The paramyxovirus simian virus 5 V protein slows progression of the cell cycle

Infection of cells by many viruses affects the cell division cycle of the host cell to favor viral replication. We examined the ability of the paramyxovirus simian parainfluenza virus 5 (SV5) to affect cell cycle progression, and we found that SV5 slows the rate of proliferation of HeLa T4 cells. The SV5-infected cells had a delayed transition from G(1) to S phase and prolonged progression through S phase, and some of the infected cells were arrested in G(2) or M phase. The levels of p53 and p21(CIP1) were not increased in SV5-infected cells compared to mock-infected cells, suggesting that the changes in the cell cycle occur through a p53-independent mechanism. However, the phosphorylation of the retinoblastoma protein (pRB) was delayed and prolonged in SV5-infected cells. The changes in the cell cycle were also observed in cells expressing the SV5 V protein but not in the cells expressing the SV5 P protein or the V protein lacking its unique C terminus (VDeltaC). The unique C terminus of the V protein of SV5 was shown previously to interact with DDB1, which is the 127-kDa subunit of the multifunctional damage-specific DNA-binding protein (DDB) heterodimer. The coexpression of DDB1 with V can partially restore the changes in the cell cycle caused by expression of the V protein.     

Sumoylation of the P protein at K254 plays an important role in growth of parainfluenza virus 5

The P protein of parainfluenza virus 5 (PIV5) is an essential cofactor of the viral RNA-dependent RNA polymerase. Phosphorylation of the P protein can positively or negatively regulate viral gene expression, depending on the precise phosphorylation sites. Sumoylation, a process of adding small ubiquitin-like modifier (SUMO) to proteins posttranslationally, plays an important role in regulating protein function. In this study, we have found that the P protein of PIV5 was sumoylated with SUMO1 in both transfected and infected cells. The K254 residue of the P protein is within a consensus sumoylation motif. Mutation of the P protein at K254 to arginine (P-K254R) reduced PIV5 minigenome activity, as well as the sumoylation level of the P protein. Incorporation of K254R into a recombinant PIV5 (rPIV5-P-K254R) resulted in a virus that grew to a lower titer and had lower levels of viral RNA synthesis and protein expression than wild-type PIV5, suggesting that sumoylation of the P protein at K254 is important for PIV5 growth. Biochemical studies did not reveal any defect of P-K254R in its interactions with viral proteins NP and L or formation of homotetramers. We propose that sumoylation of the P protein at K254 regulates PIV5 gene expression through a host protein.     

The SH integral membrane protein of the paramyxovirus simian virus 5 is required to block apoptosis in MDBK cells

In some cell types the paramyxovirus simian virus 5 (SV5) causes little cytopathic effect (CPE) and infection continues productively for long periods of time; e.g., SV5 can be produced from MDBK cells for up to 40 days with little CPE. SV5 differs from most paramyxoviruses in that it encodes a small (44-amino-acid) hydrophobic integral membrane protein (SH). When MDBK cells were infected with a recombinant SV5 containing a deletion of the SH gene (rSV5DeltaSH), the MDBK cells exhibited an increase in CPE compared to cells infected with wild-type SV5 (recovered from cDNA; rSV5). The increased CPE correlated with an increase in apoptosis in rSV5DeltaSH-infected cells over mock-infected and rSV5-infected cells when assayed for annexin V binding, DNA content (propidium iodide staining), and DNA fragmentation (terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end labeling assay). In rSV5DeltaSH-infected MDBK cells an increase in caspase-2 and caspase-3 activities was observed. By using peptide inhibitors of individual caspases it was found that caspase-2 and caspase-3 were activated separately in rSV5DeltaSH-infected cells. Expression of caspase-2 and -3 in rSV5DeltaSH-infected MDBK cells appeared not to require STAT1 protein, as STAT1 protein could not be detected in SV5-infected MDBK cells. When mutant mice homologous for a targeted disruption of STAT1 were used as a model animal system and infected with the viruses it was found that rSV5DeltaSH caused less mortality than wild-type rSV5, consistent with the notion of clearance of apoptotic cells in a host species.     

Requirements for budding of paramyxovirus simian virus 5 virus-like particles

Enveloped viruses are released from infected cells after coalescence of viral components at cellular membranes and budding of membranes to release particles. For some negative-strand RNA viruses (e.g., vesicular stomatitis virus and Ebola virus), the viral matrix (M) protein contains all of the information needed for budding, since virus-like particles (VLPs) are efficiently released from cells when the M protein is expressed from cDNA. To investigate the requirements for budding of the paramyxovirus simian virus 5 (SV5), its M protein was expressed in mammalian cells, and it was found that SV5 M protein alone could not induce vesicle budding and was not secreted from cells. Coexpression of M protein with the viral hemagglutinin-neuraminidase (HN) or fusion (F) glycoproteins also failed to result in significant VLP release. It was found that M protein in the form of VLPs was only secreted from cells, with an efficiency comparable to authentic virus budding, when M protein was coexpressed with one of the two glycoproteins, HN or F, together with the nucleocapsid (NP) protein. The VLPs appeared similar morphologically to authentic virions by electron microscopy. CsCl density gradient centrifugation indicated that almost all of the NP protein in the cells had assembled into nucleocapsid-like structures. Deletion of the F and HN cytoplasmic tails indicated an important role of these cytoplasmic tails in VLP budding. Furthermore, truncation of the HN cytoplasmic tail was found to be inhibitory toward budding, since it prevented coexpressed wild-type (wt) F protein from directing VLP budding. Conversely, truncation of the F protein cytoplasmic tail was not inhibitory and did not affect the ability of coexpressed wt HN protein to direct the budding of particles. Taken together, these data suggest that multiple viral components, including assembled nucleocapsids, have important roles in the paramyxovirus budding process.     

Apoptosis plays an important role in experimental rabies virus infection.

Cultured rat prostatic adenocarcinoma (AT3) cells infected with the challenge virus standard (CVS) strain of fixed rabies virus showed characteristic morphologic features of apoptosis, evidence of oligonucleosomal DNA fragmentation, and expression of the Bax protein. CVS-infected Bcl-2-transfected AT3 cells did not demonstrate these features. Adult ICR mice inoculated intracerebrally with CVS showed morphologic changes of apoptosis, DNA fragmentation, and increased Bax expression in neurons, with changes most marked in the hippocampus and cerebral cortex. Ultrastructurally, some neurons demonstrated morphologic features more typical of necrosis. These studies provide evidence that apoptosis plays an important role in the pathogenesis of rabies virus infection.     

PSII-Tc protein plays an important role in dimerization of photosystem II

We cloned and determined the nucleotide sequence of PSII genes, psbB and psbTc, from the thermophilic cyanobacterium, Thermosynechococcus elongatus strain BP-1. PSII-Tc, encoded by psbTc, is a small membrane-spanning subunit of the PSII core complex of cyanobacteria and plants. However, its role has not been fully elucidated. We generated an insertional disruptant of psbTc and studied the role of the PSII-Tc protein in cyanobacterial PSII. The following observations were made: (i) The psbTc disruptant could grow photoautotrophically at a rate similar to that of wild-type T. elongatus under a wide range of light conditions. (ii) Thylakoids and oxygen-evolving PSII complexes were successfully isolated from the psbTc disruptant as well as the wild type. There was no significant difference in the oxygen evolution activities of cells, thylakoids or PSII complexes between the psbTc disruptant and the wild type. This is in contrast to the lower activities in the other PSII mutants of T. elongatus. (iii) Chromatographic separation of monomeric and dimeric PSII revealed that recovery of dimeric PSII was dramatically reduced in the psbTc disruptant. (iv) SDS-urea-PAGE showed a complete loss of the 4.7-kDa band in the mutant PSII. Since this band in wild-type PSII consists of PSII-M and PSII-Tc, we assume that PSII-Tc is critical for the binding of PSII-M in the PSII complex and is involved directly and indirectly in the dimerization of PSII. These results appear to be in good agreement with the recent structural model of the dimeric PSII complex.     

The V protein of mumps virus plays a critical role in pathogenesis

Mumps virus (MuV) causes an acute infection in humans characterized by a wide array of symptoms ranging from relatively mild manifestations, such as parotitis, to more-severe complications, such as meningitis and encephalitis. Widespread mumps vaccination has reduced mumps incidence dramatically; however, outbreaks still occur in vaccinated populations. The V protein of MuV, when expressed in cell culture, blocks interferon (IFN) expression and signaling and interleukin-6 (IL-6) signaling. In this work, we generated a recombinant MuV incapable of expressing the V protein (rMuVΔV). The rescued MuV was derived from a clinical wild-type isolate from a recent outbreak in the United States (MuV(Iowa/US/06), G genotype). Analysis of the virus confirmed the roles of V protein in blocking IFN expression and signaling and IL-6 signaling. We also found that the rMuV(Iowa/US/06)ΔV virus induced high levels of IL-6 expression in vitro, suggesting that V plays a role in reducing IL-6 expression. In vivo, the rMuV(Iowa/US/06)ΔV virus was highly attenuated, indicating that the V protein plays an essential role in viral virulence.     

Appendant structure plays an important role in amyloidogenic cystatin dimerization prior to domain swapping

It has been hypothesized that prior to protein domain swapping, unfolding occurs in regions important for the stability of the native monomeric structure, which probably increases the possibility of intermolecular interaction. In order to explore the detailed information of the important unfolding regions in cystatin prior to domain swapping, 20?ns molecular dynamic simulations were performed at atomic level with typical amyloidogenic chicken cystatin (cC) mutant I66Q monomer under conditions that enable forming amyloid fibrils in biological experiments. Our results showed that I66Q mutant exhibited relatively large secondary structure changes and obvious expanding tendency of hydrophobic core compared to wild-type cC. More importantly, the appendant structure (AS) showed a large displacement and distortion towards the hydrophobic core in amyloidogenic cystatin. The structural analysis on cystatin monomer suggested that structural changes of the AS might make the hydrophobic core expand more easily. In addition, analysis on docking dimer has shown that the distorted AS was favor to intermolecular interactions between two cystatin monomers. Data from an independent theoretical derived algorithm as well as biological experiments also support this hypothesis.     

Appendant structure plays an important role in amyloidogenic cystatin dimerization prior to domain swapping

It has been hypothesized that prior to protein domain swapping, unfolding occurs in regions important for the stability of the native monomeric structure, which probably increases the possibility of intermolecular interaction. In order to explore the detailed information of the important unfolding regions in cystatin prior to domain swapping, 20?ns molecular dynamic simulations were performed at atomic level with typical amyloidogenic chicken cystatin (cC) mutant I66Q monomer under conditions that enable forming amyloid fibrils in biological experiments. Our results showed that I66Q mutant exhibited relatively large secondary structure changes and obvious expanding tendency of hydrophobic core compared to wild-type cC. More importantly, the appendant structure (AS) showed a large displacement and distortion towards the hydrophobic core in amyloidogenic cystatin. The structural analysis on cystatin monomer suggested that structural changes of the AS might make the hydrophobic core expand more easily. In addition, analysis on docking dimer has shown that the distorted AS was favor to intermolecular interactions between two cystatin monomers. Data from an independent theoretical derived algorithm as well as biological experiments also support this hypothesis.     

The N-terminal domain of RfaH plays an active role in protein fold-switching

The bacterial elongation factor RfaH promotes the expression of virulence factors by specifically binding to RNA polymerases (RNAP) paused at a DNA signal. This behavior is unlike that of its paralog NusG, the major representative of the protein family to which RfaH belongs. Both proteins have an N-terminal domain (NTD) bearing an RNAP binding site, yet NusG C-terminal domain (CTD) is folded as a β-barrel while RfaH CTD is forming an α-hairpin blocking such site. Upon recognition of the specific DNA exposed by RNAP, RfaH is activated via interdomain dissociation and complete CTD structural rearrangement into a β-barrel structurally identical to NusG CTD. Although RfaH transformation has been extensively characterized computationally, little attention has been given to the role of the NTD in the fold-switching process, as its structure remains unchanged. Here, we used Associative Water-mediated Structure and Energy Model (AWSEM) molecular dynamics to characterize the transformation of RfaH, spotlighting the sequence-dependent effects of NTD on CTD fold stabilization. Umbrella sampling simulations guided by native contacts recapitulate the thermodynamic equilibrium experimentally observed for RfaH and its isolated CTD. Temperature refolding simulations of full-length RfaH show a high success towards α-folded CTD, whereas the NTD interferes with βCTD folding, becoming trapped in a β-barrel intermediate. Meanwhile, NusG CTD refolding is unaffected by the presence of RfaH NTD, showing that these NTD-CTD interactions are encoded in RfaH sequence. Altogether, these results suggest that the NTD of RfaH favors the α-folded RfaH by specifically orienting the αCTD upon interdomain binding and by favoring β-barrel rupture into an intermediate from which fold-switching proceeds.     

Human low-density lipoprotein receptor plays an important role in hepatitis B virus infection

Hepatitis B virus (HBV) chronically infects more than 240 million people worldwide, resulting in chronic hepatitis, cirrhosis, and hepatocellular carcinoma. HBV vaccine is effective to prevent new HBV infection but does not offer therapeutic benefit to hepatitis B patients. Neither are current antiviral drugs curative of chronic hepatitis B. A more thorough understanding of HBV infection and replication holds a great promise for identification of novel antiviral drugs and design of optimal strategies towards the ultimate elimination of chronic hepatitis B. Recently, we have developed a robust HBV cell culture system and discovered that human apolipoprotein E (apoE) is enriched on the HBV envelope and promotes HBV infection and production. In the present study, we have determined the role of the low-density lipoprotein receptor (LDLR) in HBV infection. A LDLR-blocking monoclonal antibody potently inhibited HBV infection in HepG2 cells expressing the sodium taurocholate cotransporting polypeptide (NTCP) as well as in primary human hepatocytes. More importantly, small interfering RNAs (siRNAs)-mediated knockdown of LDLR expression and the CRISPR/Cas9-induced knockout of the LDLR gene markedly reduced HBV infection. A recombinant LDLR protein could block heparin-mediated apoE pulldown, suggesting that LDLR may act as an HBV cell attachment receptor via binding to the HBV-associated apoE. Collectively, these findings demonstrate that LDLR plays an important role in HBV infection probably by serving as a virus attachment receptor.     

The domain I-domain III linker plays an important role in the fusogenic conformational change of the alphavirus membrane fusion protein

The alphavirus Semliki Forest virus (SFV) infects cells through a low-pH-dependent membrane fusion reaction mediated by the virus fusion protein E1. Acidic pH initiates a series of E1 conformational changes that culminate in membrane fusion and include dissociation of the E1/E2 heterodimer, insertion of the E1 fusion loop into the target membrane, and refolding of E1 to a stable trimeric hairpin conformation. A highly conserved histidine (H3) on the E1 protein was previously shown to promote low-pH-dependent E1 refolding. An SFV mutant with an alanine substitution at this position (H3A) has a lower pH threshold and reduced efficiency of virus fusion and E1 trimer formation than wild-type SFV. Here we addressed the mechanism by which H3 promotes E1 refolding and membrane fusion. We identified E1 mutations that rescue the H3A defect. These revertants implicated a network of interactions that connect the domain I-domain III (DI-DIII) linker region with the E1 core trimer, including H3. In support of the importance of these interactions, mutation of residues in the network resulted in more acidic pH thresholds and reduced efficiencies of membrane fusion. In vitro studies of truncated E1 proteins demonstrated that the DI-DIII linker was required for production of a stable E1 core trimer on target membranes. Together, our results suggest a critical and previously unidentified role for the DI-DIII linker region during the low-pH-dependent refolding of E1 that drives membrane fusion.     

The simian immunodeficiency virus 5' untranslated leader sequence plays a role in intracellular viral protein accumulation and in RNA packaging

We investigated the role of 5' untranslated leader sequences of simian immunodeficiency virus (SIV(mac239)) in RNA encapsidation and protein expression. A series of progressively longer deletion mutants was constructed with a common endpoint six nucleotides upstream of the gag initiation codon and another endpoint at the 3' end of the primer binding site (PBS). We found that efficient intracellular Gag-Pol protein accumulation required the region between the PBS and splice donor (SD) site. Marked reduction of genomic RNA packaging was observed with all the deletion mutants that involved sequences at both the 5' and at the 3' ends of the major SD site, and increased nonspecific RNA incorporation could be detected in these mutants. RNA encapsidation was affected only modestly by a deletion of 54 nucleotides at the 3' end of the SD site when the mutant construct pDelta54 was transfected alone. In contrast, the amount of pDelta54 genomic RNA incorporated into particles was reduced more than 10-fold when this mutant was cotransfected with a construct specifying an RNA molecule with a wild-type packaging signal. Therefore, we conclude that the 175 nucleotides located 5' of the gag initiation codon are critical for efficient and selective incorporation of genomic RNA into virions. This location of the SIV Psi element provides the means for efficient discrimination between viral genomic and spliced RNAs.     

Bacillus subtilis SbcC protein plays an important role in DNA inter-strand cross-link repair

Background  

Several distinct pathways for the repair of damaged DNA exist in all cells. DNA modifications are repaired by base excision or nucleotide excision repair, while DNA double strand breaks (DSBs) can be repaired through direct joining of broken ends (non homologous end joining, NHEJ) or through recombination with the non broken sister chromosome (homologous recombination, HR). Rad50 protein plays an important role in repair of DNA damage in eukaryotic cells, and forms a complex with the Mre11 nuclease. The prokaryotic ortholog of Rad50, SbcC, also forms a complex with a nuclease, SbcD, in Escherichia coli, and has been implicated in the removal of hairpin structures that can arise during DNA replication. Ku protein is a component of the NHEJ pathway in pro- and eukaryotic cells.     

The substrate specificity of the catalytic domain of Abl plays an important role in directing phosphorylation of the adaptor protein Crk

c-Abl preferentially phosphorylates peptide substrates that contain proline at the P+3 site (relative to the phosphorylated tyrosine). We previously described a mutant form of the Abl catalytic domain (Y569W) with altered substrate specificity at the P+3 position, as measured using synthetic peptides. In this study, we examine the phosphorylation of Crk, a protein substrate of Abl that is phosphorylated in the sequence Tyr221-Ala-Gln-Pro. In vitro, phosphorylation of Crk by Y569W Abl is greatly reduced relative to wild-type Abl. Overexpression of Y569W mutant Abl in 293T kidney cells produces a similar overall pattern of tyrosine phosphorylation as wild-type Abl, indicating that not all cellular proteins depend on Pro at P+3 for Abl recognition. However, phosphorylation of Crk by Y569W Abl in these cells is markedly reduced relative to wild-type Abl. A truncated form of Abl lacking the C-terminal polyproline region is not able to phosphorylate Crk in these assay conditions. Thus, proper phosphorylation of Crk by Abl depends not only on the interaction of the Crk SH3 domain with the Abl polyproline region, but also on the recognition of amino acids surrounding tyrosine by the Abl catalytic domain.     

Gcn5p plays an important role in centromere kinetochore function in budding yeast

We report that the histone acetyltransferase Gcn5p is involved in cell cycle progression, whereas its absence induces several mitotic defects, including inefficient nuclear division, chromosome loss, delayed G₂ progression, and spindle elongation. The fidelity of chromosome segregation is finely regulated by the close interplay between the centromere and the kinetochore, a protein complex hierarchically assembled in the centromeric DNA region, while disruption of GCN5 in mutants of inner components results in sick phenotype. These synthetic interactions involving the ADA complex lay the genetic basis for the critical role of Gcn5p in kinetochore assembly and function. We found that Gcn5p is, in fact, physically linked to the centromere, where it affects the structure of the variant centromeric nucleosome. Our findings offer a key insight into a Gcn5p-dependent epigenetic regulation at centromere/kinetochore in mitosis.     

Angiopoietin-2 plays an important role in retinal angiogenesis

Angiopoietin 2 (Ang2) expression in the retina is increased during physiologic and pathologic neovascularization suggesting that it may be involved. In this study, we used Ang2-deficient mice to test that hypothesis. Mice deficient in Ang2 showed delayed and incomplete development of the superficial vascular bed of the retina, which develops primarily by vasculogenesis, and complete absence of the intermediate and deep vascular beds which develop by angiogenesis. In addition to incomplete retinal vascular development, Ang2-deficient mice showed lack of regression of the hyaloid vasculature, resulting in a phenotype that mimics infants with persistent fetal vasculature (PFV), a relatively common congenital abnormality. Exposure to high levels of oxygen resulted in partial regression of the retinal vessels, indicating that oxygen-induced regression of retinal vessels does not require Ang2. When these oxygen-exposed mice with few retinal vessels were moved to room air, there was no ischemia-induced retinal neovascularization. These data support the hypothesis that Ang2 plays a critical role in physiologic and pathologic angiogenesis, and physiologic, but not oxygen-induced vascular regression. The data also suggest that infants with PFV should be examined for genetic modifications that would be expected to cause perturbations in Tie2 signaling.