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1.  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.
PMCID: PMC3657685  PMID: 23730299
phloem-limited; plasmalemma deposit; whitefly vector; Crinivirus; quintuple gene block
2.  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.
PMCID: PMC116111  PMID: 11711606
3.  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).
PMCID: PMC3589766  PMID: 23508802
RNA virus replication; membrane vesicles; virus replication factory; endoplasmic reticulum modification; intracellular traffic
4.  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.
PMCID: PMC141117  PMID: 12551975
5.  Role of Rice Stripe Virus NSvc4 in Cell-to-Cell Movement and Symptom Development in Nicotiana benthamiana 
Our previous work has demonstrated that the NSvc4 protein of Rice stripe virus (RSV) functions as a cell-to-cell movement protein. However, the mechanisms whereby RSV traffics through plasmodesmata (PD) are unknown. Here we provide evidence that the NSvc4 moves on the actin filament and endoplasmic reticulum network, but not microtubules, to reach cell wall PD. Disruption of cytoskeleton using different inhibitors altered NSvc4 localization to PD, thus impeding RSV infection of Nicotiana benthamiana. Sequence analyses and deletion mutagenesis experiment revealed that the N-terminal 125 amino acids (AAs) of the NSvc4 determine PD targeting and that a transmembrane domain spanning AAs 106–125 is critical for PD localization. We also found that the NSvc4 protein can localize to chloroplasts in infected cells. Analyses using deletion mutants revealed that the N-terminal 73 AAs are essential for chloroplast localization. Furthermore, expression of NSvc4 from a Potato virus X (PVX) vector resulted in more severe disease symptoms than PVX alone in systemically infected N. benthamiana leaves. Expression of NSvc4 in Spodoptera frugiperda 9 cells did not elicit tubule formation, but instead resulted in punctate foci at the plasma membrane. These findings shed new light on our understanding of the movement mechanisms whereby RSV infects host plants.
PMCID: PMC3516811  PMID: 23233857
rice stripe virus; movement; chloroplast; tubules
6.  Processing and Localization of the African Swine Fever Virus CD2v Transmembrane Protein▿  
Journal of Virology  2011;85(7):3294-3305.
The African swine fever virus (ASFV)-encoded CD2v transmembrane protein is required for the hemadsorption of red blood cells around infected cells and is also required for the inhibition of bystander lymphocyte proliferation in response to mitogens. We studied the expression of CD2v by expressing the gene with a V5 tag downstream from the signal peptide near the N terminus and a hemagglutinin (HA) tag at the C terminus. In ASFV-infected cells, a full-length glycosylated form of the CD2v protein, which migrated mainly as a 89-kDa product, was detected, as well as an N-terminal glycosylated fragment of 63 kDa and a C-terminal nonglycosylated fragment of 26 kDa. All of these forms of the protein were localized in the membrane fraction of cells. The 26-kDa C-terminal fragment was also produced in infected cells treated with brefeldin A. These data indicate that the CD2v protein is cleaved within the luminal domain and that this occurs in the endoplasmic reticulum or Golgi compartments. Confocal microscopy showed that most of the expressed CD2v protein was localized within cells rather than at the cell surface. Comparison of the localization of full-length CD2v with that of a deletion mutant lacking all of the cytoplasmic tail apart from the 12 membrane-proximal amino acids indicated that signals within the cytoplasmic tail are responsible for the predominant localization of the full-length and C-terminal 26-kDa fragment within membranes around the virus factories, which contain markers for the Golgi compartment. Processing of the CD2v protein was not observed in uninfected cells, indicating that it is induced by ASFV infection.
PMCID: PMC3067853  PMID: 21248037
7.  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
8.  NS2B-3 proteinase-mediated processing in the yellow fever virus structural region: in vitro and in vivo studies. 
Journal of Virology  1994;68(6):3794-3802.
Several of the cleavages required to generate the mature nonstructural proteins from the flaviviral polyprotein are known to be mediated by a complex consisting of NS2B and a serine proteinase domain located in the N-terminal one-third of NS3. These cleavages typically occur after two basic residues followed by a short side chain residue. Cleavage at a similar dibasic site in the structural region is believed to produce the C terminus of the virion capsid protein. To study this cleavage, we developed a cell-free trans cleavage assay for yellow fever virus (YF)-specific proteolytic activity by using a substrate spanning the C protein dibasic site. Cleavage at the predicted site was observed when the substrate was incubated with detergent-solubilized lysates from YF-infected BHK cells. NS2B and the NS3 proteinase domain were the only YF-specific proteins required for this cleavage. Cell fractionation studies demonstrated that the YF-specific proteolytic activity was membrane associated and that activity could be detected only after detergent solubilization. Previous cell-free studies led to a hypothesis that processing in the C-prM region involves (i) translation of C followed by translocation and core glycosylation of prM by using an internal signal sequence, (ii) signalase cleavage to produce a membrane-anchored form of the C protein (anchC) and the N terminus of prM, and (iii) NS2B-3-mediated cleavage at the anchC dibasic site to produce the C terminus of the virion C protein. However, the results of in vivo transient-expression studies do not support this temporal cleavage order. Rather, expression of a YF polyprotein extending from C through the N-terminal one-third of NS3 revealed that C-prM processing, but not translocation, was dependent on an active NS2B-3 proteinase. This suggests that signalase-mediated cleavage in the lumen of the endoplasmic reticulum may be dependent on prior cleavage at the anchC dibasic site. Possible pathways for processing in the C-prM region are outlined and discussed.
PMCID: PMC236884  PMID: 8189517
9.  African Swine Fever Virus Structural Protein p54 Is Essential for the Recruitment of Envelope Precursors to Assembly Sites 
Journal of Virology  2004;78(8):4299-4313.
The assembly of African swine fever virus (ASFV) at the cytoplasmic virus factories commences with the formation of precursor membranous structures, which are thought to be collapsed cisternal domains recruited from the surrounding endoplasmic reticulum (ER). This report analyzes the role in virus morphogenesis of the structural protein p54, a 25-kDa polypeptide encoded by the E183L gene that contains a putative transmembrane domain and localizes at the ER-derived envelope precursors. We show that protein p54 behaves in vitro and in infected cells as a type I membrane-anchored protein that forms disulfide-linked homodimers through its unique luminal cysteine. Moreover, p54 is targeted to the ER membranes when it is transiently expressed in transfected cells. Using a lethal conditional recombinant, vE183Li, we also demonstrate that the repression of p54 synthesis arrests virus morphogenesis at a very early stage, even prior to the formation of the precursor membranes. Under restrictive conditions, the virus factories appeared as discrete electron-lucent areas essentially free of viral structures. In contrast, outside the assembly sites, large amounts of aberrant zipper-like structures formed by the unprocessed core polyproteins pp220 and pp62 were produced in close association to ER cisternae. Altogether, these results indicate that the transmembrane structural protein p54 is critical for the recruitment and transformation of the ER membranes into the precursors of the viral envelope.
PMCID: PMC374266  PMID: 15047843
10.  Mutations of the Rous sarcoma virus env gene that affect the transport and subcellular location of the glycoprotein products 
The Journal of Cell Biology  1984;99(6):2011-2023.
The envelope glycoproteins of Rous sarcoma virus (RSV), gp85 and gp37, are anchored in the membrane by a 27-amino acid, hydrophobic domain that lies adjacent to a 22-amino acid, cytoplasmic domain at the carboxy terminus of gp37. We have altered these cytoplasmic and transmembrane domains by introducing deletion mutations into the molecularly cloned sequences of a proviral env gene. The effects of the mutations on the transport and subcellular localization of the Rous sarcoma virus glycoproteins were examined in monkey (CV-1) cells using an SV40 expression vector. We found, on the one hand, that replacement of the nonconserved region of the cytoplasmic domain with a longer, unrelated sequence of amino acids (mutant C1) did not alter the rate of transport to the Golgi apparatus nor the appearance of the glycoprotein on the cell surface. Larger deletions, extending into the conserved region of the cytoplasmic domain (mutant C2), resulted in a slower rate of transport to the Golgi apparatus, but did not prevent transport to the cell surface. On the other hand, removal of the entire cytoplasmic and transmembrane domains (mutant C3) did block transport and therefore did not result in secretion of the truncated protein. Our results demonstrate that the C3 polypeptide was not transported to the Golgi apparatus, although it apparently remained in a soluble, nonanchored form in the lumen of the rough endoplasmic reticulum; therefore, it appears that this mutant protein lacks a functional sorting signal. Surprisingly, subcellular localization by internal immunofluorescence revealed that the C3 protein (unlike the wild type) did not accumulate on the nuclear membrane but rather in vesicles distributed throughout the cytoplasm. This observation suggests that the wild-type glycoproteins (and perhaps other membrane-bound or secreted proteins) are specifically transported to the nuclear membrane after their biosynthesis elsewhere in the rough endoplasmic reticulum.
PMCID: PMC2113559  PMID: 6094591
11.  Vaccinia Virus F1L Protein Is a Tail-Anchored Protein That Functions at the Mitochondria To Inhibit Apoptosis 
Journal of Virology  2005;79(2):1084-1098.
Members of the poxvirus family encode multiple immune evasion proteins, including proteins that regulate apoptosis. We recently identified one such protein, F1L, encoded by vaccinia virus, the prototypic member of the poxvirus family. F1L localizes to the mitochondria and inhibits apoptosis by interfering with the release of cytochrome c, the pivotal commitment step in the apoptotic cascade. Sequence analysis of the F1L open reading frame revealed a C-terminal motif composed of a 12-amino-acid transmembrane domain flanked by positively charged lysines, followed by an 8-amino-acid hydrophilic tail. By generating a series of F1L deletion constructs, we show that the C-terminal domain is necessary and sufficient for localization of F1L to the mitochondria. In addition, mutation of lysines 219 and 222 downstream of the C-terminal transmembrane domain resulted in altered localization of F1L to the endoplasmic reticulum. Using F1L protein generated in an in vitro transcription-translation system, we found that F1L was posttranslationally inserted into mitochondria and tightly associated with mitochondrial membranes as demonstrated by resistance to alkaline extraction. Sensitivity to protease digestion showed that the N terminus of F1L was exposed to the cytoplasm. Utilizing various F1L deletion constructs, we found that F1L localization to the mitochondria was necessary to inhibit apoptosis, since constructs that no longer localized to the mitochondria had reduced antiapoptotic ability. Our studies show that F1L is a new member of the tail-anchored protein family that localizes to mitochondria during virus infection and inhibits apoptosis as a means to enhance virus survival.
PMCID: PMC538563  PMID: 15613337
12.  Region E3 of subgroup B human adenoviruses encodes a 16-kilodalton membrane protein that may be a distant analog of the E3-6.7K protein of subgroup C adenoviruses. 
Journal of Virology  1995;69(7):4292-4298.
There is an open reading frame in the E3 transcription unit of adenovirus type 3 (Ad3) and Ad7 that could encode a protein of 16 kDa (16K protein). Ad3 and Ad7 are members of subgroup B of human adenoviruses. Using a rabbit antipeptide antiserum, we show that the 16K protein is expressed in Ad3- and Ad7-infected cells at early and late stages of infection; it is not expressed in cells infected with an Ad7 mutant that deletes the 16K gene. The 16K protein was also transcribed and translated in vitro from DNA containing the open reading frame for the 16K protein. The 16K protein has two hydrophobic domains typical of integral membrane proteins; consistent with this, we detected 16K in the crude membrane but not the cytosol cellular fractions. Although 16K has two potential sites for Asn-linked glycosylation, the protein is not glycosylated. The 16K gene is located in the same position in region E3 as the gene for the 6.7K protein of subgroup C adenoviruses (Ad2 and Ad5). E3-6.7K is an Asn-linked integral membrane glycoprotein, localized in the endoplasmic reticulum, whose function is unknown. The 16K protein has a putative transmembrane domain located in the same place in 16K as is the transmembrane domain in 6.7K, and the C-terminal portion of 16K is partially homologous to the C-terminal cytoplasmic domain of 6.7K; we suggest that these domains in 16K and 6.7K may have a similar function. The N-terminal 102 residues in 16K are not found in 6.7K; these residues may have a function that is unique to the 16K protein. In common with all known E3 proteins, the 16K protein is dispensable for virus replication in cultured cells; this suggests that the 16K protein may function in virus-host interactions.
PMCID: PMC189168  PMID: 7769690
13.  A functional link between the co-translational protein translocation pathway and the UPR 
eLife  null;4:e07426.
Upon endoplasmic reticulum (ER) stress, the transmembrane endoribonuclease Ire1α performs mRNA cleavage reactions to increase the ER folding capacity. It is unclear how the low abundant Ire1α efficiently finds and cleaves the majority of mRNAs at the ER membrane. Here, we reveal that Ire1α forms a complex with the Sec61 translocon to cleave its mRNA substrates. We show that Ire1α's key substrate, XBP1u mRNA, is recruited to the Ire1α-Sec61 translocon complex through its nascent chain, which contains a pseudo-transmembrane domain to utilize the signal recognition particle (SRP)-mediated pathway. Depletion of SRP, the SRP receptor or the Sec61 translocon in cells leads to reduced Ire1α-mediated splicing of XBP1u mRNA. Furthermore, mutations in Ire1α that disrupt the Ire1α-Sec61 complex causes reduced Ire1α-mediated cleavage of ER-targeted mRNAs. Thus, our data suggest that the Unfolded Protein Response is coupled with the co-translational protein translocation pathway to maintain protein homeostasis in the ER during stress conditions.
eLife digest
Proteins are made up of long chains of smaller building blocks called amino acids. To build this chain, a molecule called mRNA is ‘translated’ into the sequence of amino acids by a molecular machine called a ribosome. In order to work, the protein chain must then be folded into a complex shape. For many proteins, this happens inside a cell compartment called the endoplasmic reticulum.
Newly made proteins are guided to the endoplasmic reticulum by ‘signal recognition particles’, and then enter the endoplasmic reticulum through a channel protein called Sec61. If too many protein chains arrive at once, or they are folded too slowly, the accumulation of unfolded proteins can stress the endoplasmic reticulum. To fix this, cells trigger a process called the unfolded protein response.
In mammals, an enzyme called Ire1α detects when the endoplasmic reticulum is becoming stressed and responds by cleaving mRNA molecules. One particular target of Ire1α is the mRNA molecule that encodes a protein called XBP1, which can activate hundreds of genes to increase the size—and hence reduce the stress—of the endoplasmic reticulum. This protein is only made if a section of the mRNA molecule is removed from it; thus, by cleaving the mRNA, Ire1α enables the protein to be made. It remains unknown, however, how Ire1α finds and cleaves its mRNA targets.
Plumb, Zhang et al. identified the proteins that bind to Ire1α in human cells, and found that the Sec61 channel is one such protein. This interaction localizes Ire1α to the Sec61 channel in the endoplasmic reticulum membrane. The XBP1 protein is then brought to this channel by a signal recognition particle while it is still being translated—that is, when it is still attached to the ribosome and its mRNA molecule. Ire1α can then cleave the XBP1 mRNA. In cells that lack the signal recognition particle or the Sec61 channel protein, Ire1α cannot efficiently cleave the XBP1 mRNA molecule. In addition, if Ire1α is unable to interact with the channel protein, it does not efficiently cleave mRNA molecules at the endoplasmic reticulum membrane.
This work establishes a new link between the unfolded protein response and the pathway that brings new proteins to the endoplasmic reticulum membrane. It provides a basis for future studies examining the details of Ire1α signaling in mammals and, in particular, work investigating the mechanism of insulin mRNA cleavage by Ire1α, which has been implicated in type 2 diabetes.
PMCID: PMC4456659  PMID: 25993558
unfolded protein response; endoplasmic reticulum; protein translocation; human
14.  A glutamine residue in the membrane-associating domain of the bovine papillomavirus type 1 E5 oncoprotein mediates its binding to a transmembrane component of the vacuolar H(+)-ATPase. 
Journal of Virology  1992;66(1):405-413.
The 44-amino-acid E5 oncoprotein is the major transforming protein of bovine papillomavirus type 1. It is a highly hydrophobic polypeptide which dimerizes and localizes to the Golgi apparatus and endoplasmic reticulum membranes. Recent evidence suggests that E5 modulates the phosphorylation and internalization of the epidermal growth factor and colony-stimulating factor 1 receptors and constitutively activates platelet-derived growth factor receptors in C127 and FR3T3 cells. Although no direct interaction with these growth factor receptors has yet been identified, the E5 oncoprotein has been shown recently to interact with the hydrophobic 16-kDa component of the vacuolar H(+)-ATPase (16K protein) [D. J. Goldstein, M. E. Finbow, T. Andresson, P. McLean, K. Smith, V. Bubb, and R. Schlegel, Nature (London) 352:347-349, 1991]. In the current study, we have further analyzed the E5-16K protein complex by fast protein liquid chromatography and shown that each E5 dimer appears to bind two 16K proteins. In order to define the specific amino acid residues of E5 which participate in this binding, mutated E5 epitope fusion proteins were analyzed for their ability to coprecipitate 16K protein. Transformation-defective mutants containing amino acid substitutions within the short hydrophilic carboxyl-terminal domain retained the ability to associate with the 16K protein. However, E5 mutants lacking the glutamine residue in the hydrophobic domain were markedly inhibited in 16K protein binding. Most interestingly, the placement of a glutamine in several random hydrophobic sequences facilitated 16K protein binding, defining this residue as a potential binding site for the 16K protein component of the proton pump and exemplifying the critical role of hydrophilic amino acids for mediating specific interactions between transmembrane proteins.
PMCID: PMC238300  PMID: 1370089
15.  Homo-Oligomerization of the Porcine Reproductive and Respiratory Syndrome Virus Nucleocapsid Protein and the Role of Disulfide Linkages 
Journal of Virology  2003;77(8):4546-4557.
As a step toward understanding the assembly pathway of the porcine reproductive and respiratory syndrome virus (PRRSV), the oligomeric properties of the nucleocapsid (N) protein were investigated. In this study, we have demonstrated that under nonreducing conditions the N protein forms disulfide-linked homodimers. However, inclusion of an alkylating agent (N-ethylmaleimide [NEM]) prevented disulfide bond formation, suggesting that these intermolecular disulfide linkages were formed as a result of spurious oxidation during cell lysis. In contrast, N protein homodimers isolated from extracellular virions were shown to have formed NEM-resistant intermolecular disulfide linkages, the function of which is probably to impart stability to the virion. Pulse-chase analysis revealed that N protein homodimers become specifically disulfide linked within the virus-infected cell, albeit at the later stages of infection, conceivably when the virus particle buds into the oxidizing environment of the endoplasmic reticulum. Moreover, NEM-resistant disulfide linkages were shown to occur only during productive PRRSV infection, since expression of recombinant N protein did not result in the formation of NEM-resistant disulfide-linked homodimers. Mutational analysis indicated that of the three conserved cysteine residues in the N protein, only the cysteine at position 23 was involved in the formation of disulfide linkages. The N protein dimer was shown to be stable both in the presence and absence of intermolecular disulfide linkages, indicating that noncovalent interactions also play a role in dimerization. Non-disulfide-mediated N protein interactions were subsequently demonstrated both in vitro by the glutathione S-transferase (GST) pull-down assay and in vivo by the mammalian two-hybrid assay. Using a series of N protein deletion mutants fused to GST, amino acids 30 to 37 were shown to be essential for N-N interactions. Furthermore, since RNase A treatment markedly decreased N protein-binding affinity, it appears that at least in vitro, RNA may be involved in bridging N-N interactions. In cross-linking experiments, the N protein was shown to assemble into higher-order structures, including dimers, trimers, tetramers, and pentamers. Together, these findings demonstrate that the N protein possesses self-associative properties, and these likely provide the basis for PRRSV nucleocapsid assembly.
PMCID: PMC152152  PMID: 12663761
16.  Transmembrane and Coiled-Coil Domain Family 1 Is a Novel Protein of the Endoplasmic Reticulum 
PLoS ONE  2014;9(1):e85206.
The endoplasmic reticulum (ER) is a continuous membrane network in eukaryotic cells comprising the nuclear envelope, the rough ER, and the smooth ER. The ER has multiple critical functions and a characteristic structure. In this study, we identified a new protein of the ER, TMCC1 (transmembrane and coiled-coil domain family 1). The TMCC family consists of at least 3 putative proteins (TMCC1–3) that are conserved from nematode to human. We show that TMCC1 is an ER protein that is expressed in diverse human cell lines. TMCC1 contains 2 adjacent transmembrane domains near the C-terminus, in addition to coiled-coil domains. TMCC1 was targeted to the rough ER through the transmembrane domains, whereas the N-terminal region and C-terminal tail of TMCC1 were found to reside in the cytoplasm. Moreover, the cytosolic region of TMCC1 formed homo- or hetero-dimers or oligomers with other TMCC proteins and interacted with ribosomal proteins. Notably, overexpression of TMCC1 or its transmembrane domains caused defects in ER morphology. Our results suggest roles of TMCC1 in ER organization.
PMCID: PMC3891740  PMID: 24454821
17.  Intact protein folding in the glutathione-depleted endoplasmic reticulum implicates alternative protein thiol reductants 
eLife  2014;3:e03421.
Protein folding homeostasis in the endoplasmic reticulum (ER) requires efficient protein thiol oxidation, but also relies on a parallel reductive process to edit disulfides during the maturation or degradation of secreted proteins. To critically examine the widely held assumption that reduced ER glutathione fuels disulfide reduction, we expressed a modified form of a cytosolic glutathione-degrading enzyme, ChaC1, in the ER lumen. ChaC1CtoS purged the ER of glutathione eliciting the expected kinetic defect in oxidation of an ER-localized glutathione-coupled Grx1-roGFP2 optical probe, but had no effect on the disulfide editing-dependent maturation of the LDL receptor or the reduction-dependent degradation of misfolded alpha-1 antitrypsin. Furthermore, glutathione depletion had no measurable effect on induction of the unfolded protein response (UPR); a sensitive measure of ER protein folding homeostasis. These findings challenge the importance of reduced ER glutathione and suggest the existence of alternative electron donor(s) that maintain the reductive capacity of the ER.
eLife digest
Proteins are basically strings of amino acids that have folded into a specific three-dimensional shape, and this shape is often important for the protein's function. Some proteins have bonds between pairs of cysteines—an amino acid that contains sulfur—in different parts of the protein to maintain its correct shape.
In eukaryotes, such as plants and animals, these so-called ‘disulfide bonds’ are formed inside a structure within each cell called the endoplasmic reticulum, which is where many proteins are folded. Occasionally, disulfide bonds form in the wrong place in a protein, so they need to be broken and re-positioned—a process sometimes called editing—for the protein to fold correctly. It was widely assumed that a chemical called ‘reduced glutathione’ fuels the breaking of disulfide bonds in the endoplasmic reticulum, but to date few researchers have tried to test this assumption.
Tsunoda et al. have now taken an enzyme that degrades glutathione elsewhere in the cell and modified it in a way that allows it to work inside the endoplasmic reticulum. When this modified enzyme was produced in human cells grown in the laboratory, it purged the endoplasmic reticulum of glutathione. However, the lack of glutathione had no effect on the folding of a large protein with 30 disulfide bonds, many of which need to be edited at one time or another for the protein to fold correctly. The destruction of a poorly folded protein, via a process that also needs this protein's disulfide bonds to be broken down, was also not affected by a lack of reduced glutathione in the endoplasmic reticulum.
Furthermore, decreasing these levels of glutathione did not affect the unfolded protein response: a stress response in cells that are experiencing a build-up of unfolded or poorly folded proteins within the endoplasmic reticulum.
As such, the findings of Tsunoda et al. challenge the importance of reduced glutathione in the endoplasmic reticulum and suggest that other chemical processes might be involved in editing disulfide bonds. Further work is now needed to investigate the other known processes that might complete this task instead to see which, if any, are involved.
PMCID: PMC4109312  PMID: 25073928
protein folding; UPR; redox; glutathione; human
18.  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
19.  Dengue virus-induced modifications of host cell membranes. 
Journal of Virology  1975;16(4):1017-1026.
Enzymatic markers and electron microscopy were utilized to determine the cellular origin of the membrane types isolated from type 2 dengue virus-infected BHK cells by discontinuous sucrose gradient centrifugation. The results showed an apparent separation of plasma membrane, smooth and rough endoplasmic reticulum with increasing density. Virus-induced protein and RNA synthesis, as indicated by the incorporation of radiolabled precursors, was localized on the rough endoplasmic reticulum. Glycosylation, measured by the incorporation of radiolabeled glucosamine into membrane-associated proteins, was most active in the bands of intermediate and smooth endoplasmic reticulum. Polyacrylamide gel electrophoresis of isolated membrane bands, radiolabeled in the presence of actinomycin D, after pulse inhibition by cycloheximide, revealed seven virus-specific proteins associated with all membrane fractions. Viral structural protein V-3, and nonstructural proteins NV-3 and NV-2, increased with decreasing density, whereas NV-5 and NV-4 remained constant. The viral capsid protein V-2 was depleted in the intermediate and smooth endoplasmic reticulum, suggesting that these membranes may serve as the sites for viral maturation. NV-3 was the most prominent virus-specified protein found in the plasma membrane.
PMCID: PMC354764  PMID: 1165590
20.  Leader Proteinase of the Beet Yellows Closterovirus: Mutation Analysis of the Function in Genome Amplification 
Journal of Virology  2000;74(20):9766-9770.
The beet yellows closterovirus leader proteinase (L-Pro) possesses a C-terminal proteinase domain and a nonproteolytic N-terminal domain. It was found that although L-Pro is not essential for basal-level replication, deletion of its N-terminal domain resulted in a 1,000-fold reduction in RNA accumulation. Mutagenic analysis of the N-terminal domain revealed its structural flexibility except for the 54-codon-long, 5′-terminal element in the corresponding open reading frame that is critical for efficient RNA amplification at both RNA and protein levels.
PMCID: PMC112412  PMID: 11000252
21.  Membrane anchoring domain of herpes simplex virus glycoprotein gB is sufficient for nuclear envelope localization. 
Journal of Virology  1994;68(4):2272-2285.
We have used the glycoprotein gB of herpes simplex virus type 1 (gB-1), which buds from the inner nuclear membrane, as a model protein to study localization of membrane proteins in the nuclear envelope. To determine whether specific domains of gB-1 glycoprotein are involved in localization in the nuclear envelope, we have used deletion mutants of gB-1 protein as well as chimeric proteins constructed by replacing the domains of the cell surface glycoprotein G of vesicular stomatitis virus with the corresponding domains of gB. Mutant and chimeric proteins expressed in COS cells were localized by immunoelectron microscopy. A chimeric protein (gB-G) containing the ectodomain of gB and the transmembrane and cytoplasmic domains of G did not localize in the nuclear envelope. When the ectodomain of G was fused to the transmembrane and cytoplasmic domains of gB, however, the resulting chimeric protein (G-gB) was localized in the nuclear envelope. Substitution of the transmembrane domain of G with the 69 hydrophobic amino acids containing the membrane anchoring domain of gB allowed the hybrid protein (G-tmgB) to be localized in the nuclear envelope, suggesting that residues 721 to 795 of gB can promote retention of proteins in the nuclear envelope. Deletion mutations in the hydrophobic region further showed that a transmembrane segment of 21 hydrophobic amino acids, residues 774 to 795 of gB, was sufficient for localization in the nuclear envelope. Since wild-type gB and the mutant and chimeric proteins that were localized in the nuclear envelope were also retained in the endoplasmic reticulum, the membrane spanning segment of gB could also influence retention in the endoplasmic reticulum.
PMCID: PMC236703  PMID: 8139012
22.  The Transmembrane Domains of the prM and E Proteins of Yellow Fever Virus Are Endoplasmic Reticulum Localization Signals 
Journal of Virology  2004;78(22):12591-12602.
The immature flavivirus particle contains two envelope proteins, prM and E, that are associated as a heterodimer. Virion morphogenesis of the flaviviruses occurs in association with endoplasmic reticulum (ER) membranes, suggesting that there should be accumulation of the virion components in this compartment. This also implies that ER localization signals must be present in the flavivirus envelope proteins. In this work, we looked for potential subcellular localization signals in the yellow fever virus envelope proteins. Confocal immunofluorescence analysis of the subcellular localization of the E protein in yellow fever virus-infected cells indicated that this protein accumulates in the ER. Similar results were obtained with cells expressing only prM and E. Chimeric proteins containing the ectodomain of CD4 or CD8 fused to the transmembrane domains of prM or E were constructed, and their subcellular localization was studied by confocal immunofluorescence and by analyzing the maturation of their associated glycans. Although a small fraction was detected in the ER-to-Golgi intermediate and Golgi compartments, these chimeric proteins were located mainly in the ER. The C termini of prM and E form two antiparallel transmembrane α-helices. Interestingly, the first transmembrane passage contains enough information for ER localization. Taken altogether, these data indicate that, besides their role as membrane anchors, the transmembrane domains of yellow fever virus envelope proteins are ER retention signals. In addition, our data show that the mechanisms of ER retention of the flavivirus and hepacivirus envelope proteins are different.
PMCID: PMC525104  PMID: 15507646
23.  Characterization of kinectin, a kinesin-binding protein: primary sequence and N-terminal topogenic signal analysis. 
Molecular Biology of the Cell  1995;6(2):171-183.
Kinectin is a kinesin-binding protein (Toyoshima et al., 1992) that is required for kinesin-based motility (Kumar et al., 1995). A kinectin cDNA clone containing a 4.7-kilobase insert was isolated from an embryonic chick brain cDNA library by immunoscreening with a panel of monoclonal antibodies. The cDNA contained an open reading frame of 1364 amino acids encoding a protein of 156 kDa. A bacterially expressed product of the full length cDNA bound purified kinesin. Transient expression in CV-1 cells gave an endoplasmic reticulum distribution that depended upon the N-terminal domain. Analysis of the predicted amino acid sequence indicated a highly hydrophobic near N-terminal stretch of 28 amino acids and a large portion (326-1248) of predicted alpha helical coiled coils. The 30-kDa fragment containing the N-terminal hydrophobic region was produced by cell-free in vitro translation and found to assemble with canine pancreas rough microsomes. Cleavage of the N terminus was not observed confirming its role as a potential transmembrane domain. Thus, the kinectin cDNA encodes a cytoplasmic-oriented integral membrane protein that binds kinesin and is likely to be a coiled-coil dimer.
PMCID: PMC275827  PMID: 7787244
24.  Characterization of the Coronavirus Mouse Hepatitis Virus Strain A59 Small Membrane Protein E 
Journal of Virology  2000;74(5):2333-2342.
The small envelope (E) protein has recently been shown to play an essential role in the assembly of coronaviruses. Expression studies revealed that for formation of the viral envelope, actually only the E protein and the membrane (M) protein are required. Since little is known about this generally low-abundance virion component, we have characterized the E protein of mouse hepatitis virus strain A59 (MHV-A59), an 83-residue polypeptide. Using an antiserum to the hydrophilic carboxy terminus of this otherwise hydrophobic protein, we found that the E protein was synthesized in infected cells with similar kinetics as the other viral structural proteins. The protein appeared to be quite stable both during infection and when expressed individually using a vaccinia virus expression system. Consistent with the lack of a predicted cleavage site, the protein was found to become integrated in membranes without involvement of a cleaved signal peptide, nor were any other modifications of the polypeptide observed. Immunofluorescence analysis of cells expressing the E protein demonstrated that the hydrophilic tail is exposed on the cytoplasmic side. Accordingly, this domain of the protein could not be detected on the outside of virions but appeared to be inside, where it was protected from proteolytic degradation. The results lead to a topological model in which the polypeptide is buried within the membrane, spanning the lipid bilayer once, possibly twice, and exposing only its carboxy-terminal domain. Finally, electron microscopic studies demonstrated that expression of the E protein in cells induced the formation of characteristic membrane structures also observed in MHV-A59-infected cells, apparently consisting of masses of tubular, smooth, convoluted membranes. As judged by their colabeling with antibodies to E and to Rab-1, a marker for the intermediate compartment and endoplasmic reticulum, the E protein accumulates in and induces curvature into these pre-Golgi membranes where coronaviruses have been shown earlier to assemble by budding.
PMCID: PMC111715  PMID: 10666264
25.  Mapping of Functional Domains of the Lipid Kinase Phosphatidylinositol 4-Kinase Type III Alpha Involved in Enzymatic Activity and Hepatitis C Virus Replication 
Journal of Virology  2014;88(17):9909-9926.
The lipid kinase phosphatidylinositol 4-kinase III alpha (PI4KIIIα) is an endoplasmic reticulum (ER)-resident enzyme that synthesizes phosphatidylinositol 4-phosphate (PI4P). PI4KIIIα is an essential host factor for hepatitis C virus (HCV) replication. Interaction with HCV nonstructural protein 5A (NS5A) leads to kinase activation and accumulation of PI4P at intracellular membranes. In this study, we investigated the structural requirements of PI4KIIIα in HCV replication and enzymatic activity. Therefore, we analyzed PI4KIIIα mutants for subcellular localization, reconstitution of HCV replication in PI4KIIIα knockdown cell lines, PI4P induction in HCV-positive cells, and lipid kinase activity in vitro. All mutants still interacted with NS5A and localized in a manner similar to that of the full-length enzyme, suggesting multiple regions of PI4KIIIα are involved in NS5A interaction and subcellular localization. Interestingly, the N-terminal 1,152 amino acids were dispensable for HCV replication, PI4P induction, and enzymatic function, whereas further N-terminal or C-terminal deletions were deleterious, thereby defining the minimal PI4KIIIα core enzyme at a size of ca. 108 kDa. Additional deletion of predicted functional motifs within the C-terminal half of PI4KIIIα also were detrimental for enzymatic activity and for the ability of PI4KIIIα to rescue HCV replication, with the exception of a proposed nuclear localization signal, suggesting that the entire C-terminal half of PI4KIIIα is involved in the formation of a minimal enzymatic core. This view was supported by structural modeling of the PI4KIIIα C terminus, suggesting a catalytic center formed by an N- and C-terminal lobe and an armadillo-fold motif, which is preceded by three distinct alpha-helical domains probably involved in regulation of enzymatic activity.
IMPORTANCE The lipid kinase PI4KIIIα is of central importance for cellular phosphatidylinositol metabolism and is a key host cell factor of hepatitis C virus replication. However, little is known so far about the structure of this 240-kDa protein and the functional importance of specific subdomains regarding lipid kinase activity and viral replication. This work focuses on the phenotypic analysis of distinct PI4KIIIα mutants in different biochemical and cell-based assays and develops a structural model of the C-terminal enzymatic core. The results shed light on the structural and functional requirements of enzymatic activity and the determinants required for HCV replication.
PMCID: PMC4136347  PMID: 24920820

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