A reporter open reading frame (ORF) coding for a fusion of bacterial β-glucuronidase (GUS) with a proteinase domain (Pro) derived from tobacco etch potyvirus was utilized for tagging individual genes of beet yellows closterovirus (BYV). Insertion of this reporter ORF between the first and second codons of the BYV ORFs encoding the HSP70 homolog (HSP70h), a major capsid protein (CP), and a 20-kDa protein (p20) resulted in the expression of the processed GUS-Pro reporter from corresponding subgenomic RNAs. The high sensitivity of GUS assays permitted temporal analysis of reporter accumulation, revealing early expression from the HSP70h promoter, followed by the CP promoter and later the p20 promoter. The kinetics of transcription of the remaining BYV genes encoding a 64-kDa protein (p64), a minor capsid protein (CPm), and a 21-kDa protein (p21) were examined via Northern blot analysis. Taken together, the data indicated that the temporal regulation of BYV gene expression includes early (HSP70h, CPm, CP, and p21 promoters) and late (p64 and p20 promoters) phases. It was also demonstrated that the deletion of six viral genes that are nonessential for RNA amplification resulted in a dramatic increase in the level of transcription from one of the two remaining subgenomic promoters. Comparison with other positive-strand RNA viruses producing multiple subgenomic RNAs showed the uniqueness of the pattern of closterovirus transcriptional regulation.
The Hsp70 homolog (Hsp70h) of Beet yellows virus (BYV) functions in virion assembly and cell-to-cell movement and is autonomously targeted to plasmodesmata in association with the actomyosin motility system (A. I. Prokhnevsky, V. V. Peremyslov, and V. V. Dolja, J. Virol. 79:14421-14428, 2005). Myosins are a diverse category of molecular motors that possess a motor domain and a tail domain involved in cargo binding. Plants have two classes of myosins, VIII and XI, whose specific functions are poorly understood. We used dominant negative inhibition to identify myosins required for Hsp70h localization to plasmodesmata. Six full-length myosin cDNAs from the BYV host plant Nicotiana benthamiana were sequenced and shown to encode apparent orthologs of the Arabidopsis thaliana myosins VIII-1, VIII-2, VIII-B, XI-2, XI-F, and XI-K. We found that the ectopic expression of the tail domains of each of the class VIII, but not the class XI, myosins inhibited the plasmodesmatal localization of Hsp70h. In contrast, the overexpression of the motor domains or the entire molecules of the class VIII myosins did not affect Hsp70h targeting. Further mapping revealed that the minimal cargo-binding part of the myosin VIII tails was both essential and sufficient for the inhibition of the proper Hsp70h localization. Interestingly, plasmodesmatal localization of the Tobacco mosaic virus movement protein and Arabidopsis protein RGP2 was not affected by myosin VIII tail overexpression. Collectively, our data implicate class VIII myosins in protein delivery to plasmodesmata and suggest that more than one mechanism of such delivery exist in plants.
Members of the Closteroviridae and Potyviridae families of the plant positive-strand RNA viruses encode one or two papain-like leader proteinases. In addition to a C-terminal proteolytic domain, each of these proteinases possesses a nonproteolytic N-terminal domain. We compared functions of the several leader proteinases using a gene swapping approach. The leader proteinase (L-Pro) of Beet yellows virus (BYV; a closterovirus) was replaced with L1 or L2 proteinases of Citrus tristeza virus (CTV; another closterovirus), P-Pro proteinase of Lettuce infectious yellows virus (LIYV; a crinivirus), and HC-Pro proteinase of Tobacco etch virus (a potyvirus). Each foreign proteinase efficiently processed the chimeric BYV polyprotein in vitro. However, only L1 and P-Pro, not L2 and HC-Pro, were able to rescue the amplification of the chimeric BYV variants. The combined expression of L1 and L2 resulted in an increased RNA accumulation compared to that of the parental BYV. Remarkably, this L1-L2 chimera exhibited reduced invasiveness and inability to move from cell to cell. Similar analyses of the BYV hybrids, in which only the papain-like domain of L-Pro was replaced with those derived from L1, L2, P-Pro, and HC-Pro, also revealed functional specialization of these domains. In subcellular-localization experiments, distinct patterns were observed for the leader proteinases of BYV, CTV, and LIYV. Taken together, these results demonstrated that, in addition to a common proteolytic activity, the leader proteinases of closteroviruses possess specialized functions in virus RNA amplification, virus invasion, and cell-to-cell movement. The phylogenetic analysis suggested that functionally distinct L1 and L2 of CTV originated by a gene duplication event.
Systemic spread of viruses in plants involves local movement from cell to cell and long-distance transport through the vascular system. The cell-to-cell movement of the Beet yellows virus (BYV) is mediated by a movement protein that is an Hsp70 homolog (Hsp70h). This protein is required for the assembly of movement-competent virions that incorporate Hsp70h. By using the yeast two-hybrid system, in vitro coimmunoprecipitation, and in planta coexpression approaches, we show here that the Hsp70h interacts with a 20-kDa BYV protein (p20). We further demonstrate that p20 is associated with the virions presumably via binding to Hsp70h. Genetic and immunochemical analyses indicate that p20 is dispensable for assembly and cell-to-cell movement of BYV but is required for the long-distance transport of virus through the phloem. These results reveal a novel activity for the Hsp70h that provides a molecular link between the local and systemic spread of a plant virus by docking a long-distance transport factor to virions.
Criniviruses comprise one of the genera within the family Closteroviridae. Members in this family are restricted to the phloem and rely on whitefly vectors of the genera Bemisia and/or Trialeurodes for plant-to-plant transmission. All criniviruses have bipartite, positive-sense single-stranded RNA genomes, although there is an unconfirmed report of one having a tripartite genome. Lettuce infectious yellows virus (LIYV) is the type species of the genus, the best studied so far of the criniviruses and the first for which a reverse genetics system was developed. LIYV RNA 1 encodes for proteins predicted to be involved in replication, and alone is competent for replication in protoplasts. Replication results in accumulation of cytoplasmic vesiculated membranous structures which are characteristic of most studied members of the Closteroviridae. These membranous structures, often referred to as Beet yellows virus (BYV)-type vesicles, are likely sites of RNA replication. LIYV RNA 2 is replicated in trans when co-infecting cells with RNA 1, but is temporally delayed relative to RNA 1. Efficient RNA 2 replication also is dependent on the RNA 1-encoded RNA-binding protein, P34. No LIYV RNA 2-encoded proteins have been shown to affect RNA replication, but at least four, CP (major coat protein), CPm (minor coat protein), Hsp70h, and P59 are virion structural components and CPm is a determinant of whitefly transmissibility. Roles of other LIYV RNA 2-encoded proteins are largely as yet unknown, but P26 is a non-virion protein that accumulates in cells as characteristic plasmalemma deposits which in plants are localized within phloem parenchyma and companion cells over plasmodesmata connections to sieve elements. The two remaining crinivirus-conserved RNA 2-encoded proteins are P5 and P9. P5 is 39 amino acid protein and is encoded at the 5′ end of RNA 2 as ORF 1 and is part of the hallmark closterovirus gene array. The orthologous gene in BYV has been shown to play a role in cell-to-cell movement and indicated to be localized to the endoplasmic reticulum as a Type III integral membrane protein. The other small protein, P9, is encoded by ORF 4 overlaps with ORF 3 that encodes the structural protein, P59. P9 seems to be unique to viruses in the genus Crinivirus, as no similar protein has been detected in viruses of the other two genera of the Closteroviridae.
phloem-limited; plasmalemma deposit; whitefly vector; Crinivirus; quintuple gene block
A full-length cDNA clone of beet yellows closterovirus (BYV) was engineered and used to map functions involved in the replication of the viral RNA genome and subgenomic RNA formation. Among 10 open reading frames (ORFs) present in BYV, ORFs 1a and 1b suffice for RNA replication and transcription. The proteins encoded in these ORFs harbor putative methyltransferase, RNA helicase, and RNA polymerase domains common to Sindbis virus-like viruses and a large interdomain region that is unique to closteroviruses. The papain-like leader proteinase (L-Pro) encoded in the 5′-proximal region of ORF 1a was found to have a dual function in genome amplification. First, the autocatalytic cleavage between L-Pro and the remainder of the ORF 1a product was essential for replication of RNA. Second, an additional L-Pro function that was separable from proteolytic activity was required for efficient RNA accumulation. The deletion of a large, ∼5.6-kb, 3′-terminal region coding for a 6-kDa hydrophobic protein, an HSP70 homolog, a 64-kDa protein, minor and major capsid proteins, a 20-kDa protein, and a 21-kDa protein (p21) resulted in replication-competent RNA. However, examination of mutants with replacements of start codons in each of these seven 3′-terminal ORFs revealed that p21 functions as an enhancer of genome amplification. The intriguing analogies between the genome organization and replicational requirements of plant closteroviruses and animal coronavirus-like viruses are discussed.
Filamentous virions of Beet yellows virus contain a long body formed by a major capsid protein and a short tail that is assembled by a minor capsid protein (CPm), an Hsp70-homolog (Hsp70h), a 64-kDa protein (p64), and a 20-kDa protein (p20). Using mutation analysis and newly developed in planta assays, here we investigate the genetic requirements for the tail assembly. We show that the inactivation of CPm dramatically reduces incorporation of both Hsp70h and p64. Furthermore, inactivation of Hsp70h prevents incorporation of p64 into virions and vice versa. Hsp70h and p64 are each required for efficient incorporation of CPm. We also show that the tails possessing normal relative amounts of CPm, Hsp70h, and p64 can be formed in the absence of the major capsid protein and p20. Similar to the tails isolated from the wild type virions, these mutant tails encapsidate the ~700 nt-long, 5’-terminal segments of the viral RNA. Taken together, our results imply that CPm, Hsp70h and p64 act cooperatively to encapsidate a defined region of the closterovirus genome.
Virus assembly; helical virion; Closterovirus; Hsp70
In eukaryotic virus systems, infection leads to induction of membranous compartments in which replication occurs. Virus-encoded subunits of the replication complex mediate its interaction with membranes. As replication platforms, RNA viruses use the cytoplasmic surfaces of different membrane compartments, e.g., endoplasmic reticulum (ER), Golgi, endo/lysosomes, mitochondria, chloroplasts, and peroxisomes. Closterovirus infections are accompanied by formation of multivesicular complexes from cell membranes of ER or mitochondrial origin. So far the mechanisms for vesicles formation have been obscure. In the replication-associated 1a polyprotein of Beet yellows virus (BYV) and other closteroviruses, the region between the methyltransferase and helicase domains (1a central region (CR), 1a CR) is marginally conserved. Computer-assisted analysis predicts several putative membrane-binding domains in the BYV 1a CR. Transient expression of a hydrophobic segment (referred to here as CR-2) of the BYV 1a in Nicotiana benthamiana led to reorganization of the ER and formation of ~1-μm mobile globules. We propose that the CR-2 may be involved in the formation of multivesicular complexes in BYV-infected cells. This provides analogy with membrane-associated proteins mediating the build-up of “virus factories” in cells infected with diverse positive-strand RNA viruses (alpha-like viruses, picorna-like viruses, flaviviruses, and nidoviruses) and negative-strand RNA viruses (bunyaviruses).
RNA virus replication; membrane vesicles; virus replication factory; endoplasmic reticulum modification; intracellular traffic
The 66-kDa leader proteinase (L-Pro) of the Beet yellows virus (BYV) possesses a nonconserved N-terminal domain and a conserved, papain-like C-terminal domain. Previous work revealed that the N-terminal domain functions in RNA amplification, whereas the C-terminal domain is required for autoproteolysis. Alanine-scanning mutagenesis was applied to complete the functional analysis of L-Pro throughout the virus life cycle. This analysis indicated that the C-terminal domain of L-Pro, in addition to being required for proteolysis, also functions in RNA amplification and that these two functions are genetically separable. Examination of the role of L-Pro in BYV cell-to-cell movement revealed that none of the 20 examined replication-competent mutants was movement defective. In contrast, six of the L-Pro mutations affected the long-distance transport of BYV to various degrees, whereas three mutations completely abolished the transport. Because these mutations were located throughout the protein molecule, both domains of L-Pro function in virus transport. We conclude that in addition to previously identified functions of L-Pro, it also serves as the BYV long-distance transport factor.
The herpesviruses, like most other DNA viruses, replicate in the host cell nucleus. Subnuclear domains known as promyelocytic leukemia protein nuclear bodies (PML-NBs), or ND10 bodies, have been implicated in restricting early herpesviral gene expression. These viruses have evolved countermeasures to disperse PML-NBs, as shown in cells infected in vitro, but information about the fate of PML-NBs and their functions in herpesvirus infected cells in vivo is limited. Varicella-zoster virus (VZV) is an alphaherpesvirus with tropism for skin, lymphocytes and sensory ganglia, where it establishes latency. Here, we identify large PML-NBs that sequester newly assembled nucleocapsids (NC) in neurons and satellite cells of human dorsal root ganglia (DRG) and skin cells infected with VZV in vivo. Quantitative immuno-electron microscopy revealed that these distinctive nuclear bodies consisted of PML fibers forming spherical cages that enclosed mature and immature VZV NCs. Of six PML isoforms, only PML IV promoted the sequestration of NCs. PML IV significantly inhibited viral infection and interacted with the ORF23 capsid surface protein, which was identified as a target for PML-mediated NC sequestration. The unique PML IV C-terminal domain was required for both capsid entrapment and antiviral activity. Similar large PML-NBs, termed clastosomes, sequester aberrant polyglutamine (polyQ) proteins, such as Huntingtin (Htt), in several neurodegenerative disorders. We found that PML IV cages co-sequester HttQ72 and ORF23 protein in VZV infected cells. Our data show that PML cages contribute to the intrinsic antiviral defense by sensing and entrapping VZV nucleocapsids, thereby preventing their nuclear egress and inhibiting formation of infectious virus particles. The efficient sequestration of virion capsids in PML cages appears to be the outcome of a basic cytoprotective function of this distinctive category of PML-NBs in sensing and safely containing nuclear aggregates of aberrant proteins.
Many DNA viruses, including varicella-zoster virus (VZV), a herpesvirus that causes varicella (chickenpox) and zoster (shingles), replicate in the host cell nucleus. Here, we have identified an intrinsic antiviral mechanism that specifically targets newly assembled VZV capsids and contains these essential viral structures in a nuclear “safe house”. Using immuno-electron microscopy, PML (promyelocytic leukemia) protein fibers that formed filamentous spherical cages were shown to trap virion capsids very efficiently, preventing their transport out of the nucleus and inhibiting the formation of infectious virus particles. PML cages containing virion capsids were found in VZV-infected neurons and satellite cells in human sensory ganglia and in skin cells, which are major targets during VZV pathogenesis. Similar PML nuclear bodies that sequester abnormal proteins have been reported in neurodegenerative disorders, like Huntington's disease. We found that cages formed by PML isoform IV sequestered both the virion capsids of VZV, which is a neurotropic herpesvirus, and the mutant Huntington's disease protein. This work provides the first evidence that PML, which is abundant in mammalian cell nuclei, can function both to contain potentially damaging cellular protein aggregates and as an intrinsic host defense against a herpesvirus during nuclear virion assembly.
A cell lysate prepared from MA104 cells that had been infected with human rotavirus KUN strain (HRV-KUN) contained a 35-kilodalton protein capable of binding to MA104 cells. The binding of the 35-kilodalton protein was inhibited by a serotype 2-specific antiserum but not by antisera to other serotypes. Not only trypsin-treated, infectious HRV-KUN but also untreated, noninfectious virions effectively competed with the 35-kilodalton protein for the same cell surface binding sites. One monoclonal anti-VP7 (AH6) absorbed the 35-kilodalton protein from the HRV-KUN-infected cell lysate, whereas another monoclonal anti-VP7 (S2-2G10) inhibited the virions to compete with the 35-kilodalton protein for the cell surface binding sites. Both anti-VP7 (S2-2G10) and anti-VP3 (K-1532, K-376) monoclonal antibodies had the virus-neutralization activity, but only anti-VP7 inhibited virus adsorption. On the other hand, anti-VP3 monoclonal antibodies were capable of completely inhibiting the infection of preadsorbed HRV-KUN as long as virions were not yet internalized. Subsequent studies with [35S]methionine-labeled and purified HRV-KUN showed that not only trypsin-treated, infectious virions but also untreated, noninfectious virions were capable of efficient target cell binding and internalization. The internalization modes of these two HRV-KUN preparations were, however, quite different. Only the components of the inner capsid were internalized from trypsin-treated virions, whereas no such selective internalization was seen with untreated virions. Furthermore, anti-VP3 inhibited this selective internalization of the inner capsid from the infectious virions. From these results we conclude that VP7 is the HRV-KUN cell attachment protein and that adsorption of HRV-KUN via VP7 is independent of trypsin treatment, whereas the limited cleavage of VP3 by trypsin, which is essential for the development of HRV-KUN infectivity, is needed for the selective internalization of the inner capsid components, a process that is apparently essential for HRV-KUN infection.
Incorporation of the human immunodeficiency virus type 1 (HIV-1) Gag-Pol into virions is thought to be mediated by the N-terminal Gag domain via interaction with the Gag precursor. However, one recent study has demonstrated that the murine leukemia virus Pol can be incorporated into virions independently of Gag-Pol expression, implying a possible interaction between the Pol and Gag precursor. To test whether the HIV-1 Pol can be incorporated into virions on removal of the N-terminal Gag domain and to define sequences required for the incorporation of Gag-Pol into virions in more detail, a series of HIV Gag-Pol expression plasmids with various extensive deletions in the region upstream of the reverse transcriptase (RT) domain was constructed, and viral incorporation of the Gag-Pol deletion mutants was examined by cotransfecting 293T cells with a plasmid expressing Pr55gag. Analysis indicated that deletion of the N-terminal two-thirds of the gag coding region did not significantly affect the incorporation of Gag-Pol into virions. In contrast, Gag-Pol proteins with deletions covering the capsid (CA) major homology regions and the adjacent C-terminal CA regions were impaired with respect to assembly into virions. However, Gag-Pol with sequences deleted upstream of the protease, or of the RT domain but retaining 15 N-terminal gag codons, could still be rescued into virions at a level about 20% of the wild-type level. When assayed in a nonmyristylated Gag-Pol context, all of the Gag-Pol deletion mutants were incorporated into virions at a level comparable to their myristylated counterparts, suggesting that the incorporation of the Gag-Pol deletion mutants into virions is independent of the N-terminal myristylation signal.
Extensive studies of orthoretroviral capsids have shown that many regions of the CA protein play unique roles at different points in the virus life cycle. The N-terminal domain (NTD) flexible-loop (FL) region is one such example: exposed on the outer capsid surface, it has been implicated in Gag-mediated particle assembly, capsid maturation, and early replication events. We have now defined the contributions of charged residues in the FL region of the Rous sarcoma virus (RSV) CA to particle assembly. Effects of mutations on assembly were assessed in vivo and in vitro and analyzed in light of new RSV Gag lattice models. Virus replication was strongly dependent on the preservation of charge at a few critical positions in Gag-Gag interfaces. In particular, a cluster of charges at the beginning of FL contributes to an extensive electrostatic network that is important for robust Gag assembly and subsequent capsid maturation. Second-site suppressor analysis suggests that one of these charged residues, D87, has distal influence on interhexamer interactions involving helix α7. Overall, the tolerance of FL to most mutations is consistent with current models of Gag lattice structures. However, the results support the interpretation that virus evolution has achieved a charge distribution across the capsid surface that (i) permits the packing of NTD domains in the outer layer of the Gag shell, (ii) directs the maturational rearrangements of the NTDs that yield a functional core structure, and (iii) supports capsid function during the early stages of virus infection.
IMPORTANCE The production of infectious retrovirus particles is a complex process, a choreography of protein and nucleic acid that occurs in two distinct stages: formation and release from the cell of an immature particle followed by an extracellular maturation phase during which the virion proteins and nucleic acids undergo major rearrangements that activate the infectious potential of the virion. This study examines the contributions of charged amino acids on the surface of the Rous sarcoma virus capsid protein in the assembly of appropriately formed immature particles and the maturational transitions that create a functional virion. The results provide important biological evidence in support of recent structural models of the RSV immature virions and further suggest that immature particle assembly and virion maturation are controlled by an extensive network of electrostatic interactions and long-range communication across the capsid surface.
During herpes simplex virus 1 (HSV1) egress in neurons, viral particles travel from the neuronal cell body along the axon towards the synapse. Whether HSV1 particles are transported as enveloped virions as proposed by the ‘married’ model or as non-enveloped capsids suggested by the ‘separate’ model is controversial. Specific viral proteins may form a recruitment platform for microtubule motors that catalyze such transport. However, their subviral location has remained elusive. Here we established a system to analyze herpesvirus egress by cryo electron tomography. At 16 h post infection, we observed intra-axonal transport of progeny HSV1 viral particles in dissociated hippocampal neurons by live-cell fluorescence microscopy. Cryo electron tomography of frozen-hydrated neurons revealed that most egressing capsids were transported independently of the viral envelope. Unexpectedly, we found not only DNA-containing capsids (cytosolic C-capsids), but also capsids lacking DNA (cytosolic A-/B-capsids) in mid-axon regions. Subvolume averaging revealed lower amounts of tegument on cytosolic A-/B-capsids than on C-capsids. Nevertheless, all capsid types underwent active axonal transport. Therefore, even few tegument proteins on the capsid vertices seemed to suffice for transport. Secondary envelopment of capsids was observed at axon terminals. On their luminal face, the enveloping vesicles were studded with typical glycoprotein-like spikes. Furthermore, we noted an accretion of tegument density at the concave cytosolic face of the vesicle membrane in close proximity to the capsids. Three-dimensional analysis revealed that these assembly sites lacked cytoskeletal elements, but that filamentous actin surrounded them and formed an assembly compartment. Our data support the ‘separate model’ for HSV1 egress, i.e. progeny herpes viruses being transported along axons as subassemblies and not as complete virions within transport vesicles.
Herpes simplex virus 1 (HSV1) establishes lifelong latent infections in the peripheral nervous system. After reactivation, progeny viral particles travel within sensory neurons towards sites of initial infection. There are conflicting reports what type of viral structures are transported: some studies observed non-enveloped capsids traveling while others reported transport of fully enveloped viruses within vesicles. Here, we used cryo electron tomography to analyze the three-dimensional architecture of HSV1 in axons of hippocampal neurons. In mid-axonal regions we found predominantly non-enveloped capsids. Interestingly, we observed both genome-containing and empty capsids that differed significantly in the amount of bound proteins. Viral protein recruitment thus varied between the different cytosolic capsid types, but effective transport occurred despite these differences. Furthermore, we observed three-dimensional snapshots of secondary capsid envelopment in axon terminals. Altogether, this study provides valuable structural detail on axonal HSV1 particles supporting the notion that viral subassemblies are conveyed along the axons to be assembled only after axonal transport.
Modeling how PRD1, a dsDNA membrane-containing virus, packages its genome using electron cryo-microscopy.
Two crucial steps in the virus life cycle are genome encapsidation to form an infective virion and genome exit to infect the next host cell. In most icosahedral double-stranded (ds) DNA viruses, the viral genome enters and exits the capsid through a unique vertex. Internal membrane-containing viruses possess additional complexity as the genome must be translocated through the viral membrane bilayer. Here, we report the structure of the genome packaging complex with a membrane conduit essential for viral genome encapsidation in the tailless icosahedral membrane-containing bacteriophage PRD1. We utilize single particle electron cryo-microscopy (cryo-EM) and symmetry-free image reconstruction to determine structures of PRD1 virion, procapsid, and packaging deficient mutant particles. At the unique vertex of PRD1, the packaging complex replaces the regular 5-fold structure and crosses the lipid bilayer. These structures reveal that the packaging ATPase P9 and the packaging efficiency factor P6 form a dodecameric portal complex external to the membrane moiety, surrounded by ten major capsid protein P3 trimers. The viral transmembrane density at the special vertex is assigned to be a hexamer of heterodimer of proteins P20 and P22. The hexamer functions as a membrane conduit for the DNA and as a nucleating site for the unique vertex assembly. Our structures show a conformational alteration in the lipid membrane after the P9 and P6 are recruited to the virion. The P8-genome complex is then packaged into the procapsid through the unique vertex while the genome terminal protein P8 functions as a valve that closes the channel once the genome is inside. Comparing mature virion, procapsid, and mutant particle structures led us to propose an assembly pathway for the genome packaging apparatus in the PRD1 virion.
The life cycle of a virus involves serial coordination of viral molecular machines. These machines facilitate functions such as membrane fusion and genome delivery during infection, and capsid formation and genome packaging during replication and shedding. Icosahedral dsDNA viruses use one genome-translocation machine for both genome delivery and packaging. The genome-translocation machine of the membrane-containing bacterial virus PRD1 is composed of four packaging protein species at a unique vertex. Because these proteins do not follow the dominating icosahedral symmetry of the viral capsid, the structure of this vertex has remained elusive. In this study, we localize the unique vertex in the virus from raw electron cryo-microscopy images of the virus. We show that the genome-packaging complex of PRD1 replaces the regular 5-fold structure at the unique vertex and contains a transmembrane conduit as a genome translocation channel. We extend our structural studies to the procapsid—a precursor of the virus—and three packaging mutant particles, allowing us to localize all individual protein species in the complex. Based on these structures, we propose a model of the molecular mechanism of assembly and packaging in the life cycle of the PRD1 virus.
Thermus thermophilus bacteriophage P23-77 is the type member of a new virus family of icosahedral, tailless, inner-membrane-containing double-stranded DNA (dsDNA) viruses infecting thermophilic bacteria and halophilic archaea. The viruses have a unique capsid architecture consisting of two major capsid proteins assembled in various building blocks. We analyzed the function of the minor capsid protein VP11, which is the third known capsid component in bacteriophage P23-77. Our findings show that VP11 is a dynamically elongated dimer with a predominantly α-helical secondary structure and high thermal stability. The high proportion of basic amino acids in the protein enables electrostatic interaction with negatively charged molecules, including nucleic acid and large unilamellar lipid vesicles (LUVs). The plausible biological function of VP11 is elucidated by demonstrating the interactions of VP11 with Thermus-derived LUVs and with the major capsid proteins by means of the dynamic-light-scattering technique. In particular, the major capsid protein VP17 was able to link VP11-complexed LUVs into larger particles, whereas the other P23-77 major capsid protein, VP16, was unable to link VP11-comlexed LUVs. Our results rule out a previously suggested penton function for VP11. Instead, the electrostatic membrane association of VP11 triggers the binding of the major capsid protein VP17, thus facilitating a controlled incorporation of the two different major protein species into the assembling capsid.
IMPORTANCE The study of thermophilic viruses with inner membranes provides valuable insights into the mechanisms used for stabilization and assembly of protein-lipid systems at high temperatures. Our results reveal a novel way by which an internal membrane and outer capsid shell are linked in a virus that uses two different major protein species for capsid assembly. We show that a positive protein charge is important in order to form electrostatic interactions with the lipid surface, thereby facilitating the incorporation of other capsid proteins on the membrane surface. This implies an alternative function for basic proteins present in the virions of other lipid-containing thermophilic viruses, whose proposed role in genome packaging is based on their capability to bind DNA. The unique minor capsid protein of bacteriophage P23-77 resembles in its characteristics the scaffolding proteins of tailed phages, though it constitutes a substantial part of the mature virion.
Human cytomegalovirus (HCMV) protein pUL48 is closely associated with the capsid and has a deubiquitinating protease (DUB) activity in its N-terminal region. Although this DUB activity moderately increases virus replication in cultured fibroblast cells, the requirements of the N-terminal region of pUL48 in the viral replication cycle are not fully understood. In this study, we characterized the recombinant viruses encoding UL48(ΔDUB/NLS), which lacks the DUB domain and the adjacent nuclear localization signal (NLS), UL48(ΔDUB), which lacks only the DUB, and UL48(Δ360–1200), which lacks the internal region (amino acids 360 to 1200) downstream of the DUB/NLS. While ΔDUB/NLS and Δ360–1200 mutant viruses did not grow in fibroblasts, the ΔDUB virus replicated to titers 100-fold lower than those for wild-type virus and showed substantially reduced viral gene expression at low multiplicities of infection. The DUB domain contained ubiquitination sites, and DUB activity reduced its own proteasomal degradation in trans. Deletion of the DUB domain did not affect the nuclear and cytoplasmic localization of pUL48, whereas the internal region (360–1200) was necessary for cytoplasmic distribution. In coimmunoprecipitation assays, pUL48 interacted with three tegument proteins (pUL47, pUL45, and pUL88) and two capsid proteins (pUL77 and pUL85) but the DUB domain contributed to only pUL85 binding. Furthermore, we found that the ΔDUB virus showed reduced virion stability and less efficiently delivered its genome into the cell than the wild-type virus. Collectively, our results demonstrate that the N-terminal DUB domain of pUL48 contributes to efficient viral growth by regulating its own stability and promoting virion stabilization and virus entry.
IMPORTANCE HCMV pUL48 and its herpesvirus homologs play key roles in virus entry, regulation of immune signaling pathways, and virion assembly. The N terminus of pUL48 contains the DUB domain, which is well conserved among all herpesviruses. Although studies using the active-site mutant viruses revealed that the DUB activity promotes viral growth, the exact role of this region in the viral life cycle is not fully understood. In this study, using the mutant virus lacking the entire DUB domain, we demonstrate that the DUB domain of pUL48 contributes to viral growth by regulating its own stability via autodeubiquitination and promoting virion stability and virus entry. This report is the first to demonstrate the characteristics of the mutant virus with the entire DUB domain deleted, which, along with information on the functions of this region, is useful in dissecting the functions associated with pUL48.
Retrovirus assembly is driven by polymerization of the Gag polyprotein as nascent virions bud from host cells. Gag is then processed proteolytically, releasing the capsid protein (CA) to assemble de novo inside maturing virions. CA has N-terminal and C-terminal domains (NTDs and CTDs, respectively) whose folds are conserved, although their sequences are divergent except in the 20-residue major homology region (MHR) in the CTD. The MHR is thought to play an important role in assembly, and some mutations affecting it, including the F167Y substitution, are lethal. A temperature-sensitive second-site suppressor mutation in the NTD, A38V, restores infectivity. We have used cryoelectron tomography to investigate the morphotypes of this double mutant. Virions produced at the nonpermissive temperature do not assemble capsids, although Gag is processed normally; moreover, they are more variable in size than the wild type and have fewer glycoprotein spikes. At the permissive temperature, virions are similar in size and spike content as in the wild type and capsid assembly is restored, albeit with altered polymorphisms. The mutation F167Y-A38V (referred to as FY/AV in this paper) produces fewer tubular capsids than wild type and more irregular polyhedra, which tend to be larger than in the wild type, containing ∼30% more CA subunits. It follows that FY/AV CA assembles more efficiently in situ than in the wild type and has a lower critical concentration, reflecting altered nucleation properties. However, its infectivity is lower than that of the wild type, due to a 4-fold-lower budding efficiency. We conclude that the wild-type CA protein sequence represents an evolutionary compromise between competing requirements for optimization of Gag assembly (of the immature virion) and CA assembly (in the maturing virion).
Interferon alpha (IFN-α) is an approved medication for chronic hepatitis B therapy. Besides acting as an immunomodulator, IFN-α elicits a pleiotropic antiviral state in hepatitis B virus (HBV)-infected hepatocytes, but whether or not IFN-α impedes the late steps of the HBV life cycle, such as HBV secretion, remains elusive. Here we report that IFN-α treatment of HepAD38 cells with established HBV replication selectively reduced HBV virion release without altering intracellular viral replication or the secretion of HBV subviral particles and nonenveloped capsids. In search of the interferon-stimulated gene(s) that is responsible for the reduction of HBV virion release, we found that tetherin, a broad-spectrum antiviral transmembrane protein that inhibits the egress of a variety of enveloped viruses, was highly induced by IFN-α in HepAD38 cells and in primary human hepatocytes. We further demonstrated that the expression of full-length tetherin, but not the C-terminal glycosylphosphatidylinositol (GPI) anchor-truncated form, inhibited HBV virion egress from HepAD38 cells. In addition, GPI anchor-truncated tetherin exhibited a dominant-negative effect and was incorporated into the liberated virions. We also found colocalization of tetherin and HBV L protein at the intracellular multivesicular body, where the budding of HBV virions takes place. In line with this, electron microscopy demonstrated that HBV virions were tethered in the lumen of the cisterna membrane under tetherin expression. Finally, knockdown of tetherin or overexpression of dominant negative tetherin attenuated the IFN-α-mediated reduction of HBV virion release. Taken together, our study suggests that IFN-α inhibits HBV virion egress from hepatocytes through the induction of tetherin.
IMPORTANCE Tetherin is a host restriction factor that blocks the egress of a variety of enveloped viruses through tethering the budding virions on the cell surface with its membrane anchor domains. Here we report that interferon directly and selectively inhibits the secretion of HBV virions, but not subviral particles or nonenveloped capsids, through the induction of tetherin in hepatocyte-derived cells. The antiviral function of tetherin requires the carboxyl-terminal GPI anchor, while the GPI anchor deletion mutant exhibits dominant negative activity and attaches to liberated HBV virions. Consistent with the fact that HBV is an intracellular budding virus, microscopy analyses demonstrated that the tethering of HBV virions occurs in the intracellular cisterna and that tetherin colocalizes with HBV virions on the multivesicular body, which is the HBV virion budding site. Our study not only expands the antiviral spectrum of tetherin but also sheds light on the mechanisms of interferon-elicited anti-HBV responses.
Papillomaviruses are a family of nonenveloped DNA viruses that infect the skin or mucosa of their vertebrate hosts. The viral life cycle is closely tied to the differentiation of infected keratinocytes. Papillomavirus virions are released into the environment through a process known as desquamation, in which keratinocytes lose structural integrity prior to being shed from the surface of the skin. During this process, virions are exposed to an increasingly oxidative environment, leading to their stabilization through the formation of disulfide cross-links between neighboring molecules of the major capsid protein, L1. We used time-lapse cryo-electron microscopy and image analysis to study the maturation of HPV16 capsids assembled in mammalian cells and exposed to an oxidizing environment after cell lysis. Initially, the virion is a loosely connected procapsid that, under in vitro conditions, condenses over several hours into the more familiar 60-nm-diameter papillomavirus capsid. In this process, the procapsid shrinks by ~5% in diameter, its pentameric capsomers change in structure (most markedly in the axial region), and the interaction surfaces between adjacent capsomers are consolidated. A C175S mutant that cannot achieve normal inter-L1 disulfide cross-links shows maturation-related shrinkage but does not achieve the fully condensed 60-nm form. Pseudoatomic modeling based on a 9-Å resolution reconstruction of fully mature capsids revealed C-terminal disulfide-stabilized “suspended bridges” that form intercapsomeric cross-links. The data suggest a model in which procapsids exist in a range of dynamic intermediates that can be locked into increasingly mature configurations by disulfide cross-linking, possibly through a Brownian ratchet mechanism.
Human papillomaviruses (HPVs) cause nearly all cases of cervical cancer, a major fraction of cancers of the penis, vagina/vulva, anus, and tonsils, and genital and nongenital warts. HPV types associated with a high risk of cancer, such as HPV16, are generally transmitted via sexual contact. The nonenveloped virion of HPVs shows a high degree of stability, allowing the virus to persist in an infectious form in environmental fomites. In this study, we used cryo-electron microscopy to elucidate the structure of the HPV16 capsid at different stages of maturation. The fully mature capsid adopts a rigid, highly regular structure stabilized by intermolecular disulfide bonds. The availability of a pseudoatomic model of the fully mature HPV16 virion should help guide understanding of antibody responses elicited by HPV capsid-based vaccines.
Herpesvirus nascent capsids, after assembly in the nucleus, must acquire a variety of tegument proteins during maturation. However, little is known about the identity of the tegument proteins that are associated with capsids in the nucleus or the molecular mechanisms involved in the nuclear egress of capsids into the cytoplasm, especially for the two human gammaherpesviruses Epstein-Barr virus (EBV) and Kaposi's sarcoma-associated herpesvirus (KSHV), due to a lack of efficient lytic replication systems. Murine gammaherpesvirus 68 (MHV-68) is genetically related to human gammaherpesviruses and serves as an excellent model to study the de novo lytic replication of gammaherpesviruses. We have previously shown that open reading frame 33 (ORF33) of MHV-68 is a tegument protein of mature virions and is essential for virion assembly and egress. However, it remains unclear how ORF33 is incorporated into virions. In this study, we first show that the endogenous ORF33 protein colocalizes with capsid proteins at discrete areas in the nucleus during viral infection. Cosedimentation analysis as well as an immunoprecipitation assay demonstrated that ORF33 is associated with both nuclear and cytoplasmic capsids. An immunogold labeling experiment using an anti-ORF33 monoclonal antibody revealed that ORF33-rich areas in the nucleus are surrounded by immature capsids. Moreover, ORF33 is associated with nucleocapsids prior to primary envelopment as well as with mature virions in the cytoplasm. Finally, we show that ORF33 interacts with two capsid proteins, suggesting that nucleocapsids may interact with ORF33 in a direct manner. In summary, we identified ORF33 to be a tegument protein that is associated with intranuclear capsids prior to primary envelopment, likely through interacting with capsid proteins in a direct manner.
IMPORTANCE Morphogenesis is an essential step in virus propagation that leads to the generation of progeny virions. For herpesviruses, this is a complicated process that starts in the nucleus. Although the process of capsid assembly and genome packaging is relatively well understood, how capsids acquire tegument (the layer between the capsid and the envelope in a herpesvirus virion) and whether the initial tegumentation process takes place in the nucleus remain unclear. We previously showed that ORF33 of MHV-68 is a tegument protein and functions in both the nuclear egress of capsids and final virion maturation in the cytoplasm. In the present study, we show that ORF33 is associated with intranuclear capsids prior to primary envelopment and identify novel interactions between ORF33 and two capsid proteins. Our work provides new insights into the association between tegument proteins and nucleocapsids at an early stage of the virion maturation process for herpesviruses.
Heat shock protein 70 (Hsp70) is incorporated within the membrane of primate lentiviral virions. Here we demonstrate that Hsp70 is also incorporated into oncoretroviral virions and that it remains associated with membrane-stripped human immunodeficiency virus type 1 (HIV-1) virion cores. To determine if Hsp70 promotes virion infectivity, we attempted to generate Hsp70-deficient virions with gag deletion mutations, Hsp70 transdominant mutants, or RNA interference, but these efforts were confounded, largely because they disrupt virion assembly. Given that polypeptide substrates are bound and released by Hsp70 in an ATP-hydrolytic reaction cycle, we supposed that incubation of HIV-1 virions with ATP would perturb Hsp70 interaction with substrates in the virion and thereby decrease infectivity. Treatment with ATP or ADP had no observable effect, but ATPγS and GTPγS, nucleotide triphosphate analogues resistant to Hsp70 hydrolysis, dramatically reduced the infectivity of HIV-1 and murine leukemia virus virions. ATPγS-treated virions were competent for fusion with susceptible target cells, but viral cDNA synthesis was inhibited to an extent that correlated with the magnitude of decrease in infectivity. Intravirion reverse transcription by HIV-1, simian immunodeficiency virus, or murine leukemia virus was also inhibited by ATPγS. The effects of ATPγS on HIV-1 reverse transcription appeared to be indirect, resulting from disruption of virion core morphology that was evident by transmission electron microscopy. Consistent with effects on capsid conformation, ATPγS-treated viruslike particles failed to saturate host antiviral restriction activity. Our observations support a model in which the catalytic activity of virion-associated Hsp70 is required to maintain structural integrity of the virion core.
Adeno-associated virus (AAV) is gaining momentum as a gene therapy vector for human applications. However, there remain impediments to the development of this virus as a vector. One of these is the incomplete understanding of the biology of the virus, including nuclear targeting of the incoming virion during initial infection, as well as assembly of progeny virions from structural components in the nucleus. Toward this end, we have identified four basic regions (BR) on the AAV2 capsid that represent possible nuclear localization sequence (NLS) motifs. Mutagenesis of BR1 (120QAKKRVL126) and BR2 (140PGKKRPV146) had minor effects on viral infectivity (∼4- and ∼10-fold, respectively), whereas BR3 (166PARKRLN172) and BR4 (307RPKRLN312) were found to be essential for infectivity and virion assembly, respectively. Mutagenesis of BR3, which is located in Vp1 and Vp2 capsid proteins, does not interfere with viral production or trafficking of intact AAV capsids to the nuclear periphery but does inhibit transfer of encapsidated DNA into the nucleus. Substitution of the canine parvovirus NLS rescued the BR3 mutant to wild-type (wt) levels, supporting the role of an AAV NLS motif. In addition, rAAV2 containing a mutant form of BR3 in Vp1 and a wt BR3 in Vp2 was found to be infectious, suggesting that the function of BR3 is redundant between Vp1 and Vp2 and that Vp2 may play a role in infectivity. Mutagenesis of BR4 was found to inhibit virion assembly in the nucleus of transfected cells. This affect was not completely due to the inefficient nuclear import of capsid subunits based on Western blot analysis. In fact, aberrant capsid foci were observed in the cytoplasm of transfected cells, compared to the wild type, suggesting a defect in early viral assembly or trafficking. Using three-dimensional structural analysis, the lysine- and arginine-to-asparagine change disrupts hydrogen bonding between these basic residues and adjacent beta strand glutamine residues that may prevent assembly of intact virions. Taken together, these data support that the BR4 domain is essential for virion assembly. Each BR was also found to be conserved in serotypes 1 to 11, suggesting that these regions are significant and function similarly in each serotype. This study establishes the importance of two BR motifs on the AAV2 capsid that are essential for infectivity and virion assembly.
The L1 major capsid protein of human papillomavirus type 11 (HPV-11) was expressed in Escherichia coli, and the soluble recombinant protein was purified to near homogeneity. The recombinant L1 protein bound DNA as determined by the Southwestern assay method, and recombinant mutant L1 proteins localized the DNA-binding domain to the carboxy-terminal 11 amino acids of L1. Trypsin digestion of the full-length L1 protein yielded a discrete 42-kDa product (trpL1), determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, resulting from cleavage at R415, 86 amino acids from the L1 carboxy terminus. Sucrose gradient sedimentation analysis demonstrated that trpL1 sedimented at 11S, while L1 proteins with amino-terminal deletions of 29 and 61 residues sedimented at 4S. Electron microscopy showed that the full-length L1 protein appeared as pentameric capsomeres which self-assembled into capsid-like particles. The trpL1 protein also had a pentameric morphology but was unable to assemble further. In an enzyme-linked immunosorbent assay, the trpL1 and L1 capsids reacted indistinguishably from virus-like particles purified after expression of HPV-11 L1 in insect cells. The carboxy terminus of L1 therefore constitutes the interpentamer linker arm responsible for HPV-11 capsid formation, much like the carboxy-terminal domain of the polyomavirus VP1 protein. The trypsin susceptibility of HPV-11 L1 capsids suggests a possible mechanism for virion disassembly.
Despite their high genomic diversity, all known viruses are structurally constrained to a limited number of virion morphotypes. One morphotype of viruses infecting bacteria, archaea, and eukaryotes is the tailless icosahedral morphotype with an internal membrane. Although it is considered an abundant morphotype in extreme environments, only seven such archaeal viruses are known. Here, we introduce Haloarcula californiae icosahedral virus 1 (HCIV-1), a halophilic euryarchaeal virus originating from salt crystals. HCIV-1 also retains its infectivity under low-salinity conditions, showing that it is able to adapt to environmental changes. The release of progeny virions resulting from cell lysis was evidenced by reduced cellular oxygen consumption, leakage of intracellular ATP, and binding of an indicator ion to ruptured cell membranes. The virion contains at least 12 different protein species, lipids selectively acquired from the host cell membrane, and a 31,314-bp-long linear double-stranded DNA (dsDNA). The overall genome organization and sequence show high similarity to the genomes of archaeal viruses in the Sphaerolipoviridae family. Phylogenetic analysis based on the major conserved components needed for virion assembly—the major capsid proteins and the packaging ATPase—placed HCIV-1 along with the alphasphaerolipoviruses in a distinct, well-supported clade. On the basis of its virion morphology and sequence similarities, most notably, those of its core virion components, we propose that HCIV-1 is a member of the PRD1-adenovirus structure-based lineage together with other sphaerolipoviruses. This addition to the lineage reinforces the notion of the ancient evolutionary links observed between the viruses and further highlights the limits of the choices found in nature for formation of a virion.
Under conditions of extreme salinity, the majority of the organisms present are archaea, which encounter substantial selective pressure, being constantly attacked by viruses. Regardless of the enormous viral sequence diversity, all known viruses can be clustered into a few structure-based viral lineages based on their core virion components. Our description of a new halophilic virus-host system adds significant insights into the largely unstudied field of archaeal viruses and, in general, of life under extreme conditions. Comprehensive molecular characterization of HCIV-1 shows that this icosahedral internal membrane-containing virus exhibits conserved elements responsible for virion organization. This places the virus neatly in the PRD1-adenovirus structure-based lineage. HCIV-1 further highlights the limited diversity of virus morphotypes despite the astronomical number of viruses in the biosphere. The observed high conservation in the core virion elements should be considered in addressing such fundamental issues as the origin and evolution of viruses and their interplay with their hosts.