Cytoplasm Of Infected Cells Research Articles

Interaction of the Mycobacterial Heparin-Binding Hemagglutinin with Actin, as Evidenced by Single-Molecule Force Spectroscopy

Although Mycobacterium tuberculosis and related species are considered to be typical endosomal pathogens, recent studies have suggested that mycobacteria can be present in the cytoplasm of infected cells and cause cytoskeleton rearrangements, the mechanisms of which remain unknown. Here, we used single-molecule force spectroscopy to demonstrate that the heparin-binding hemagglutinin (HBHA), a surface adhesin from Mycobacterium tuberculosis displaying sequence similarities with actin-binding proteins, is able to bind to actin. Force curves recorded between actin and the coiled-coil, N-terminal domain of HBHA showed a bimodal distribution of binding forces reflecting the detection of single and double HBHA-actin interactions. Force curves obtained between actin and the lysine-rich C-terminal domain of HBHA showed a broader distribution of binding events, suggesting they originate primarily from intermolecular electrostatic bridges between cationic HBHA domains and anionic actin residues. We also explored the dynamics of the HBHA-actin interaction, showing that the binding force and binding frequency increased with the pulling speed and contact time, respectively. Taken together, our data indicate that HBHA is able to specifically bind actin, via both its N-terminal and C-terminal domains, strongly suggesting a role of the HBHA-actin interaction in the pathogenesis of mycobacterial diseases.

Structural and Functional Characterization of Nonstructural Protein 2 for Its Role in Hepatitis C Virus Assembly

The hepatitis C virus (HCV) is a flavivirus replicating in the cytoplasm of infected cells. The HCV genome is a single-stranded RNA encoding a polyprotein that is cleaved by cellular and viral proteases into 10 different products. While the structural proteins core protein, envelope protein 1 (E1) and E2 build up the virus particle, most nonstructural (NS) proteins are required for RNA replication. One of the least studied proteins is NS2, which is composed of a C-terminal cytosolic protease domain and a highly hydrophobic N-terminal domain. It is assumed that the latter is composed of three trans-membrane segments (TMS) that tightly attach NS2 to intracellular membranes. Taking advantage of a system to study HCV assembly in a hepatoma cell line, in this study we performed a detailed characterization of NS2 with respect to its role for virus particle assembly. In agreement with an earlier report ( Jones, C. T., Murray, C. L., Eastman, D. K., Tassello, J., and Rice, C. M. (2007) J. Virol. 81, 8374-8383 ), we demonstrate that the protease domain, but not its enzymatic activity, is required for infectious virus production. We also show that serine residue 168 in NS2, implicated in the phosphorylation and stability of this protein, is dispensable for virion formation. In addition, we determined the NMR structure of the first TMS of NS2 and show that the N-terminal segment (amino acids 3-11) forms a putative flexible helical element connected to a stable alpha-helix (amino acids 12-21) that includes an absolutely conserved helix side in genotype 1b. By using this structure as well as the amino acid conservation as a guide for a functional study, we determined the contribution of individual amino acid residues in TMS1 for HCV assembly. We identified several residues that are critical for virion formation, most notably a central glycine residue at position 10 of TMS1. Finally, we demonstrate that mutations in NS2 blocking HCV assembly can be rescued by trans-complementation.

The molecular and cellular basis of infection—Perspectives from the first advanced summer school in Africa

The molecular and cellular basis of infection—Perspectives from the first advanced summer school in Africa

African swine fever virus protein p30 interaction with heterogeneous nuclear ribonucleoprotein K (hnRNP-K) during infection

Heterogeneous nuclear ribonucleoprotein K (hnRNP-K) was identified as interacting cellular protein with the abundant immediate early protein p30 from African swine fever virus (ASFV) in a macrophage cDNA library screening. The interacting regions of hnRNP-K with p30 were established within residues 35–197, which represent KH1 and KH2 domains responsible for RNA binding. Colocalization of hnRNP-K and p30 was observed mainly in the nucleus, but not in the cytoplasm of infected cells and infection modified hnRNP-K subcellular distribution and decreased the incorporation of 5-fluorouridine into nascent RNA. Since similar effects were observed in cells transiently expressing p30, this interaction provides new insights into p30 function and could represent a possible additional mechanism by which ASFV downregulates host cell mRNA translation. Structured summaryMINT-6742660:hnRNP-K (uniprotkb:P61978) physically interacts (MI:0218)with p30 (uniprotkb:Q8V1E7) by pull down (MI:0096)MINT-6742673, MINT-6742696, MINT-6742729:hnRNP-K (uniprotkb:P61978) physically interacts (MI:0218)with p30 (uniprotkb:Q8V1E7) by two hybrid (MI:0018)MINT-6742711:p30 (uniprotkb:Q8V1E7) and hnRNP-K (uniprotkb:P61978) colocalize (MI:0403) by fluorescence microscopy (MI:0416)

SARS-Coronavirus Replication Is Supported by a Reticulovesicular Network of Modified Endoplasmic Reticulum

Positive-strand RNA viruses, a large group including human pathogens such as SARS-coronavirus (SARS-CoV), replicate in the cytoplasm of infected host cells. Their replication complexes are commonly associated with modified host cell membranes. Membrane structures supporting viral RNA synthesis range from distinct spherular membrane invaginations to more elaborate webs of packed membranes and vesicles. Generally, their ultrastructure, morphogenesis, and exact role in viral replication remain to be defined. Poorly characterized double-membrane vesicles (DMVs) were previously implicated in SARS-CoV RNA synthesis. We have now applied electron tomography of cryofixed infected cells for the three-dimensional imaging of coronavirus-induced membrane alterations at high resolution. Our analysis defines a unique reticulovesicular network of modified endoplasmic reticulum that integrates convoluted membranes, numerous interconnected DMVs (diameter 200–300 nm), and “vesicle packets” apparently arising from DMV merger. The convoluted membranes were most abundantly immunolabeled for viral replicase subunits. However, double-stranded RNA, presumably revealing the site of viral RNA synthesis, mainly localized to the DMV interior. Since we could not discern a connection between DMV interior and cytosol, our analysis raises several questions about the mechanism of DMV formation and the actual site of SARS-CoV RNA synthesis. Our data document the extensive virus-induced reorganization of host cell membranes into a network that is used to organize viral replication and possibly hide replicating RNA from antiviral defense mechanisms. Together with biochemical studies of the viral enzyme complex, our ultrastructural description of this “replication network” will aid to further dissect the early stages of the coronavirus life cycle and its virus-host interactions.

Structural Rearrangement within an Enveloped Virus upon Binding to the Host Cell

We have made the surprising discovery that the interactions of herpes simplex virus with its initial cell attachment receptor induce a rapid and highly efficient structural change in the tegument, the region of the virion situated between the membrane and the capsid. It has been known for nearly a decade that viruses can trigger host signaling pathways when they bind to receptors on the cell surface; however, until now there has been no evidence that a signal can be sent in reverse--from the "outside in"--across a viral membrane. Evidence for this signaling event was found during studies of UL16, a tegument protein that is conserved among all the herpesviruses. Previous work has demonstrated that UL16 is bound to capsids isolated from the cytoplasm of infected cells, but this interaction is destabilized during subsequent egress steps, leading to release of the extracellular virion. Pretreatment with N-ethylmaleimide, a small, membrane-permeating compound that covalently modifies free cysteines, restabilizes the interaction, thereby permitting the capsid-UL16 complex to be isolated following disruption of virions with NP-40. In the experiments described here, we found that the natural signal for release of UL16 from capsids is sent when virions merely bind to cells at 4 degrees C. The internal change was also observed upon binding to immobilized heparin in a manner that requires viral glycoprotein C. This represents the first example of signaling across a viral envelope following receptor binding.

Structural and functional analysis of virus factories purified from Rabbit vesivirus-infected Vero cells

Rabbit vesivirus infection induces membrane modifications and accumulation of vesicular structures in the cytoplasm of infected Vero cells. Crude RaV replication complexes (RCs) have been purified and their structural and functional properties have been characterized. We show that calnexin, an ER-resident protein, RaV non-structural proteins 2AB-, 2C-, 3A-, 3B- and 3CD-like as well as viral RNAs co-localize within membranous structures which are able to replicate the endogenous RNA templates. The purified virus factories protected their viral RNA contents from microccocal nuclease degradation and were inaccessible to exogenously added synthetic transcripts. In addition, we have shown that RCs can be used to investigate uridylylation of native endogenous VPg. In contrast to the observation that the virus factories were inaccessible to RNAs, RCs were accessible to added recombinant VPg which was subsequently nucleotidylylated. Nevertheless no elongation of an RNA chain attached to native or recombinant VPg could be demonstrated.

VSV-G pseudotyping rescues HIV-1 CA mutations that impair core assembly or stability

BackgroundThe machinery of early HIV-1 replication still remains to be elucidated. Recently the viral core was reported to persist in the infected cell cytoplasm as an assembled particle, giving rise to the reverse transcription complex responsible for the synthesis of proviral DNA and its transport to the nucleus. Numerous studies have demonstrated that reverse transcription of the HIV-1 genome into proviral DNA is tightly dependent upon proper assembly of the capsid (CA) protein into mature cores that display appropriate stability. The functional impact of structural properties of the core in early replicative steps has yet to be determined.ResultsHere, we show that infectivity of HIV-1 mutants bearing S149A and S178A mutations in CA can be efficiently restored when pseudotyped with vesicular stomatitis virus envelope glycoprotein, that addresses the mutant cores through the endocytic pathway rather than by fusion at the plasma membrane. The mechanisms by which these mutations disrupt virus infectivity were investigated. S149A and S178A mutants were unable to complete reverse transcription and/or produce 2-LTR DNA. Morphological analysis of viral particles and in vitro uncoating assays of isolated cores demonstrated that infectivity defects resulted from disruption of the viral core assembly and stability for S149A and S178A mutants, respectively. Consistent with these results, both mutants failed to saturate TRIM-antiviral restriction activity.ConclusionDefects generated at the level of core assembly and stability by S149A and S178A mutations are sensitive to the way of delivery of viral nucleoprotein complexes into the target cell. Addressing CA mutants through the endocytic pathway may compensate for defects generated at the reverse transcription/nuclear import level subsequent to impairment of core assembly or stability.

Varicella-zoster virus ORF1 gene product is a tail-anchored membrane protein localized to plasma membrane and trans-Golgi network in infected cells

Varicella-zoster virus (VZV) encodes five genes that do not have herpes simplex virus homologs. One of these genes, VZV open reading frame 1 (ORF1), encodes a membrane protein with a hydrophobic domain at its C-terminus that is predicted to be the transmembrane domain. However, the detailed characterization of ORF1 protein in infected cells has not been reported. Here, we produced mono-specific antibodies against ORF1 protein and characterized the gene products in infected cells. Western blot analyses showed the ORF1 polypeptides had apparent molecular masses of approximately 14–17 kDa. Furthermore, ORF1 was found to be a phosphoprotein by immunoprecipitation assay. In immunofluorescence assays, the VZV ORF1 protein was detected at both the plasma membrane and trans-Golgi network in both VZV-infected and ORF1-transfected cells. Moreover, ORF1 proteins associated with each other to form homodimer, and were incorporated into viral particles. The C-terminal hydrophobic domain was required for the association of ORF1 with the membrane structures, indicating that ORF1 protein is anchored to the membrane thorough its C-terminus, which is a transmembrane domain. Because ORF1 possesses a C-terminal transmembrane domain without an N-terminal signal sequence for its translocation to the ER lumen, ORF1 can be classified as a tail-anchored membrane protein. These results show that the N terminus of ORF1 protein faces the cytoplasm in infected cells and the tegument region in mature virions.

Identification of envelope protein pORF81 of koi herpesvirus

Koi herpesvirus (KHV), an emerging pathogen causing mass mortality in koi and common carp, possesses the largest known herpesvirus genome of 295 kbp predicted to encode 156 different proteins. However, none of them has been identified or functionally characterized up to now. In this study, a rabbit antiserum was prepared against a bacterial fusion protein that permitted detection of the predicted type III membrane protein encoded by ORF81 of KHV. In Western blot analyses, the abundant ORF81 gene product of KHV exhibited an apparent mass of 26 kDa and appeared to be non-glycosylated. It could be localized in the cytoplasm of infected cells and in virion envelopes by indirect immunofluorescence and immunoelectron microscopy, respectively. The antiserum was also suitable for the detection of pORF81 in sections of gills, kidneys, hepatopancreas and skin of KHV-infected carp by immunohistochemistry.

1H, 13C, and 15N resonance assignment of the N-terminal domain of Mason-Pfizer monkey virus capsid protein, CA 1-140

Mason-Pfizer monkey virus (M-PMV) belongs to the family of betaretroviruses characterized by the assembly of immature particles within cytoplasm of infected cells in contrast to other retroviruses (e.g. HIV, RSV) that assemble their immature particles at a plasma membrane. Simultaneously with or shortly after budding a virus-encoded protease is activated and the Gag polyprotein is cleaved into three major structural proteins: matrix (MA), capsid (CA), and nucleocapsid (NC) protein. Mature retroviral CA proteins consist of two independently folded structural domains: N-terminal domain (NTD) and C-terminal dimerization domain (CTD), separated by a flexible linker. As a first step toward the solution structure elucidation, we present nearly complete backbone and side-chain 1H, 13C and 15N resonance assignment of the M-PMV NTD CA.

Mutation in murine coronavirus replication protein nsp4 alters assembly of double membrane vesicles

Coronaviruses are positive-strand RNA viruses that replicate in the cytoplasm of infected cells by generating a membrane-associated replicase complex. The replicase complex assembles on double membrane vesicles (DMVs). Here, we studied the role of a putative replicase anchor, nonstructural protein 4 (nsp4), in the assembly of murine coronavirus DMVs. We used reverse genetics to generate infectious clone viruses (icv) with an alanine substitution at nsp4 glycosylation site N176 or N237, or an asparagine to threonine substitution (nsp4-N258T), which is proposed to confer a temperature sensitive phenotype. We found that nsp4-N237A is lethal and nsp4-N258T generated a virus (designated Alb ts6 icv) that is temperature sensitive for viral replication. Analysis of Alb ts6 icv-infected cells revealed that there was a dramatic reduction in DMVs and that both nsp4 and nsp3 partially localized to mitochondria when cells were incubated at the non-permissive temperature. These results reveal a critical role of nsp4 in directing coronavirus DMV assembly.

Remodelling of the nuclear lamina during human cytomegalovirus infection: role of the viral proteins pUL50 and pUL53

A fundamental step in the efficient production of human cytomegalovirus (HCMV) progeny is viral egress from the nucleus to the cytoplasm of infected cells. In the family Herpesviridae, this process involves alteration of nuclear lamina components by two highly conserved proteins, whose homologues in HCMV are named pUL50 and pUL53. This study showed that HCMV infection induced the mislocalization of nuclear lamins and that pUL50 and pUL53 play a role in this event. At late stages of infection, both lamin A/C and lamin B showed an irregular distribution on the nuclear rim, coincident with areas of pUL53 accumulation. No variations in the total amount of nuclear lamins could be detected, supporting the view that HCMV induces a qualitative, rather than a quantitative, alteration of these cellular components, as has been suggested previously for other herpesviruses. Interestingly, pUL53, in the absence of other viral products, localized diffusely in the nucleus, whilst the co-expression and interaction of pUL53 with its partner, pUL50, restored its nuclear rim localization in distinct patches, thus indicating that pUL50 is sufficient to induce the localization of pUL53 observed during virus infection. Importantly, analysis of the nuclear lamina in the presence of pUL50-pUL53 complexes at the nuclear boundary and in the absence of other viral products showed that the two viral proteins were sufficient to promote alterations of lamins, strongly resembling those observed during HCMV infection. These results suggest that pUL50 and pUL53 may play an important role in the exit of virions from the nucleus by inducing structural modifications of the nuclear lamina.

Role of Sindbis Virus Capsid Protein Region II in Nucleocapsid Core Assembly and Encapsidation of Genomic RNA

Sindbis virus is an enveloped positive-sense RNA virus in the alphavirus genus. The nucleocapsid core contains the genomic RNA surrounded by 240 copies of a single capsid protein. The capsid protein is multifunctional, and its roles include acting as a protease, controlling the specificity of RNA that is encapsidated into nucleocapsid cores, and interacting with viral glycoproteins to promote the budding of mature virus and the release of the genomic RNA into the newly infected cell. The region comprising amino acids 81 to 113 was previously implicated in two processes, the encapsidation of the viral genomic RNA and the stable accumulation of nucleocapsid cores in the cytoplasm of infected cells. In the present study, specific amino acids within this region responsible for the encapsidation of the genomic RNA have been identified. The region that is responsible for nucleocapsid core accumulation has considerable overlap with the region that controls encapsidation specificity.

The human cytomegalovirus regulatory protein UL69 and its effect on mRNA export

One of the characteristic features of herpesviruses is that most of their genes are intronless. Thus, their replication relies on the selective nuclear export of intronless viral mRNAs, which have to compete with the nuclear export of bulk spliced cellular mRNAs. Therefore, the regulation of nuclear mRNA export is crucial for the replication and pathogenesis of herpesviruses. Besides the thymidine kinase transcript of herpes simplex virus type 1, which contains specific sequences to facilitate the nuclear export of intronless mRNA, other cis-acting RNA elements for nuclear mRNA export have not yet been identified in the rest of herpesviral intronless mRNAs. Instead, emerging studies show that herpesviruses encode viral mRNA export factors, which interact with components of the major cellular mRNA export pathway, the RNA polymerase II transcription complex and specific splicing factors to selectively export intronless herpesviral mRNAs to the cytoplasm in infected cells. These herpesviral mRNA export factors are members of a conserved gene family that can be found in all herpesviruses. The human cytomegalovirus transactivator protein UL69, which has been demonstrated to belong to this conserved protein family, shares common features with its herpesviral homologues but also possesses unique properties that will be discussed in this review.

Bacterial Actin Assembly Requires Toca-1 to Relieve N-WASP Autoinhibition

Actin polymerization in the mammalian cytosol can be locally activated by mechanisms that relieve the autoinhibited state of N-WASP, an initiator of actin assembly, a process that also requires the protein Toca-1. Several pathogenic bacteria, including Shigella, exploit this host feature to infect and disseminate efficiently. The Shigella outer membrane protein IcsA recruits N-WASP, which upon activation at the bacterial surface mediates localized actin polymerization. The molecular role of Toca-1 in N-WASP activation during physiological or pathological actin assembly processes in intact mammalian cells remains unclear. We show that actin tail initiation by S. flexneri requires Toca-1 for the conversion of N-WASP from a closed inactive conformation to an open active one. While N-WASP recruitment is dependent on IcsA, Toca-1 recruitment is instead mediated by S. flexneri type III secretion effectors. Thus, S. flexneri independently hijacks two nodes of the N-WASP actin assembly pathway to initiate localized actin tail assembly.

New mRNAs Are Preferentially Translated during Vesicular Stomatitis Virus Infection

During vesicular stomatitis virus (VSV) infection, host protein synthesis is inhibited, while synthesis of viral proteins increases. VSV infection causes inhibition of host transcription and RNA transport. Therefore, most host mRNAs in the cytoplasm of infected cells were synthesized before infection. However, viral mRNAs are synthesized throughout infection and are newer than preexisting host mRNAs. To determine if the timing of appearance of mRNAs in the cytoplasm affected their translation during VSV infection, we transfected reporter mRNAs into cells at various times relative to the time of infection and measured their rate of translation in mock- and VSV-infected cells. We found that translation of mRNAs transfected during infection was not inhibited but that translation of mRNAs transfected prior to infection was inhibited during VSV infection. Based on these data, we conclude that the timing of viral mRNA appearance in the cytoplasm is responsible, at least in part, for the preferential translation of VSV mRNAs. A time course measuring translation efficiencies of viral and host mRNAs showed that the translation efficiencies of viral mRNAs increased between 4 and 8 h postinfection, while translation efficiencies of host mRNAs decreased. The increased translation efficiency of viral mRNAs occurred in cells infected with an M protein mutant virus that is defective in host shutoff, demonstrating that the enhanced translation of viral mRNA is genetically separable from inhibition of translation of host mRNA.

Characterization of a late gene, ORF67 from Bombyx mori nucleopolyhedrovirus

Open reading frame 67 of Bombyx mori nucleopolyhedrovirus (BmORF67) is a homologue of Autographa californica multiple NPV ORF81. The gene is conserved among all baculoviruses and is thus considered a baculovirus core gene. The transcript of BmORF67 was detected at 18–72 h post-infection (p.i.). Polyclonal antiserum raised to a His-BmORF67 fusion protein recognized BmORF67 in infected cell lysates from 24 to 72 h p.i., suggesting that BmORF67 is a late gene. BmORF67 was not detected either in budded viruses or occlusion-derived virus. Immunofluoresence analysis showed that the protein located in the cytoplasm and interacted with host protein actin A3. In conclusion, BmORF67 is a late protein localized in the cytoplasm of infected cells that interacts with host protein.

Design of beta-hairpin peptidomimetics that inhibit binding of alpha-helical HIV-1 Rev protein to the rev response element RNA.

The virally encoded Rev protein of human immunodeficiency virus type-1 (HIV-1) plays a critical role in viral replication by regulating the transport of unspliced and partially spliced viral RNA from the nucleus to the cytoplasm of infected cells.[1, 2] Rev binds first to a specific region of the HIV-1 mRNA, called the Rev responsive element (RRE) of stem loop IIb (Figure 1); subsequent to this initial binding event, approximately 10 additional Rev molecules oligomerize through protein–protein and protein–RNA interactions and coat the entire RRE.[3] Because of its essential role in viral replication, the interaction between Rev protein and the high-affinity RRE binding site represents an attractive yet unexploited target for antiviral therapy.

The p26 gene of the Autographa californica nucleopolyhedrovirus: Timing of transcription, and cellular localization and dimerization of product

p26, an early AcMNPV gene, codes for a 240-amino acid polypeptide of unknown function. Primer extension analysis showed that the p26 transcripts, initiating at three clustered start sites, accumulated between 2 and 12h post-infection, after which these transcripts declined in quantity. Indirect immunofluorescence studies detected the p26 protein primarily dispersed in the cytoplasm of infected cells, although some staining of the nucleus was also observed. Immunoblots of infected cell fractions also detected p26 primarily in the cytoplasm. A yeast two-hybrid assay detected p26 in association only with other p26 molecules. Biochemical analysis showed that p26 forms dimers under physiological conditions.