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1.  Class VIII Myosins Are Required for Plasmodesmatal Localization of a Closterovirus Hsp70 Homolog▿  
Journal of Virology  2008;82(6):2836-2843.
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.
doi:10.1128/JVI.02246-07
PMCID: PMC2258991  PMID: 18199648
2.  The 64-Kilodalton Capsid Protein Homolog of Beet Yellows Virus Is Required for Assembly of Virion Tails 
Journal of Virology  2003;77(4):2377-2384.
The filamentous virion of the closterovirus Beet yellows virus (BYV) consists of a long body formed by the major capsid protein (CP) and a short tail composed of the minor capsid protein (CPm) and the virus-encoded Hsp70 homolog. By using nano-liquid chromatography-tandem mass spectrometry and biochemical analyses, we show here that the BYV 64-kDa protein (p64) is the fourth integral component of BYV virions. The N-terminal domain of p64 is exposed at the virion surface and is accessible to antibodies and mild trypsin digestion. In contrast, the C-terminal domain is embedded in the virion and is inaccessible to antibodies or trypsin. The C-terminal domain of p64 is shown to be homologous to CP and CPm. Mutation of the signature motifs of capsid proteins of filamentous RNA viruses in p64 results in the formation of tailless virions, which are unable to move from cell to cell. These results reveal the dual function of p64 in tail assembly and BYV motility and support the concept of the virion tail as a specialized device for BYV cell-to-cell movement.
doi:10.1128/JVI.77.4.2377-2384.2003
PMCID: PMC141117  PMID: 12551975
3.  Crinivirus replication and host interactions 
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.
doi:10.3389/fmicb.2013.00099
PMCID: PMC3657685  PMID: 23730299
phloem-limited; plasmalemma deposit; whitefly vector; Crinivirus; quintuple gene block
4.  Leader Proteinase of Beet Yellows Virus Functions in Long-Distance Transport 
Journal of Virology  2003;77(5):2843-2849.
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.
doi:10.1128/JVI.77.5.2843-2849.2003
PMCID: PMC149760  PMID: 12584307
5.  Functional Specialization and Evolution of Leader Proteinases in the Family Closteroviridae 
Journal of Virology  2001;75(24):12153-12160.
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.
doi:10.1128/JVI.75.24.12153-12160.2001
PMCID: PMC116111  PMID: 11711606
6.  Recruitment of the Host Plant Heat Shock Protein 70 by Tomato Yellow Leaf Curl Virus Coat Protein Is Required for Virus Infection 
PLoS ONE  2013;8(7):e70280.
A functional capsid protein (CP) is essential for host plant infection and insect transmission of Tomato yellow leaf curl virus (TYLCV) and other monopartite begomoviruses. We have previously shown that TYLCV CP specifically interacts with the heat shock protein 70 (HSP70) of the virus insect vector, Bemisia tabaci. Here we demonstrate that during the development of tomato plant infection with TYLCV, a significant amount of HSP70 shifts from a soluble form into insoluble aggregates. CP and HSP70 co-localize in these aggregates, first in the cytoplasm, then in the nucleus of cells associated with the vascular system. CP-HSP70 interaction was demonstrated by co-immunopreciptation in cytoplasmic - but not in nuclear extracts from leaf and stem. Inhibition of HSP70 expression by quercetin caused a decrease in the amount of nuclear CP aggregates and a re-localization of a GFP-CP fusion protein from the nucleus to the cytoplasm. HSP70 inactivation resulted in a decrease of TYLCV DNA levels, demonstrating the role of HSP70 in TYLCV multiplication in planta. The current study reveals for the first time the involvement of plant HSP70 in TYLCV CP intracellular movement. As described earlier, nuclear aggregates contained TYLCV DNA-CP complexes and infectious virions. Showing that HSP70 localizes in these large nuclear aggregates infers that these structures operate as nuclear virus factories.
doi:10.1371/journal.pone.0070280
PMCID: PMC3720902  PMID: 23894631
7.  Beet yellows virus replicase and replicative compartments: parallels with other RNA viruses 
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).
doi:10.3389/fmicb.2013.00038
PMCID: PMC3589766  PMID: 23508802
RNA virus replication; membrane vesicles; virus replication factory; endoplasmic reticulum modification; intracellular traffic
8.  Actin Cytoskeleton Is Involved in Targeting of a Viral Hsp70 Homolog to the Cell Periphery 
Journal of Virology  2005;79(22):14421-14428.
The cell-to-cell movement of plant viruses involves translocation of virus particles or nucleoproteins to and through the plasmodesmata (PDs). As we have shown previously, the movement of the Beet yellows virus requires the concerted action of five viral proteins including a homolog of cellular ∼70-kDa heat shock proteins (Hsp70h). Hsp70h is an integral component of the virus particles and is also found in PDs of the infected cells. Here we investigate subcellular distribution of Hsp70h using transient expression of Hsp70h fused to three spectrally distinct fluorescent proteins. We found that fluorophore-tagged Hsp70h forms motile granules that are associated with actin microfilaments, but not with microtubules. In addition, immobile granules were observed at the cell periphery. A pairwise appearance of these granules at the opposite sides of cell walls and their colocalization with the movement protein of Tobacco mosaic virus indicated an association of Hsp70h with PDs. Treatment with various cytoskeleton-specific drugs revealed that the intact actomyosin motility system is required for trafficking of Hsp70h in cytosol and its targeting to PDs. In contrast, none of the drugs interfered with the PD localization of Tobacco mosaic virus movement protein. Collectively, these findings suggest that Hsp70h is translocated and anchored to PDs in association with the actin cytoskeleton.
doi:10.1128/JVI.79.22.14421-14428.2005
PMCID: PMC1280222  PMID: 16254376
9.  An interdomain sector mediating allostery in Hsp70 molecular chaperones 
The Hsp70 family of molecular chaperones provides a well defined and experimentally powerful model system for understanding allosteric coupling between different protein domains.New extensions to the statistical coupling analysis (SCA) method permit identification of a group of co-evolving amino-acid positions—a sector—in the Hsp70 that is associated with allosteric function.Literature-based and new experimental studies support the notion that the protein sector identified through SCA underlies the allosteric mechanism of Hsp70.This work extends the concept of protein sectors by showing that two non-homologous protein domains can share a single sector when the underlying biological function is defined by the coupled activity of the two domains.
Allostery is a biologically critical property by which distantly positioned functional surfaces on proteins functionally interact. This property remains difficult to elucidate at a mechanistic level (Smock and Gierasch, 2009) because long-range coupling within proteins arises from the cooperative action of groups of amino acids. As a case study, consider the Hsp70 molecular chaperones, a large and diverse family of two-domain allosteric proteins required for cellular viability in nearly every organism (Figure 1) (Mayer and Bukau, 2005). In the ADP-bound state, the two domains act independently, the C-terminal substrate-binding domain displays a stable configuration in which the so-called ‘lid' region is docked against the β-sandwich subdomain, and substrates bind with relatively high affinity (Figure 1A) (Moro et al, 2003; Swain et al, 2007; Bertelsen et al, 2009). Exchange of ADP for ATP in the N-terminal nucleotide-binding domain causes significant local and propagated conformational change, formation of an interface with the substrate-binding domain, opening of the lid subdomain, and a decrease in the binding affinity for substrates (Figure 1B) (Rist et al, 2006; Swain et al, 2007). Upon ATP hydrolysis by the nucleotide-binding domain, Hsp70 is returned to the ADP-bound configuration suitable for another round of substrate binding and release. This process of cyclical substrate binding and release underlies all biological functions of Hsp70 proteins.
What is the structural basis for the long-range functional coupling within Hsp70? When allostery is a conserved property of a protein family, one approach to this problem is to analyze the correlated evolution of amino acids in the family—the expected statistical signature of cooperative action of protein residues (Lockless and Ranganathan, 1999; Kass and Horovitz, 2002; Suel et al, 2003). Previous work using an implementation of this concept (the statistical coupling analysis or SCA) showed that proteins contain sparse networks of co-evolving amino acids termed ‘sectors' that link protein active sites with distinct functional surfaces through the protein core (Halabi et al, 2009). This architecture is consistent with known allosteric mechanisms in protein domains (Suel et al, 2003; Halabi et al, 2009).
However, the principle of co-evolution of protein residues need not be limited to the study of individual protein domains. Indeed, conserved allosteric coupling between two (or more) non-homologous domains implies the existence of shared sectors that span functional sites on different domains. Here, we test this concept by extending the SCA method to consider the allosteric mechanism acting between the two domains of the Hsp70 proteins. Hsp70-like proteins include not only the allosteric Hsp70s, but also the Hsp110s—homologs that contain both domains and are regarded as structural models for Hsp70s, but that do not exhibit allosteric coupling. In this study, we take advantage of the functional divergence between the Hsp70s and Hsp110s to reveal patterns of co-evolution between amino acids that are specifically associated with the allosteric mechanism.
To identify the allosteric sector in Hsp70, we used SCA to compute a weighted correlation matrix, C̃, that describes the co-evolution of every pair of amino-acids positions in a sequence alignment of 926 members of the Hsp70/110 family. We then applied a mathematical method known as singular value decomposition to simultaneously evaluate the pattern of divergence between sequences and the pattern of co-evolution between amino-acid positions. The basic idea is that if the pattern of sequence divergence is able to classify members of a protein family into distinct functional subgroups, then we can rigorously identify the group of co-evolving residues that correspond to the underlying mechanism. Figure 2A shows the principal axis of sequence variation in the Hsp70/110 family, showing a clear separation of the allosteric (Hsp70) and non-allosteric (Hsp110) members of this family. The corresponding axis of co-evolution between amino-acid positions reveals a subset of Hsp70/110 positions (∼20%, 115 residues out of 605 total) that underlie the divergence of Hsp70 and Hsp110 proteins (Figure 2B). These positions derive roughly equally from the nucleotide-binding domain (in blue, 56 positions) and the substrate-binding domain (in green, 59 positions) and are more conserved within the Hsp70 sub-family. These results define a protein sector that is predicted to underlie the allosteric mechanism of Hsp70.
What is the structural arrangement of the putative allosteric sector within the Hsp70 protein? Consistent with a function in allosteric coupling, the 115 sector residues form a physically contiguous network of atoms, linking the ATP-binding site on the nucleotide-binding domain to the substrate recognition site on the substrate-binding domain through the interdomain interface (Figure 2C). The physical connectivity is remarkable given that only ∼20% of overall Hsp70 residues is involved (Figure 2B). Thus, functionally coupled but non-homologous protein domains can share a single sector of co-evolving residues that connects their respective functional sites.
We compared the Hsp70 sector mapping with the large body of biochemical studies that have been carried out in this family. We find strong experimental support for the involvement of sector positions in the Hsp70 allosteric mechanism in several regions: (1) within the ATP-binding site, (2) at the interface linking the two domains, and (3) within the β-sandwich core of the substrate-binding domain. The sector analysis also makes predictions about the involvement of some previously untested residues; we show that mutations at two such sites in fact reduce the allosteric coupling within Hsp70 in vitro and fail to complement a DnaK knockout strain of E. coli in a stress-response assay. Taken together, we conclude that sector positions are associated with the allosteric mechanism of Hsp70.
This work also adds a new finding with regard to the concept of protein sectors. Previous work showed that multiple quasi-independent sectors, each of which contributes a different aspect of function, are possible within a single protein domain (Halabi et al, 2009). This work shows that a single sector can also span two different protein domains when biological function (here, nucleotide-dependent substrate binding) arises from their coupled action. This result emphasizes the point that sectors are units of functional selection and are not obviously related to traditional hierarchies of structural organization in proteins. An interesting possibility is that evolution of allostery between proteins might evolve through the joining of protein sectors, a conjecture that can be tested in future work.
Allosteric coupling between protein domains is fundamental to many cellular processes. For example, Hsp70 molecular chaperones use ATP binding by their actin-like N-terminal ATPase domain to control substrate interactions in their C-terminal substrate-binding domain, a reaction that is critical for protein folding in cells. Here, we generalize the statistical coupling analysis to simultaneously evaluate co-evolution between protein residues and functional divergence between sequences in protein sub-families. Applying this method in the Hsp70/110 protein family, we identify a sparse but structurally contiguous group of co-evolving residues called a ‘sector', which is an attribute of the allosteric Hsp70 sub-family that links the functional sites of the two domains across a specific interdomain interface. Mutagenesis of Escherichia coli DnaK supports the conclusion that this interdomain sector underlies the allosteric coupling in this protein family. The identification of the Hsp70 sector provides a basis for further experiments to understand the mechanism of allostery and introduces the idea that cooperativity between interacting proteins or protein domains can be mediated by shared sectors.
doi:10.1038/msb.2010.65
PMCID: PMC2964120  PMID: 20865007
allostery; chaperone; co-evolution; SCA; sector
10.  Virion Tails of Beet Yellows Virus: Coordinated Assembly by Three Structural Proteins 
Virology  2006;359(1):220-226.
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.
doi:10.1016/j.virol.2006.09.007
PMCID: PMC1847569  PMID: 17027895
Virus assembly; helical virion; Closterovirus; Hsp70
11.  Genes Required for Replication of the 15.5-Kilobase RNA Genome of a Plant Closterovirus 
Journal of Virology  1998;72(7):5870-5876.
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.
PMCID: PMC110390  PMID: 9621048
12.  Regulation of Closterovirus Gene Expression Examined by Insertion of a Self-Processing Reporter and by Northern Hybridization 
Journal of Virology  1999;73(10):7988-7993.
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.
PMCID: PMC112813  PMID: 10482546
13.  Capsid Protein-Mediated Recruitment of Host DnaJ-Like Proteins Is Required for Potato Virus Y Infection in Tobacco Plants▿  
Journal of Virology  2007;81(21):11870-11880.
The capsid protein (CP) of potyviruses is required for various steps during plant infection, such as virion assembly, cell-to-cell movement, and long-distance transport. This suggests a series of compatible interactions with putative host factors which, however, are largely unknown. By using the yeast two-hybrid system the CP from Potato virus Y (PVY) was found to interact with a novel subset of DnaJ-like proteins from tobacco, designated NtCPIPs. Mutational analysis identified the CP core region, previously shown to be essential for virion formation and plasmodesmal trafficking, as the interacting domain. The ability of NtCPIP1 and NtCPIP2a to associate with PVY CP could be confirmed in vitro and was additionally verified in planta by bimolecular fluorescence complementation. The biological significance of the interaction was assayed by PVY infection of agroinfiltrated leaves and transgenic tobacco plants that expressed either full-length or J-domain-deficient variants of NtCPIPs. Transient expression of truncated dominant-interfering NtCPIP2a but not of the functional protein resulted in strongly reduced accumulation of PVY in the inoculated leaf. Consistently, stable overexpression of J-domain-deficient variants of NtCPIP1 and NtCPIP2a dramatically increased the virus resistance of various transgenic lines, indicating a critical role of functional NtCPIPs during PVY infection. The negative effect of impaired NtCPIP function on viral pathogenicity seemed to be the consequence of delayed cell-to-cell movement, as visualized by microprojectile bombardment with green fluorescent protein-tagged PVY. Therefore, we propose that NtCPIPs act as important susceptibility factors during PVY infection, possibly by recruiting heat shock protein 70 chaperones for viral assembly and/or cellular spread.
doi:10.1128/JVI.01525-07
PMCID: PMC2168797  PMID: 17715215
14.  N-terminal basic amino acid residues of Beet black scorch virus capsid protein play a critical role in virion assembly and systemic movement 
Virology Journal  2013;10:200.
Background
Beet black scorch virus (BBSV) is a small single-stranded, positive-sense RNA plant virus belonging to the genus Necrovirus, family Tombusviridae. Its capsid protein (CP) contains a 13 amino acid long basic region at the N-terminus, rich in arginine and lysine residues, which is thought to interact with viral RNA to initiate virion assembly.
Results
In the current study, a series of BBSV mutants containing amino acid substitutions as well as deletions within the N-terminal region were generated and examined for their effects on viral RNA replication, virion assembly, and long distance spread in protoplasts and whole host plants of BBSV. The RNA-binding activities of the mutated CPs were also evaluated in vitro. These experiments allowed us to identify two key basic amino acid residues in this region that are responsible for initiating virus assembly through RNA-binding. Proper assembly of BBSV particles is in turn needed for efficient viral systemic movement.
Conclusions
We have identified two basic amino acid residues near the N-terminus of the BBSV CP that bind viral RNA with high affinity to initiate virion assembly. We further provide evidence showing that systemic spread of BBSV in infected plants requires intact virions. This study represents the first in-depth investigation of the role of basic amino acid residues within the N-terminus of a necroviral CP.
doi:10.1186/1743-422X-10-200
PMCID: PMC3691604  PMID: 23786675
Beet black scorch virus; Capsid protein; RNA binding; Virion assembly; Viral systemic movement
15.  Comparative genomics and evolution of the HSP90 family of genes across all kingdoms of organisms 
BMC Genomics  2006;7:156.
Background
HSP90 proteins are essential molecular chaperones involved in signal transduction, cell cycle control, stress management, and folding, degradation, and transport of proteins. HSP90 proteins have been found in a variety of organisms suggesting that they are ancient and conserved. In this study we investigate the nuclear genomes of 32 species across all kingdoms of organisms, and all sequences available in GenBank, and address the diversity, evolution, gene structure, conservation and nomenclature of the HSP90 family of genes across all organisms.
Results
Twelve new genes and a new type HSP90C2 were identified. The chromosomal location, exon splicing, and prediction of whether they are functional copies were documented, as well as the amino acid length and molecular mass of their polypeptides. The conserved regions across all protein sequences, and signature sequences in each subfamily were determined, and a standardized nomenclature system for this gene family is presented. The proeukaryote HSP90 homologue, HTPG, exists in most Bacteria species but not in Archaea, and it evolved into three lineages (Groups A, B and C) via two gene duplication events. None of the organellar-localized HSP90s were derived from endosymbionts of early eukaryotes. Mitochondrial TRAP and endoplasmic reticulum HSP90B separately originated from the ancestors of HTPG Group A in Firmicutes-like organisms very early in the formation of the eukaryotic cell. TRAP is monophyletic and present in all Animalia and some Protista species, while HSP90B is paraphyletic and present in all eukaryotes with the exception of some Fungi species, which appear to have lost it. Both HSP90C (chloroplast HSP90C1 and location-undetermined SP90C2) and cytosolic HSP90A are monophyletic, and originated from HSP90B by independent gene duplications. HSP90C exists only in Plantae, and was duplicated into HSP90C1 and HSP90C2 isoforms in higher plants. HSP90A occurs across all eukaryotes, and duplicated into HSP90AA and HSP90AB in vertebrates. Diplomonadida was identified as the most basal organism in the eukaryote lineage.
Conclusion
The present study presents the first comparative genomic study and evolutionary analysis of the HSP90 family of genes across all kingdoms of organisms. HSP90 family members underwent multiple duplications and also subsequent losses during their evolution. This study established an overall framework of information for the family of genes, which may facilitate and stimulate the study of this gene family across all organisms.
doi:10.1186/1471-2164-7-156
PMCID: PMC1525184  PMID: 16780600
16.  The N-Terminal Region of Wheat Streak Mosaic Virus Coat Protein Is a Host- and Strain-Specific Long-Distance Transport Factor▿  
Journal of Virology  2010;85(4):1718-1731.
Understanding the genetics underlying host range differences among plant virus strains can provide valuable insights into viral gene functions and virus-host interactions. In this study, we examined viral determinants and mechanisms of differential infection of Zea mays inbred line SDp2 by Wheat streak mosaic virus (WSMV) isolates. WSMV isolates Sidney 81 (WSMV-S81) and Type (WSMV-T) share 98.7% polyprotein sequence identity but differentially infect SDp2: WSMV-S81 induces a systemic infection, but WSMV-T does not. Coinoculation and sequential inoculation of SDp2 with WSMV-T and/or WSMV-S81 did not affect systemic infection by WSMV-S81, suggesting that WSMV-T does not induce a restrictive defense response but that virus-encoded proteins may be involved in differential infection of SDp2. The viral determinant responsible for strain-specific host range was mapped to the N terminus of coat protein (CP) by systematic exchanges of WSMV-S81 sequences with those of WSMV-T and by reciprocal exchanges of CP or CP codons 1 to 74. Green fluorescent protein (GFP)-tagged WSMV-S81 with CP or CP residues 1 to 74 from WSMV-T produced similar numbers of infection foci and genomic RNAs and formed virions in inoculated leaves as those produced with WSMV-S81, indicating that failure to infect SDp2 systemically is not due to defects in replication, cell-to-cell movement, or virion assembly. However, these GFP-tagged hybrids showed profound defects in long-distance transport of virus through the phloem. Furthermore, we found that four of the five differing amino acids in the N terminus of CP between the WSMV-S81 and WSMV-T isolates were collectively involved in systemic infection of SDp2. Taken together, these results demonstrate that the N-terminal region of tritimoviral CP functions in host- and strain-specific long-distance movement.
doi:10.1128/JVI.02044-10
PMCID: PMC3028878  PMID: 21147925
17.  Short distance movement of genomic negative strands in a host and nonhost for Sugarcane mosaic virus (SCMV) 
Virology Journal  2011;8:15.
Background
In order to obtain an initial and preliminary understanding of host and nonhost resistance in the initial step of potyvirus replication, both positive and negative Sugarcane mosaic virus (SCMV) strands where traced in inoculated and systemic leaves in host and nonhost resistant maize and sugarcane for one Mexican potyviral isolate (SCMV-VER1). Intermediary replication forms, such as the negative viral strand, seem to only move a short distance as surveyed by RT-PCR analysis and ELISA in different leaves. Virus purification was also done in leaves and stems.
Results
Susceptible maize plants allowed for viral SCMV replication, cell-to-cell, and long distance movement, as indicated by the presence of the coat protein along the plant. In the host resistant maize plants for the SCMV-VER1 isolate, the virus was able to establish the disease though the initial steps of virus replication, as detected by the presence of negative strands, in the basal area of the inoculated leaves at six and twelve days post inoculation. The nonhost sugarcane for SCMV-VER1 and the host sugarcane for SCMV-CAM6 also allowed the initial steps of viral replication for the VER1 isolate in the local inoculated leaf. SCMV-VER1 virions could be extracted from stems of susceptible maize with higher titers than leaves.
Conclusion
Nonhost and host resistance allow the initial steps of potyvirus SCMV replication, as shown by the negative strands' presence. Furthermore, both hosts allow the negative viral strands' local movement, but not their systemic spread through the stem. The presence of larger amounts of extractable virions from the stem (as compared to the leaves) in susceptible maize lines suggests their long distance movement as assembled particles. This will be the first report suggesting the long distance movement of a monocot potyvirus as a virion.
doi:10.1186/1743-422X-8-15
PMCID: PMC3036626  PMID: 21232133
18.  Contribution of Host Intracellular Transport Machineries to Intercellular Movement of Turnip Mosaic Virus 
PLoS Pathogens  2013;9(10):e1003683.
The contribution of different host cell transport systems in the intercellular movement of turnip mosaic virus (TuMV) was investigated. To discriminate between primary infections and secondary infections associated with the virus intercellular movement, a gene cassette expressing GFP-HDEL was inserted adjacent to a TuMV infectious cassette expressing 6K2:mCherry, both within the T-DNA borders of the binary vector pCambia. In this system, both gene cassettes were delivered to the same cell by a single binary vector and primary infection foci emitted green and red fluorescence while secondarily infected cells emitted only red fluorescence. Intercellular movement was measured at 72 hours post infiltration and was estimated to proceed at an average rate of one cell being infected every three hours over an observation period of 17 hours. To determine if the secretory pathway were important for TuMV intercellular movement, chemical and protein inhibitors that blocked both early and late secretory pathways were used. Treatment with Brefeldin A or Concanamycin A or expression of ARF1 or RAB-E1d dominant negative mutants, all of which inhibit pre- or post-Golgi transport, reduced intercellular movement by the virus. These treatments, however, did not inhibit virus replication in primary infected cells. Pharmacological interference assays using Tyrphostin A23 or Wortmannin showed that endocytosis was not important for TuMV intercellular movement. Lack of co-localization by endocytosed FM4-64 and Ara7 (AtRabF2b) with TuMV-induced 6K2-tagged vesicles further supported this conclusion. Microfilament depolymerizing drugs and silencing expression of myosin XI-2 gene, but not myosin VIII genes, also inhibited TuMV intercellular movement. Expression of dominant negative myosin mutants confirmed the role played by myosin XI-2 as well as by myosin XI-K in TuMV intercellular movement. Using this dual gene cassette expression system and transport inhibitors, components of the secretory and actomyosin machinery were shown to be important for TuMV intercellular spread.
Author Summary
Plant viruses move from the initially infected cell to neighboring cells during local movement and then over long distances through vascular tissue to establish a systemic infection in the plant. Virus intercellular transport requires viral and host factors to move viral RNA-protein complexes through plasmodesmata (PDs). Virus intercellular movement is normally assessed by assays that cannot always differentiate between reduced viral RNA replication and intercellular movement. By using a dual cassette of genes encoding fluorescent proteins that can differentiate between primary infected cells and cells infected after intercellular transport, we provide evidence that turnip mosaic virus (TuMV) needs a functional secretory pathway where pre- and post-Golgi trafficking and the actomyosin network are important for its movement. Interestingly, disruption of these host transport machineries had no impact on TuMV accumulation in initially infected cells. These results support the idea that virus replication activities can be influenced separately from those involved in other virus activities such as movement, although aspects of both are likely coordinated.
doi:10.1371/journal.ppat.1003683
PMCID: PMC3789768  PMID: 24098128
19.  Association of heat shock protein 70 with enterovirus capsid precursor P1 in infected human cells. 
Journal of Virology  1992;66(3):1520-1527.
Members of the human heat shock (HSP) family of related proteins are involved in the intracellular folding, transport, and assembly of proteins and protein complexes. We have observed that human heat shock protein 70 (HSP70) is associated with the capsid precursor P1 of poliovirus and coxsackievirus B1 in infected HeLa cells. Antiserum generated against HSP70 coimmunoprecipitated the poliovirus protein P1, an intermediate in capsid assembly. Similarly, alpha-virion serum coimmunoprecipitated HSP70 from virus-infected cell extracts, but not from mock-infected cell extracts. The HSP70-P1 complex was stable in high-salt medium but was sensitive to incubation with 2 mM ATP, which is a characteristic of other known functional complexes between HSP70 and cellular proteins. The P1 in the complex was predominantly newly synthesized, and the half-life of complexed P1 was nearly twice as long as that of total P1. The HSP70-P1 complex was found to sediment at 3S to 6S, suggesting that it may be part of, or a precursor to, the "5S promoter particles" thought to be an assembly intermediate of picornaviruses. The finding that HSP70 was associated with the capsid precursors of at least two enteroviruses may suggest a functional role of these complexes in the viral life cycles.
Images
PMCID: PMC240877  PMID: 1310763
20.  FTIP1 Is an Essential Regulator Required for Florigen Transport 
PLoS Biology  2012;10(4):e1001313.
FT-INTERACTING PROTEIN 1 is a novel protein that is involved in transporting florigen, a long-known mobile signal that induces flowering in plants in response to day length, from companion cells to sieve elements in the phloem of Arabidopsis.
The capacity to respond to day length, photoperiodism, is crucial for flowering plants to adapt to seasonal change. The photoperiodic control of flowering in plants is mediated by a long-distance mobile floral stimulus called florigen that moves from leaves to the shoot apex. Although the proteins encoded by FLOWERING LOCUS T (FT) in Arabidopsis and its orthologs in other plants are identified as the long-sought florigen, whether their transport is a simple diffusion process or under regulation remains elusive. Here we show that an endoplasmic reticulum (ER) membrane protein, FT-INTERACTING PROTEIN 1 (FTIP1), is an essential regulator required for FT protein transport in Arabidopsis. Loss of function of FTIP1 exhibits late flowering under long days, which is partly due to the compromised FT movement to the shoot apex. FTIP1 and FT share similar mRNA expression patterns and subcellular localization, and they interact specifically in phloem companion cells. FTIP1 is required for FT export from companion cells to sieve elements, thus affecting FT transport through the phloem to the SAM. Our results provide a mechanistic understanding of florigen transport, demonstrating that FT moves in a regulated manner and that FTIP1 mediates FT transport to induce flowering.
Author Summary
The transition to flowering is the most dramatic phase change in flowering plants and is crucial for reproductive success. Such a transition from vegetative to reproductive growth is controlled by seasonal changes in day length. Studies originally performed in the 1930s were the first to suggest that day length is perceived by a plant's leaves; by contrast, flower formation takes place in the shoot apical meristem (the tip of the shoot that gives rise to plant organs, such as leaves and flowers). The term “florigen” was later proposed to describe a mobile floral stimulus that moves from leaves to the shoot apical meristem to induce flowering. It is only recently that FLOWERING LOCUS T (FT) in Arabidopsis, and its orthologs in various other plant species, was identified as being florigen, but how florigen is transported in plants remains completely unknown. Here, we report that a novel ER membrane protein, FT-INTERACTING PROTEIN 1 (FTIP1), interacts with FT in companion cells of the phloem (a specialized type of parenchyma cell in the phloem of the plant's vascular system) and mediates FT protein movement from companion cells to sieve elements (the conducting cells of the phloem), thus affecting FT transport to the shoot apical meristem in Arabidopsis. To our knowledge, this study reveals the first regulator that is required for florigen transport and offers new insights into possible florigen transport mechanisms in other flowering plants.
doi:10.1371/journal.pbio.1001313
PMCID: PMC3328448  PMID: 22529749
21.  Reverse Genetic Analysis of Ourmiaviruses Reveals the Nucleolar Localization of the Coat Protein in Nicotiana benthamiana and Unusual Requirements for Virion Formation ▿ † ‡  
Journal of Virology  2011;85(10):5091-5104.
Ourmia melon virus (OuMV) is the type member of the genus Ourmiavirus. These viruses have a trisegmented genome, each part of which encodes a single protein. Ourmiaviruses share a distant similarity with other plant viruses only in their movement proteins (MP), whereas their RNA-dependent RNA polymerase (RdRP) shares features only with fungal viruses of the family Narnaviridae. Thus, ourmiaviruses are in a unique phylogenetic position among existing plant viruses. Here, we developed an agroinoculation system to launch infection in Nicotiana benthamiana plants. Using different combinations of the three segments, we demonstrated that RNA1 is necessary and sufficient for cis-acting replication in the agroinfiltrated area. RNA2 and RNA3, encoding the putative movement protein and the coat protein (CP), respectively, are both necessary for successful systemic infection of N. benthamiana. The CP is dispensable for long-distance transport of the virus through vascular tissues, but its absence prevents efficient systemic infection at the exit sites. Virion formation occurred only when the CP was translated from replication-derived RNA3. Transient expression of a green fluorescent protein-MP (GFP-MP) fusion via agroinfiltration showed that the MP is present in cytoplasmic connections across plant cell walls; in protoplasts the GFP-MP fusion stimulates the formation of tubular protrusions. Expression through agroinfiltration of a GFP-CP fusion displays most of the fluorescence inside the nucleus and within the nucleolus in particular. Nuclear localization of the CP was also confirmed through Western blot analysis of purified nuclei. The significance of several unusual properties of OuMV for replication, virion assembly, and movement is discussed in relation to other positive-strand RNA viruses.
doi:10.1128/JVI.02565-10
PMCID: PMC3126195  PMID: 21411534
22.  Citrus tristeza virus-host interactions 
Citrus tristeza virus (CTV) is a phloem-limited virus whose natural host range is restricted to citrus and related species. Although the virus has killed millions of trees, almost destroying whole industries, and continually limits production in many citrus growing areas, most isolates are mild or symptomless in most of their host range. There is little understanding of how the virus causes severe disease in some citrus and none in others. Movement and distribution of CTV differs considerably from that of well-studied viruses of herbaceous plants where movement occurs largely through adjacent cells. In contrast, CTV systemically infects plants mainly by long-distance movement with only limited cell-to-cell movement. The virus is transported through sieve elements and occasionally enters an adjacent companion or phloem parenchyma cell where virus replication occurs. In some plants this is followed by cell-to-cell movement into only a small cluster of adjacent cells, while in others there is no cell-to-cell movement. Different proportions of cells adjacent to sieve elements become infected in different plant species. This appears to be related to how well viral gene products interact with specific hosts. CTV has three genes (p33, p18, and p13) that are not necessary for infection of most of its hosts, but are needed in different combinations for infection of certain citrus species. These genes apparently were acquired by the virus to extend its host range. Some specific viral gene products have been implicated in symptom induction. Remarkably, the deletion of these genes from the virus genome can induce large increases in stem pitting (SP) symptoms. The p23 gene, which is a suppressor of RNA silencing and a regulator of viral RNA synthesis, has been shown to be the cause of seedling yellows (SY) symptoms in sour orange. Most isolates of CTV in nature are populations of different strains of CTV. The next frontier of CTV biology is the understanding how the virus variants in those mixtures interact with each other and cause diseases.
doi:10.3389/fmicb.2013.00088
PMCID: PMC3653117  PMID: 23717303
Citrus tristeza virus; citrus; disease; host-interactions; stem pitting; seedling yellows
23.  Hsp90/Cdc37 Chaperone/co-chaperone complex, a novel junction anticancer target elucidated by the mode of action of herbal drug Withaferin A 
BMC Bioinformatics  2011;12(Suppl 1):S30.
Background
HSPs (Heat shock proteins) are highly conserved ubiquitous proteins among species which are involved in maintaining appropriate folding and conformation of other proteins and are thus referred to as molecular chaperones. Hsp90 (Heat-shock protein 90 kDa) is one of a group of molecular chaperones responsible for managing protein folding and quality control in cell environment. However it is also involved in the maturation and stabilization of a wide range of oncogenic client proteins which are crucial for oncogenesis and malignant progression. Hsp90 requires a series of co-chaperones to assemble into a super-chaperone complex for its function. These co-chaperones bind and leave the complex at various stages to regulate the chaperoning process. Arresting the chaperone cycle at these stages by targeting different co-chaperone/Hsp90 interactions seems to be quite a viable alternative and is likely to achieve similar consequences as that of Hsp90 direct inhibition with added favors of high specificity and reduced side effect profile. The study conducted here is an attempt to explore the potential of Withania somnifera’s major constituent WA (Withaferin A) in attenuating the Hsp90/Cdc37 chaperone/co-chaperone interactions for enhanced tumor arresting activity and to elucidate the underlying mode of action using computational approaches.
Results
Formation of active Hsp90/Cdc37 complex is one of the essential steps for facilitation of chaperone client interaction, non-assembly of which can lead to prevention of the chaperone-client association resulting in apoptosis of tumor cells. From our flexible docking analysis of WA into active Hsp90/Cdc37 complex in which key interfacing residues of the complex were kept flexible, disruption of the active association complex can be discerned. While docking of WA into segregated Hsp90 leaves the interface residues untouched. Thus the molecular docking analysis of WA into Hsp90 and active Hsp90/Cdc37 complex conducted in this study provides significant evidence in support of the proposed mechanism of chaperone assembly suppression by inhibition or disruption of active Hsp90/Cdc37 complex formation being accounted by non-assembly of the catalytically active Hsp90/Cdc37 complex. Results from the molecular dynamics simulations in water show that the trajectories of the protein complexed with ligand WA are stable over a considerably long time period of 4 ns, with the energies of the complex being lowered in comparison to the un-docked association complex, suggesting the thermodynamic stability of WA complexed Hsp90/Cdc37.
Conclusions
The molecular chaperone Hsp90 has been a promising target for cancer therapy. Cancer is a disease marked by genetic instability. Thus specific inhibition of individual proteins or signalling pathways holds a great potential for subversion of this genetic plasticity of cancers. This study is a step forward in this direction. Our computational analysis provided a rationalization to the ability of naturally occurring WA to alter the chaperone signalling pathway. The large value of binding energy involved in binding of WA to the active Hsp90/Cdc37 complex consolidates the thermodynamic stability of the binding. Our docking results obtained substantiate the hypothesis that WA has the potential to inhibit the association of chaperone (Hsp90) to its co-chaperone (Cdc37) by disrupting the stability of attachment of Hsp90 to Cdc37. Conclusively our results strongly suggest that withaferin A is a potent anticancer agent as ascertained by its potent Hsp90-client modulating capability.
doi:10.1186/1471-2105-12-S1-S30
PMCID: PMC3044286  PMID: 21342561
24.  Implication of Bemisia tabaci Heat Shock Protein 70 in Begomovirus-Whitefly Interactions 
Journal of Virology  2012;86(24):13241-13252.
The whitefly Bemisia tabaci (Gennadius) is a major cosmopolitan pest capable of feeding on hundreds of plant species and transmits several major plant viruses. The most important and widespread viruses vectored by B. tabaci are in the genus Begomovirus, an unusual group of plant viruses owing to their small, single-stranded DNA genome and geminate particle morphology. B. tabaci transmits begomoviruses in a persistent circulative nonpropagative manner. Evidence suggests that the whitefly vector encounters deleterious effects following Tomato yellow leaf curl virus (TYLCV) ingestion and retention. However, little is known about the molecular and cellular basis underlying these coevolved begomovirus-whitefly interactions. To elucidate these interactions, we undertook a study using B. tabaci microarrays to specifically describe the responses of the transcriptomes of whole insects and dissected midguts following TYLCV acquisition and retention. Microarray, real-time PCR, and Western blot analyses indicated that B. tabaci heat shock protein 70 (HSP70) specifically responded to the presence of the monopartite TYLCV and the bipartite Squash leaf curl virus. Immunocapture PCR, protein coimmunoprecipitation, and virus overlay protein binding assays showed in vitro interaction between TYLCV and HSP70. Fluorescence in situ hybridization and immunolocalization showed colocalization of TYLCV and the bipartite Watermelon chlorotic stunt virus virions and HSP70 within midgut epithelial cells. Finally, membrane feeding of whiteflies with anti-HSP70 antibodies and TYLCV virions showed an increase in TYLCV transmission, suggesting an inhibitory role for HSP70 in virus transmission, a role that might be related to protection against begomoviruses while translocating in the whitefly.
doi:10.1128/JVI.00880-12
PMCID: PMC3503126  PMID: 23015709
25.  ATPγS Disrupts Human Immunodeficiency Virus Type 1 Virion Core Integrity 
Journal of Virology  2005;79(9):5557-5567.
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.
doi:10.1128/JVI.79.9.5557-5567.2005
PMCID: PMC1082765  PMID: 15827170

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