DNA replication, whereas physical protein-protein interactions of purified protein complexes are not host specific in the absence of viral origin DNA (29, 42).Consistent with other polyomaviruses, analyses of BKV TAg-dependent DNA replication recently revealed that BKV DNA cannot be replicated in murine cells and that cell extracts are able to mimic this behavior (28). Furthermore, a BKV DNA replication system with the purified human proteins Pol-prim, RPA, topoisomerase I, and BKV TAg was inhibited by murine extracts, whereas SV40 DNA replication was not. Further investigations revealed that the presence of inhibitory activities (IAs) in extracts from murine cells blocks BKV DNA replication at an early step of TAg-mediated unwinding of the BKV origin of replication. Detailed analyses using the BKV monopolymerase DNA replication system, which we report here, show that Pol-prim functions as a species-specific factor associated with core origin functions. In addition, we reveal that the inhibitory activities in murine extracts, which are associated with origin-flanking sequence-dependent factor(s), regulate BKV DNA replication in murine cell extracts in a Pol-prim-independent manner.     

Mutations Abrogating VP35 Interaction with Double-Stranded RNA Render Ebola Virus Avirulent in Guinea Pigs

Ebola virus (EBOV) protein VP35 is a double-stranded RNA (dsRNA) binding inhibitor of host interferon (IFN)-α/β responses that also functions as a viral polymerase cofactor. Recent structural studies identified key features, including a central basic patch, required for VP35 dsRNA binding activity. To address the functional significance of these VP35 structural features for EBOV replication and pathogenesis, two point mutations, K319A/R322A, that abrogate VP35 dsRNA binding activity and severely impair its suppression of IFN-α/β production were identified. Solution nuclear magnetic resonance (NMR) spectroscopy and X-ray crystallography reveal minimal structural perturbations in the K319A/R322A VP35 double mutant and suggest that loss of basic charge leads to altered function. Recombinant EBOVs encoding the mutant VP35 exhibit, relative to wild-type VP35 viruses, minimal growth attenuation in IFN-defective Vero cells but severe impairment in IFN-competent cells. In guinea pigs, the VP35 mutant virus revealed a complete loss of virulence. Strikingly, the VP35 mutant virus effectively immunized animals against subsequent wild-type EBOV challenge. These in vivo studies, using recombinant EBOV viruses, combined with the accompanying biochemical and structural analyses directly correlate VP35 dsRNA binding and IFN inhibition functions with viral pathogenesis. Moreover, these studies provide a framework for the development of antivirals targeting this critical EBOV virulence factor.Ebola viruses (EBOVs) are zoonotic, enveloped negative-strand RNA viruses belonging to the family Filoviridae which cause lethal viral hemorrhagic fever in humans and nonhuman primates (47). Currently, information regarding EBOV-encoded virulence determinants remains limited. This, coupled with our lack of understanding of biochemical and structural properties of virulence factors, limits efforts to develop novel prophylactic or therapeutic approaches toward these infections.It has been proposed that EBOV-encoded mechanisms to counter innate immune responses, particularly interferon (IFN) responses, are critical to EBOV pathogenesis (7). However, a role for viral immune evasion functions in the pathogenesis of lethal EBOV infection has yet to be demonstrated. Of the eight major EBOV gene products, two viral proteins have been demonstrated to counter host IFN responses. The VP35 protein is a viral polymerase cofactor and structural protein that also inhibits IFN-α/β production by preventing the activation of interferon regulatory factor (IRF)-3 and -7 (3, 4, 8, 24, 27, 34, 41). VP35 also inhibits the activation of PKR, an IFN-induced, double-stranded RNA (dsRNA)-activated kinase with antiviral activity, and inhibits RNA silencing (17, 20, 48). The VP24 protein is a minor structural protein implicated in virus assembly and regulation of viral RNA synthesis, and changes in VP24 coding sequences are also associated with adaptation of EBOVs to mice and guinea pigs (2, 13, 14, 27, 32, 37, 50, 52). Further, VP24 inhibits cellular responses to both IFN-α/β and IFN-γ by preventing the nuclear accumulation of tyrosine-phosphorylated STAT1 (44, 45). The functions of VP35 and VP24 proteins are manifested in EBOV-infected cells by the absence of IRF-3 activation, impaired production of IFN-α/β, and severely reduced expression of IFN-induced genes, even after treatment of infected cells with IFN-α (3, 19, 21, 22, 24, 25, 28).Previous studies proposed that VP35 basic residues 305, 309, and 312 are required for VP35 dsRNA binding activity (26). VP35 residues K309 and R312 were subsequently identified as critical for binding to dsRNA, and mutation of these residues impaired VP35 suppression of IFN-α/β production (8). In vivo, an EBOV engineered to carry a VP35 R312A point mutation exhibited reduced replication in mice (23). However, because the parental recombinant EBOV into which the mutation was built did not cause disease in these animals, the impact of the mutation on viral pathogenesis could not be fully evaluated. Further, the lack of available structural and biochemical data to explain how the R312A mutation affects VP35 function limited avenues for the therapeutic targeting of critical VP35 functions. Recent structural analyses of the VP35 carboxy-terminal interferon inhibitory domain (IID) suggested that additional residues from the central basic patch may contribute to VP35 dsRNA binding activity and IFN-antagonist function (30). However, a direct correlation between dsRNA and IFN inhibitory functions of VP35 with viral pathogenesis is currently lacking.In order to further define the molecular basis for VP35 dsRNA binding and IFN-antagonist function and to define the contribution of these functions to EBOV pathogenesis, an integrated molecular, structural, and virological approach was taken. The data presented below identify two VP35 carboxy-terminal basic amino acids, K319 and R322, as required for its dsRNA binding and IFN-antagonist functions. Interestingly, these residues are outside the region originally identified as being important for dsRNA binding and IFN inhibition (26). However, they lie within the central basic patch identified by prior structural studies (26, 30). Introduction of these mutations (VP35 with these mutations is designated KRA) into recombinant EBOV renders this otherwise fully lethal virus avirulent in guinea pigs. KRA-infected animals also develop EBOV-specific antibodies and become fully resistant to subsequent challenge with wild-type (WT) virus. Our data further reveal that the KRA EBOV is immunogenic and likely replicates to low levels early after infection in vivo. However, the mutant virus is subsequently cleared by host immune responses. These data demonstrate that the VP35 central basic patch is important not only for IFN-antagonist function but also for EBOV immune evasion and pathogenesis in vivo. High-resolution structural analysis, coupled with our in vitro and in vivo analyses of the recombinant Ebola viruses, provides the molecular basis for loss of function by the VP35 mutant and highlights the therapeutic potential of targeting the central basic patch with small-molecule inhibitors and for future vaccine development efforts.     

Conserved Motifs within Ebola and Marburg Virus VP40 Proteins Are Important for Stability,Localization, and Subsequent Budding of Virus-Like Particles

The filovirus VP40 protein is capable of budding from mammalian cells in the form of virus-like particles (VLPs) that are morphologically indistinguishable from infectious virions. Ebola virus VP40 (eVP40) contains well-characterized overlapping L domains, which play a key role in mediating efficient virus egress. L domains represent only one component required for efficient budding and, therefore, there is a need to identify and characterize additional domains important for VP40 function. We demonstrate here that the ₉₆LPLGVA₁₀₁ sequence of eVP40 and the corresponding ₈₄LPLGIM₈₉ sequence of Marburg virus VP40 (mVP40) are critical for efficient release of VP40 VLPs. Indeed, deletion of these motifs essentially abolished the ability of eVP40 and mVP40 to bud as VLPs. To address the mechanism by which the ₉₆LPLGVA₁₀₁ motif of eVP40 contributes to egress, a series of point mutations were introduced into this motif. These mutants were then compared to the eVP40 wild type in a VLP budding assay to assess budding competency. Confocal microscopy and gel filtration analyses were performed to assess their pattern of intracellular localization and ability to oligomerize, respectively. Our results show that mutations disrupting the ₉₆LPLGVA₁₀₁ motif resulted in both altered patterns of intracellular localization and self-assembly compared to wild-type controls. Interestingly, coexpression of either Ebola virus GP-WT or mVP40-WT with eVP40-ΔLPLGVA failed to rescue the budding defective eVP40-ΔLPLGVA mutant into VLPs; however, coexpression of eVP40-WT with mVP40-ΔLPLGIM successfully rescued budding of mVP40-ΔLPLGIM into VLPs at mVP40-WT levels. In sum, our findings implicate the LPLGVA and LPLGIM motifs of eVP40 and mVP40, respectively, as being important for VP40 structure/stability and budding.Ebola and Marburg viruses are members of the family Filoviridae. Filoviruses are filamentous, negative-sense, single-stranded RNA viruses that cause lethal hemorrhagic fevers in both humans and nonhuman primates (5). Filoviruses encode seven viral proteins including: NP (major nucleoprotein), VP35 (phosphoprotein), VP40 (matrix protein), GP (glycoprotein), VP30 (minor nucleoprotein), VP24 (secondary matrix protein), and L (RNA-dependent RNA polymerase) (2, 5, 10, 12, 45). Numerous studies have shown that expression of Ebola virus VP40 (eVP40) alone in mammalian cells leads to the production of virus-like particles (VLPs) with filamentous morphology which is indistinguishable from infectious Ebola virus particles (12, 17, 18, 25, 26, 27, 30, 31, 34, 49). Like many enveloped viruses such as rhabdovirus (11) and arenaviruses (44), Ebola virus encodes late-assembly or L domains, which are sequences required for the membrane fission event that separates viral and cellular membranes to release nascent virion particles (1, 5, 7, 10, 12, 18, 25, 27, 34). Thus far, four classes of L domains have been identified which were defined by their conserved amino acid core sequences: the Pro-Thr/Ser-Ala-Pro (PT/SAP) motif (25, 27), the Pro-Pro-x-Tyr (PPxY) motif (11, 12, 18, 19, 41, 53), the Tyr-x-x-Leu (YxxL) motif (3, 15, 27, 37), and the Phe-Pro-Ile-Val (FPIV) motif (39). Both PTAP and the PPxY motifs are essential for efficient particle release for eVP40 (25, 27, 48, 49), whereas mVP40 contains only a PPxY motif. L domains are believed to act as docking sites for the recruitment of cellular proteins involved in endocytic trafficking and multivesicular body biogenesis to facilitate virus-cell separation (8, 13, 14, 16, 28, 29, 33, 36, 43, 50, 51).In addition to L domains, oligomerization, and plasma-membrane localization of VP40 are two functions of the protein that are critical for efficient budding of VLPs and virions. Specific sequences involved in self-assembly and membrane localization have yet to be defined precisely. However, recent reports have attempted to identify regions of VP40 that are important for its overall function in assembly and budding. For example, the amino acid region ₂₁₂KLR₂₁₄ located at the C-terminal region was found to be important for efficient release of eVP40 VLPs, with Leu₂₁₃ being the most critical (30). Mutation of the ₂₁₂KLR₂₁₄ region resulted in altered patterns of cellular localization and oligomerization of eVP40 compared to those of the wild-type genotype (30). In addition, the proline at position 53 was also implicated as being essential for eVP40 VLP release and plasma-membrane localization (54).In a more recent study, a YPLGVG motif within the M protein of Nipah virus (NiV) was shown to be important for stability, membrane binding, and budding of NiV VLPs (35). Whether this NiV M motif represents a new class of L domain remains to be determined. However, it is clear that this YPLGVG motif of NiV M is important for budding, perhaps involving a novel mechanism (35). Our rationale for investigating the corresponding, conserved motifs present within the Ebola and Marburg virus VP40 proteins was based primarily on these findings with NiV. In addition, Ebola virus VP40 motif maps close to the hinge region separating the N- and C-terminal domains of VP40 (4). Thus, the ₉₆LPLGVA₁₀₁ motif of eVP40 is predicted to be important for the overall stability and function of VP40 during egress. Findings presented here indicate that disruption of these filovirus VP40 motifs results in a severe defect in VLP budding, due in part to impairment in overall VP40 structure, stability and/or intracellular localization.     

Divergent Evolution of Norovirus GII/4 by Genome Recombination from May 2006 to February 2009 in Japan

Norovirus GII/4 is a leading cause of acute viral gastroenteritis in humans. We examined here how the GII/4 virus evolves to generate and sustain new epidemics in humans, using 199 near-full-length GII/4 genome sequences and 11 genome segment clones from human stool specimens collected at 19 sites in Japan between May 2006 and February 2009. Phylogenetic studies demonstrated outbreaks of 7 monophyletic GII/4 subtypes, among which a single subtype, termed 2006b, had continually predominated. Phylogenetic-tree, bootscanning-plot, and informative-site analyses revealed that 4 of the 7 GII/4 subtypes were mosaics of recently prevalent GII/4 subtypes and 1 was made up of the GII/4 and GII/12 genotypes. Notably, single putative recombination breakpoints with the highest statistical significance were constantly located around the border of open reading frame 1 (ORF1) and ORF2 (P ≤ 0.000001), suggesting outgrowth of specific recombinant viruses in the outbreaks. The GII/4 subtypes had many unique amino acids at the time of their outbreaks, especially in the N-term, 3A-like, and capsid proteins. Unique amino acids in the capsids were preferentially positioned on the outer surface loops of the protruding P2 domain and more abundant in the dominant subtypes. These findings suggest that intersubtype genome recombination at the ORF1/2 boundary region is a common mechanism that realizes independent and concurrent changes on the virion surface and in viral replication proteins for the persistence of norovirus GII/4 in human populations.Norovirus (NoV) is a nonenveloped RNA virus that belongs to the family Caliciviridae and can cause acute gastroenteritis in humans. The NoV genome is a single-stranded, positive-sense, polyadenylated RNA that encodes three open reading frames, ORF1, ORF2, and ORF3 (68). ORF1 encodes a long polypeptide (∼200 kDa) that is cleaved in the cells by the viral proteinase (3C^pro) into six proteins (4). These proteins function in NoV replication in host cells (19). ORF2 encodes a viral capsid protein, VP1. The capsid gene evolved at a rate of 4.3 × 10⁻³ nucleotide substitutions/site/year (7), which is comparable to the substitution rates of the envelope and capsid genes of human immunodeficiency virus (30). The capsid protein of NoV consists of a shell (S) and two protruding (P) domains: P1 and P2 (47). The S domain is relatively conserved within the same genetic lineages of NoVs (38) and is responsible for the assembly of VP1 (6). The P1 subdomain is also relatively conserved (38) and has a role in enhancing the stability of virus particles (6). The P2 domain is positioned at the most exposed surface of the virus particle (47) and forms binding clefts for putative infection receptors, such as human histo-blood group antigens (HBGA) (8, 13, 14, 60). The P2 domain also contains epitopes for neutralizing antibodies (27, 33) and is consistently highly variable even within the same genetic lineage of NoVs (38). ORF3 encodes a VP2 protein that is suggested to be a minor structural component of virus particles (18) and to be responsible for the expression and stabilization of VP1 (5).Thus far, the NoVs found in nature are classified into five genogroups (GI to GV) and multiple genotypes on the basis of the phylogeny of capsid sequences (71). Among them, genogroup II genotype 4 (GII/4), which was present in humans in the mid-1970s (7), is now the leading cause of NoV-associated acute gastroenteritis in humans (54). The GII/4 is further subclassifiable into phylogenetically distinct subtypes (32, 38, 53). Notably, the emergence and spread of a new GII/4 subtype with multiple amino acid substitutions on the capsid surface are often associated with greater magnitudes of NoV epidemics (53, 54). In 2006 and 2007, a GII/4 subtype, termed 2006b, prevailed globally over preexisting GII/4 subtypes in association with increased numbers of nonbacterial acute gastroenteritis cases in many countries, including Japan (32, 38, 53). The 2006b subtype has multiple unique amino acid substitutions that occur most preferentially in the protruding subdomain of the capsid, the P2 subdomain (32, 38, 53). Together with information on human population immunity against NoV GII/4 subtypes (12, 32), it has been postulated that the accumulation of P2 mutations gives rise to antigenic drift and plays a key role in new epidemics of NoV GII/4 in humans (32, 38, 53).Genetic recombination is common in RNA viruses (67). In NoV, recombination was first suggested by the phylogenetic analysis of an NoV genome segment clone: a discordant branching order was noted with the trees of the 3D^pol and capsid coding regions (21). Subsequently, many studies have reported the phylogenetic discordance using sequences from various epidemic sites in different study periods (1, 10, 11, 16, 17, 22, 25, 40, 41, 44-46, 49, 51, 57, 63, 64, 66). These results suggest that genome recombination frequently occurs among distinct lineages of NoV variants in vivo. However, the studies were done primarily with direct sequencing data of the short genome portion, and information on the cloned genome segment or full-length genome sequences is very limited (21, 25). Therefore, we lack an overview of the structural and temporal dynamics of viral genomes during NoV epidemics, and it remains unclear whether NoV mosaicism plays a role in these events.To clarify these issues, we collected 199 near-full-length genome sequences of GII/4 from NoV outbreaks over three recent years in Japan, divided them into monophyletic subtypes, analyzed the temporal and geographical distribution of the subtypes, collected phylogenetic evidence for the viral genome mosaicism of the subtypes, identified putative recombination breakpoints in the genomes, and isolated mosaic genome segments from the stool specimens. We also performed computer-assisted sequence and structural analyses with the identified subtypes to address the relationship between the numbers of P2 domain mutations at the times of the outbreaks and the magnitudes of the epidemics. The obtained data suggest that intersubtype genome recombination at the ORF1/2 boundary region is common in the new GII/4 outbreaks and promotes the effective acquisition of mutation sets of heterogeneous capsid surface and viral replication proteins.     

Tryptophan Residues in the Portal Protein of Herpes Simplex Virus 1 Critical to the Interaction with Scaffold Proteins and Incorporation of the Portal into Capsids

Incorporation of the herpes simplex virus 1 (HSV-1) portal vertex into the capsid requires interaction with a 12-amino-acid hydrophobic domain within capsid scaffold proteins. The goal of this work was to identify domains and residues in the U_L6-encoded portal protein pU_L6 critical to the interaction with scaffold proteins. We show that whereas the wild-type portal and scaffold proteins readily coimmunoprecipitated with one another in the absence of other viral proteins, truncation beyond the first 18 or last 36 amino acids of the portal protein precluded this coimmunoprecipitation. The coimmunoprecipitation was also precluded by mutation of conserved tryptophan (W) residues to alanine (A) at positions 27, 90, 127, 163, 241, 262, 532, and 596 of U_L6. All of these W-to-A mutations precluded the rescue of a viral deletion mutant lacking U_L6, except W163A, which supported replication poorly, and W596A, which fully rescued replication. A recombinant virus bearing the W596A mutation replicated and packaged DNA normally, and scaffold proteins readily coimmunoprecipitated with portal protein from lysates of infected cells. Thus, viral functions compensated for the W596A mutation''s detrimental effects on the portal-scaffold interaction seen during transient expression of portal and scaffold proteins. In contrast, the W27A mutation precluded portal-scaffold interactions in infected cell lysates, reduced the solubility of pU_L6, decreased incorporation of the portal into capsids, and abrogated viral-DNA cleavage and packaging.Immature herpesvirus capsids or procapsids consist of two shells: an inner shell, or scaffold, and an outer shell that is roughly spherical and largely composed of the major capsid protein VP5 (24, 38).The capsid scaffold consists of a mixture of the U_L26.5 and U_L26 gene products, with the U_L26.5 gene product (pU_L26.5, ICP35, or VP22a) being the most abundant (1, 12, 20, 21, 32, 38). The U_L26.5 open reading frame shares its coding frame and C terminus with the U_L26 gene but initiates at codon 307 of U_L26 (17). The extreme C termini of both VP22a and the UL26-encoded protein (pU_L26) interact with the N terminus of VP5 (7, 14, 26, 40, 41). Capsid assembly likely initiates when the portal binds VP5/VP22a and/or VP5/pU_L26 complexes (22, 25). The addition of more of these complexes to growing capsid shells eventually produces a closed sphere bearing a single portal. pU_L26 within the scaffold contains a protease that cleaves itself between amino acids 247 and 248, separating pU_L26 into an N-terminal protease domain called VP24 and a C-terminal domain termed VP21 (4, 5, 8, 9, 28, 42). The protease also cleaves 25 amino acids from pU_L26 and VP22a to release VP5 (5, 8, 9). VP21 and VP22a are replaced with DNA when the DNA is packaged (12, 29).When capsids undergo maturation, the outer protein shell angularizes to become icosahedral (13). One fivefold-symmetrical vertex in the angularized outer capsid shell is biochemically distinct from the other 11 and is called the portal vertex because it serves as the channel through which DNA is inserted as it is packaged (23). In herpes simplex virus (HSV), the portal vertex is composed of 12 copies of the portal protein encoded by U_L6 (2, 23, 39). We and others have shown that interactions between scaffold and portal proteins are critical for incorporation of the portal into the capsid (15, 33, 44, 45). Twelve amino acids of scaffold proteins are sufficient to interact with the portal protein, and tyrosine and proline resides within this domain are critical for the interaction with scaffold proteins and incorporation of the portal into capsids (45).One goal of the current study was to map domains and residues within the U_L6-encoded portal protein that mediate interaction with scaffold proteins. We show that the portal-scaffold interaction requires all but the first 18 and last 36 amino acids of pU_L6, as well as several tryptophan residues positioned throughout the portal protein.     

A Single Coxsackievirus B2 Capsid Residue Controls Cytolysis and Apoptosis in Rhabdomyosarcoma Cells

Coxsackievirus B2 (CVB2), one of six human pathogens of the group B coxsackieviruses within the enterovirus genus of Picornaviridae, causes a wide spectrum of human diseases ranging from mild upper respiratory illnesses to myocarditis and meningitis. The CVB2 prototype strain Ohio-1 (CVB2O) was originally isolated from a patient with summer grippe in the 1950s. Later on, CVB2O was adapted to cytolytic replication in rhabdomyosarcoma (RD) cells. Here, we present analyses of the correlation between the adaptive mutations of this RD variant and the cytolytic infection in RD cells. Using reverse genetics, we identified a single amino acid change within the exposed region of the VP1 protein (glutamine to lysine at position 164) as the determinant for the acquired cytolytic trait. Moreover, this cytolytic virus induced apoptosis, including caspase activation and DNA degradation, in RD cells. These findings contribute to our understanding of the host cell adaptation process of CVB2O and provide a valuable tool for further studies of virus-host interactions.Virus infections depend on complex interactions between viral and cellular proteins. Consequently, the nature of these interactions has important implications for viral cell type specificity, tissue tropism, and pathogenesis. Group B coxsackieviruses (CVB1 to CVB6), members of the genus Enterovirus within the family of Picornaviridae, are human pathogens that cause a broad spectrum of diseases, ranging from mild upper respiratory illnesses to more severe infections of the central nervous system, heart, and pancreas (61). These viruses have also been associated with certain chronic muscle diseases and myocardial infarction (2, 3, 12, 13, 22).The positive single-stranded RNA genome (approximately 7,500 nucleotides in length) of CVBs is encapsidated within a small T=1, icosahedral shell (30 nm in diameter) comprised of repeating identical subunits made up of four structural proteins (VP1 to VP4). Parts of VP1, VP2, and VP3 are exposed on the outer surface of the capsid, whereas VP4 is positioned on the interior. The virion morphology is characterized by a star-shaped mesa at each 5-fold icosahedral symmetry axis, surrounded by a narrow depression referred to as the “canyon” (69). All six serotypes of CVB can use the coxsackie and adenovirus receptor (CAR) for cell attachment and entry (9, 55, 82). Some strains of CVB1, -3, and -5 also use decay accelerating factor ([DAF] CD55) for initial attachment to the host cell; however, binding to DAF alone is insufficient to permit entry into the cell (10, 54, 76).Picornaviruses are generally characterized by their cytolytic nature in cell culture. However, several in vivo and in vitro studies have shown that some picornaviruses, e.g., poliovirus, Theiler''s murine encephalomyelitis virus, foot-and-mouth disease virus, CVB3, CVB4, and CVB5, may also establish persistent, noncytolytic infections (4, 29, 35, 39, 62, 74). Recently, it has been shown that the diverse outcomes of picornaviral infections may depend on interactions between the virus and the apoptotic machinery of the infected cell (14, 30, 71). Several picornaviral proteins have been identified as inducers of an apoptotic response, including viral capsid proteins VP1, VP2, and VP3, as well as nonstructural proteins 2A and 3C (7, 20, 32, 33, 42, 50, 63). In addition, antiapoptotic activity has been assigned to the nonstructural proteins 2B and 3A (16, 59).Picornaviruses have the potential to adapt rapidly to new host environments. Virus features affecting adaptability include high mutation rates, short replication times, large populations, and frequent incidences of recombination (25-27, 53). Consequently, picornaviruses exist as genetically heterogenous populations, referred to as viral quasispecies (25, 26).Previously, the CVB2 prototype strain Ohio-1 (CVB2O) was adapted to cytolytic replication in rhabdomyosarcoma (RD) cells (66). Two amino acid changes were identified in the capsid-coding region, and one was identified in the 2C-coding region of the adapted virus. Further characterization of the virus-host interaction showed that the infection was not affected by anti-DAF antibodies, indicating the use of an alternative receptor.In this study, the amino acid substitutions associated with the adaptation of CVB2O to cytolytic infection of RD cells were evaluated. Site-directed mutagenesis studies showed that a single amino acid change in the VP1 capsid protein was responsible for the cytolytic RD phenotype. In addition, as indicated by caspase activation and DNA degradation, the apoptotic pathway was activated in RD cells infected by the cytolytic virus.     

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

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

Characterization of a Putative Ancestor of Coxsackievirus B5

Like other RNA viruses, coxsackievirus B5 (CVB5) exists as circulating heterogeneous populations of genetic variants. In this study, we present the reconstruction and characterization of a probable ancestral virion of CVB5. Phylogenetic analyses based on capsid protein-encoding regions (the VP1 gene of 41 clinical isolates and the entire P1 region of eight clinical isolates) of CVB5 revealed two major cocirculating lineages. Ancestral capsid sequences were inferred from sequences of these contemporary CVB5 isolates by using maximum likelihood methods. By using Bayesian phylodynamic analysis, the inferred VP1 ancestral sequence dated back to 1854 (1807 to 1898). In order to study the properties of the putative ancestral capsid, the entire ancestral P1 sequence was synthesized de novo and inserted into the replicative backbone of an infectious CVB5 cDNA clone. Characterization of the recombinant virus in cell culture showed that fully functional infectious virus particles were assembled and that these viruses displayed properties similar to those of modern isolates in terms of receptor preferences, plaque phenotypes, growth characteristics, and cell tropism. This is the first report describing the resurrection and characterization of a picornavirus with a putative ancestral capsid. Our approach, including a phylogenetics-based reconstruction of viral predecessors, could serve as a starting point for experimental studies of viral evolution and might also provide an alternative strategy for the development of vaccines.The group B coxsackieviruses (CVBs) (serotypes 1 to 6) were discovered in the 1950s in a search for new poliovirus-like viruses (33, 61). Infections caused by CVBs are often asymptomatic but may occasionally result in severe diseases of the heart, pancreas, and central nervous system (99). CVBs are small icosahedral RNA viruses belonging to the Human enterovirus B (HEV-B) species within the family Picornaviridae (89). In the positive single-stranded RNA genome, the capsid proteins VP1 to VP4 are encoded within the P1 region, whereas the nonstructural proteins required for virus replication are encoded within the P2 and P3 regions (4). The 30-nm capsid has an icosahedral symmetry and consists of 60 copies of each of the four structural proteins. The VP1, VP2, and VP3 proteins are surface exposed, whereas the VP4 protein lines the interior of the virus capsid (82). The coxsackievirus and adenovirus receptor (CAR), a cell adhesion molecule of the immunoglobulin superfamily, serves as the major cell surface attachment molecule for all six serotypes of CVB (5, 6, 39, 60, 98). Some strains of CVB1, CVB3 and CVB5 also interact with the decay-accelerating factor (DAF) (CD55), a member of the family of proteins that regulate the complement cascade. However, the attachment of CVBs to DAF alone does not permit the infection of cells (6, 7, 59, 85).Picornaviruses exist as genetically highly diverse populations within their hosts, referred to as quasispecies (20, 57). This genetic plasticity enables these viruses to adapt rapidly to new environments, but at the same time, it may compromise the structural integrity and enzymatic functionality of the virus. The selective constraints imposed on the picornavirus genome are reflected in the different regions used for different types of evolutionary studies. The highly conserved RNA-dependent RNA polymerase (3D^pol) gene is used to establish phylogenetic relationships between more-distantly related viruses (e.g., viruses belonging to different genera) (38), whereas the variable genomic sequence encoding the VP1 protein is used for the classification of serotypes (13, 14, 69, 71, 72).In 1963, Pauling and Zuckerkandl proposed that comparative analyses of contemporary protein sequences can be used to predict the sequences of their ancient predecessors (73). Experimental reconstruction of ancestral character states has been applied to evolutionary studies of several different proteins, e.g., galectins (49), G protein-coupled receptors (52), alcohol dehydrogenases (95), rhodopsins (15), ribonucleases (46, 88, 110), elongation factors (32), steroid receptors (10, 96, 97), and transposons (1, 45, 87). In the field of virology, reconstructed ancestral or consensus protein sequences have been used in attempts to develop vaccine candidates for human immunodeficiency virus type 1 (21, 51, 66, 81) but rarely to examine general phenotypic properties.In this study, a CVB5 virus with a probable ancestral virion (CVB5-P1anc) was constructed and characterized. We first analyzed in detail the evolutionary relationships between structural genes of modern CVB5 isolates and inferred a time scale for their evolutionary history. An ancestral virion sequence was subsequently inferred by using a maximum likelihood (ML) method. This sequence was then synthesized de novo, cloned into a replicative backbone of an infectious CVB5 cDNA clone, and transfected into HeLa cells. The hypothetical CVB5-P1anc assembled into functional virus particles that displayed phenotypic properties similar to those of contemporary clinical isolates. This is the first report describing the reconstruction and characterization of a fully functional picornavirus with a putative ancestral capsid.     

Vacuolar Protein Sorting Pathway Contributes to the Release of Marburg Virus

VP40, the major matrix protein of Marburg virus, is the main driving force for viral budding. Additionally, cellular factors are likely to play an important role in the release of progeny virus. In the present study, we characterized the influence of the vacuolar protein sorting (VPS) pathway on the release of virus-like particles (VLPs), which are induced by Marburg virus VP40. In the supernatants of HEK 293 cells expressing VP40, different populations of VLPs with either a vesicular or a filamentous morphology were detected. While the filaments were almost completely composed of VP40, the vesicular particles additionally contained considerable amounts of cellular proteins. In contrast to that in the vesicles, the VP40 in the filaments was regularly organized, probably inducing the elimination of cellular proteins from the released VLPs. Vesicular particles were observed in the supernatants of cells even in the absence of VP40. Mutation of the late-domain motif in VP40 resulted in reduced release of filamentous particles, and likewise, inhibition of the VPS pathway by expression of a dominant-negative (DN) form of VPS4 inhibited the release of filamentous particles. In contrast, the release of vesicular particles did not respond significantly to the expression of DN VPS4. Like the budding of VLPs, the budding of Marburg virus particles was partially inhibited by the expression of DN VPS4. While the release of VLPs from VP40-expressing cells is a valuable tool with which to investigate the budding of Marburg virus particles, it is important to separate filamentous VLPs from vesicular particles, which contain many cellular proteins and use a different budding mechanism.In recent years, virus-like particles (VLPs), which are formed upon recombinant expression of the viral matrix and/or surface glycoproteins, have been recognized as representing powerful tools for developing novel vaccines and investigating certain aspects of the viral replication cycle (24, 44, 59, 63). Matrix proteins from many enveloped RNA viruses, including retroviruses, rhabdoviruses, filoviruses, paramyxoviruses, orthomyxoviruses, and arenaviruses, are able to induce VLPs (10, 14, 18, 28-30, 48, 49, 52). Increasing evidence also indicates that budding activity, and thus the release of VLPs, is often influenced by a complex interplay with components of the endosomal sorting complexes required for transport (ESCRTs), which mainly constitute the vacuolar protein sorting (VPS) pathway (16, 38, 42, 54). ESCRTs trigger the formation and budding of vesicles into the lumina of multivesicular bodies (MVBs), and the constituents of the ESCRTs are recycled by the activity of VPS4, an AAA-type ATPase. Expression of dominant-negative (DN) VPS4 mutants, which lack the ability to bind or hydrolyze ATP, blocks recycling of the ESCRTs and induces the formation of enlarged endosomes lacking internal vesicle accumulation (2, 3, 7). The inward budding of vesicles into the MVBs is topologically similar to the budding of viruses, since the vesicles bud away from the cytosol and into the lumen (reviewed in references 1, 20, and 26). Therefore, it is not entirely surprising that viruses use the cellular ESCRT machinery to organize the budding of viral progeny. Interactions between viral matrix proteins and ESCRTs occur through tetrapeptide motifs, known as late domains, which were first identified in retroviruses. Known late domains consist of the amino acid sequence P(T/S)AP, PPxY, or YxxL, where “x” represents any amino acid (19, 25, 62). The P(T/S)AP motif, for example, mediates interaction with tumor susceptibility gene 101 (Tsg101) (16, 36, 57); the PPxY motif mediates binding to WW domains of Nedd4-like ubiquitin ligases (9, 22); and the YxxL motif mediates interaction with AIP1/Alix (35, 47, 58). Recently, a novel late-domain motif, FPIV, has been identified in paramyxoviruses (46), and it is thought that additional late-domain motifs remain to be discovered (for a review, see reference 5).Inhibition of the VPS pathway has been shown to inhibit the budding of various viruses that are released with the help of ESCRTs. However, the budding of viruses and VLPs depends on the activity of ESCRTs to different degrees. Downregulation of Tsg101, a member of the ESCRT-I complex, inhibited the release of VLPs mediated by lymphocytic choriomeningitis virus Z protein and Marburg virus (MARV) VP40 (42, 54) but did not substantially inhibit the release of Gag-induced VLPs of Moloney murine leukemia virus and Rous sarcoma virus or that of matrix protein-induced VLPs of rabies virus (16, 27, 38). Expression of DN VPS4 inhibited the release of VLPs induced by the Gag proteins of Rous sarcoma virus and Moloney murine leukemia virus (16, 38) as well as that of VLPs induced by Lassa virus Z protein (55) but had no effect on the budding of rabies virus and cytomegalovirus (13, 27). These data indicate that in spite of the presence of late-domain motifs, a block in the VPS pathway may not always be critical for the budding of VLPs. In addition, the lack of known late domains in many enveloped viruses raises the question of whether they use other entry points into the VPS pathway or whether they exploit entirely different mechanisms of budding (60). To date, knowledge of how viral matrix proteins engage cellular machineries, such as the VPS pathway, to induce viral budding at the plasma membrane is very limited (8).The matrix protein VP40 of MARV contains only one known late-domain motif, PPPY, and a recent study showed that mutation of this late domain inhibited the release of VP40-induced VLPs. In addition, depletion of Tsg101 reduced the release of VP40-induced VLPs, suggesting that ESCRT-I is involved in this process (54). Whether a functional VPS pathway is important for the release of MARV VP40-induced VLPs or MARV particles remains unknown.VLPs induced by many viral matrix proteins have a morphology similar to that of cellular vesicles, which makes it difficult to separate the spherical VLPs from released cellular vesicles (4, 17, 53). In contrast, VLPs induced by the filovirus matrix protein VP40 are elongated and similar in morphology to viral particles (30, 49). Nevertheless, we observed that the supernatants of cells expressing VP40 contained various populations of particles with different morphologies. This raised the questions of whether the different particles are released by the same mechanism, whether they are all induced by VP40, and whether they are dependent on the same cellular pathways.The aim of the present study was to analyze the populations of particles released from cells expressing the MARV matrix protein VP40 and to gain further insights into the interaction between MARV and the cellular machinery involved in the budding of VLPs and MARV particles.We found that cells expressing VP40 released vesicular and filamentous particles, which could be separated by gradient centrifugation. Fractions with mainly vesicular particles represented a mixture of vesicles containing exclusively cellular proteins and vesicles also containing VP40 and few short filamentous particles. Longer filamentous particles, whose morphology resembled that of MARV particles but which displayed a much higher variability in length (400 nm to 5 μm), were found in denser gradient fractions. Filamentous VP40-induced VLPs were able to sort out cellular proteins efficiently. Release of VP40-induced filamentous VLPs was supported by the late-domain motif present in VP40, and inhibition of the cellular ESCRT machinery reduced the amount of these VLPs in the supernatant. Interestingly, the release of VLPs induced by a mutant of VP40 lacking the late domain was also reduced by inhibition of the cellular ESCRT machinery. Expression of a DN mutant of VPS4 diminished the budding of infectious MARV particles by 50%, a finding consistent with the idea that the activity of the ESCRT machinery supports viral budding but is not essential.     

Effects of Major Capsid Proteins,Capsid Assembly,and DNA Cleavage/Packaging on the pUL17/pUL25 Complex of Herpes Simplex Virus 1

The U_L17 and U_L25 proteins (pU_L17 and pU_L25, respectively) of herpes simplex virus 1 are located at the external surface of capsids and are essential for DNA packaging and DNA retention in the capsid, respectively. The current studies were undertaken to determine whether DNA packaging or capsid assembly affected the pU_L17/pU_L25 interaction. We found that pU_L17 and pU_L25 coimmunoprecipitated from cells infected with wild-type virus, whereas the major capsid protein VP5 (encoded by the U_L19 gene) did not coimmunoprecipitate with these proteins under stringent conditions. In addition, pU_L17 (i) coimmunoprecipitated with pU_L25 in the absence of other viral proteins, (ii) coimmunoprecipitated with pU_L25 from lysates of infected cells in the presence or absence of VP5, (iii) did not coimmunoprecipitate efficiently with pU_L25 in the absence of the triplex protein VP23 (encoded by the U_L18 gene), (iv) required pU_L25 for proper solubilization and localization within the viral replication compartment, (v) was essential for the sole nuclear localization of pU_L25, and (vi) required capsid proteins VP5 and VP23 for nuclear localization and normal levels of immunoreactivity in an indirect immunofluorescence assay. Proper localization of pU_L25 in infected cell nuclei required pU_L17, pU_L32, and the major capsid proteins VP5 and VP23, but not the DNA packaging protein pU_L15. The data suggest that VP23 or triplexes augment the pU_L17/pU_L25 interaction and that VP23 and VP5 induce conformational changes in pU_L17 and pU_L25, exposing epitopes that are otherwise partially masked in infected cells. These conformational changes can occur in the absence of DNA packaging. The data indicate that the pU_L17/pU_L25 complex requires multiple viral proteins and functions for proper localization and biochemical behavior in the infected cell.Immature herpes simplex virus (HSV) capsids, like those of all herpesviruses, consist of two protein shells. The outer shell comprises 150 hexons, each composed of six copies of VP5, and 11 pentons, each containing five copies of VP5 (23, 29, 47). One vertex of fivefold symmetry is composed of 12 copies of the protein encoded by the U_L6 gene and serves as the portal through which DNA is inserted (22, 39). The pentons and hexons are linked together by 320 triplexes composed of two copies of the U_L18 gene product, VP23, and one copy of the U_L38 gene product, VP19C (23). Each triplex arrangement has two arms contacting neighboring VP5 subunits (47). The internal shell of the capsid consists primarily of more than 1,200 copies of the scaffold protein ICP35 (VP22a) and a smaller number of protease molecules encoded by the U_L26 open reading frame, which self-cleaves to form VP24 and VP21 derived from the amino and carboxyl termini, respectively (11, 12, 19, 25; reviewed in reference 31). The outer shell is virtually identical in the three capsid types found in HSV-infected cells, termed types A, B, and C (5, 6, 7, 29, 43, 48). It is believed that all three are derived from the immature procapsid (21, 38). Type C capsids contain DNA in place of the internal shell, type B capsids contain both shells, and type A capsids consist only of the outer shell (15, 16). Cleavage of viral DNA to produce type C capsids requires not only the portal protein, but all of the major capsid proteins and the products of the U_L15, U_L17, U_L28, U_L32, and U_L33 genes (2, 4, 10, 18, 26, 28, 35, 46). Only C capsids go on to become infectious virions (27).The outer capsid shell contains minor capsid proteins encoded by the U_L25 and U_L17 open reading frames (1, 17, 20). These proteins are located on the external surface of the viral capsid (24, 36, 44) and are believed to form a heterodimer arranged as a linear structure, termed the C capsid-specific complex (CCSC), located between pentons and hexons (41). This is consistent with the observation that levels of pU_L25 are increased in C capsids as opposed to in B capsids (30). On the other hand, other studies have indicated that at least some U_L17 and U_L25 proteins (pU_L17 and pU_L25, respectively) associate with all capsid types, and pU_L17 can associate with enveloped light particles, which lack capsid and capsid proteins but contain a number of viral tegument proteins (28, 36, 37). How the U_L17 and U_L25 proteins attach to capsids is not currently known, although the structure of the CCSC suggests extensive contact with triplexes (41). It is also unclear when pU_L17 and pU_L25 become incorporated into the capsid during the assembly pathway. Less pU_L25 associates with pU_L17(−) capsids, suggesting that the two proteins bind capsids either cooperatively or sequentially, although this could also be consequential to the fact that less pU_L25 associates with capsids lacking DNA (30, 36).Both pU_L25 and pU_L17 are necessary for proper nucleocapsid assembly, but their respective deletion generates different phenotypes. Deletion of pU_L17 precludes DNA packaging and induces capsid aggregation in the nuclei of infected cells, suggesting a critical early function (28, 34), whereas deletion of pU_L25 precludes correct cleavage or retention of full-length cleaved DNA within the capsid (8, 20, 32), thus suggesting a critical function later in the assembly pathway.The current studies were undertaken to determine how pU_L17 and pU_L25 associate with capsids by studying their interaction and localization in the presence and absence of other capsid proteins.