Bacterial intermediate filaments: in vivo assembly,organization, and dynamics of crescentin

Crescentin, which is the founding member of a rapidly growing family of bacterial cytoskeletal proteins, was previously proposed to resemble eukaryotic intermediate filament (IF) proteins based on structural prediction and in vitro polymerization properties. Here, we demonstrate that crescentin also shares in vivo properties of assembly and dynamics with IF proteins by forming stable filamentous structures that continuously incorporate subunits along their length and that grow in a nonpolar fashion. De novo assembly of crescentin is biphasic and involves a cell size-dependent mechanism that controls the length of the structure by favoring lateral insertion of crescentin subunits over bipolar longitudinal extension when the structure ends reach the cell poles. The crescentin structure is stably anchored to the cell envelope, and this cellular organization requires MreB function, identifying a new function for MreB and providing a parallel to the role of actin in IF assembly and organization in metazoan cells. Additionally, analysis of an MreB localization mutant suggests that cell wall insertion during cell elongation normally occurs along two helices of opposite handedness, each counterbalancing the other''s torque.     

New biological research and understanding of Papanicolaou's test

The development of the Papanicolaou smear test by Dr. George Nicholas Papanicolaou (1883‐1962) is one of the most significant achievements in screening for disease and cancer prevention in history. The Papanicolaou smear has been used for screening of cervical cancer since the 1950s. The test is technically straightforward and practical and based on a simple scientific observation: malignant cells have an aberrant nuclear morphology that can be distinguished from benign cells. Here, we review the scientific understanding that has been achieved and continues to be made on the causes and consequences of abnormal nuclear morphology, the basis of Dr. Papanicolaou's invention. The deformed nuclear shape is caused by the loss of lamina and nuclear envelope structural proteins. The consequences of a nuclear envelope defect include chromosomal numerical instability, altered chromatin organization and gene expression, and increased cell mobility because of a malleable nuclear envelope. HPV (Human Papilloma Virus) infection is recognized as the key etiology in the development of cervical cancer. Persistent HPV infection causes disruption of the nuclear lamina, which presents as a change in nuclear morphology detectable by a Papanicolaou smear. Thus, the causes and consequences of nuclear deformation are now linked to the mechanisms of viral carcinogenesis, and are still undergoing active investigation to reveal the details. Recently a statue was installed in front of the Papanicolaou's Cancer Research Building to honor the inventor. Remarkably, the invention nearly 60 years ago by Dr. Papanicolaou still exerts clinical impacts and inspires scientific inquiries.     

Homeostasis of T cell diversity

T cell homeostasis commonly refers to the maintenance of relatively stable T cell numbers in the peripherallymphoid organs.Among the large numbers of T cells in the periphery,T cells exhibit structural diversity,i.e.,theexpression of a diverse repertoire of T cell receptors(TCRs),and functional diversity,i.e.,the presence of T cells atnave,effector,and memory developmental stages.Although the homeostasis of T cell numbers has been extensivelystudied,investigation of the mechanisms underlying the maintenance of structural and functional diversity of Tceils is still at an early stage.The fundamental feature throughout T cell development is the interaction between theTCR and either self or foreign peptides in association with MHC molecules.In this review,we present evidenceshowing that homeostasis of T cell number and diversity is mediated through competition for limiting resources.The number of T cells is maintained through competition for limiting cytokines,whereas the diversity of T cells ismaintained by competition for self-peptide-MHC complexes.In other words,diversity of the self-peptide repertoirelimits the structural(TCR)diversity of a T cell population.We speculate that cognate low affinity self-peptides,acting as weak agonists and antagonists,regulate the homeostasis of T cell diversity whereas non-cognate or nullpeptides which are extremely abundant for any given TCR,may contribute to the homeostasis of T cell number byproviding survival signals.Moreover,self-peptides and cytokines may form specialized niches for the regulation ofT cell homeostasis.Cellular & Molecular Immunology.2005;2(1):1-10.     

Louping ill virus envelope protein expressed by recombinant baculovirus and vaccinia virus fails to stimulate protective immunity.

We have constructed recombinant baculoviruses and vaccinia viruses containing cloned DNA, encoding either the envelope protein alone or all of the structural proteins (core, membrane and envelope) of louping ill virus. Glycosylated viral envelope protein, presented both inside and on the surface of insect and mammalian cells, was expressed by all four recombinant viruses. Differences in antigenic presentation of the envelope protein were observed between the envelope protein and structural protein constructs as well as between the insect and mammalian cell expression systems. Despite the expression of epitopes known to elicit neutralizing and protective antibodies when present in authentic antigen, the recombinant envelope protein expressed by either vector failed to induce, in mice or rabbits, either neutralizing or protective antibodies against louping ill virus.     

Multiple envelope proteins are involved in white spot syndrome virus (WSSV) infection in crayfish

Summary. White spot syndrome virus (WSSV) is a devastating viral pathogen of cultured shrimp worldwide. Previous studies have shown that the intact virion consists of at least 39 structural proteins and, among them, six were identified as envelope proteins involved in the virus infection. In this paper, the structural proteins VP36A, VP36B and VP31 (J Virol 2004; 78: 11360–11370), containing the RGD motif, were expressed in Escherichia coli and used to produce specific antibodies. Western blot confirmed that VP36A is a newly reported envelope protein. A neutralization assay with these three antibodies demonstrated that VP36A, VP36B and VP31 could significantly delay the initial infection of crayfish, but mortality still reached 100% at day 11 post-injection. However, a neutralization assay with the combination of antibodies against different envelope proteins showed that a combination of VP36B and VP31 antibodies could strongly inhibit WSSV infection in crayfish. These results revealed that multiple envelope proteins are involved in WSSV infection in crayfish and that VP36B and VP31 play a key role during this process.     

T4 gene 40 mutants. II. Phenotypic properties.

Evidence is presented that p40 affects the solubility of p20 inside the T4-infected cell. In the absence of p20 and p40 function at high temperature, p20 is located specifically and exclusively in the cell envelope. It is possible that p40 may interact with p20 before head assembly can be initiated. Cleavage of the major head protein in gene 40 mutant-infected cells is also inhibited. In addition to producing single-layered polyheads and phage at 30°, am restrictive cells infected with gene 40 am mutants produce some empty capsids. It is likely that p40 function is only strictly required for phage growth at high temperature. Electron microscopy of thin sections of crude cell envelopes showed few recognizable structural components, although most phage structural proteins were present upon sodium dodecyl sulfate-gel electrophoresis of envelope preparations. Extraction with sodium chloride selectively removes some of the viral proteins from the crude cell envelope preparation, while extraction with 4 M guanidine · HCl greatly reduces the amount of T4 protein in the cell envelopes. The envelope association of most of these proteins appears to be nonspecific.     

Analysis of a non-structural gene reveals evidence of possible hepatitis C virus (HCV) compartmentalization

Viral diversity is a hallmark of hepatitis C virus (HCV) infection; however, only limited data are available regarding HCV variability in extrahepatic sites, and none have systematically compared diversity in non‐structural and structural genomic regions. Therefore, HCV diversity in the NS5B and envelope 1 (E1) hypervariable region 1 (HVR1) genes was evaluated in matched sera and peripheral blood mononuclear cells (PBMCs) obtained from 13 HCV‐infected women. Multiple clonal sequences were compared to evaluate quasispecies diversity and viral compartmentalization in PBMCs. Genetic distances were higher for E1/HVR1 compared to NS5B in both the sera and PBMCs (P = 0.0511 and 0.0284). Genetic distances were higher in serum NS5B compared to PBMC NS5B (P = 0.0003); however, they were not different when comparing E1/HVR1 in sera to PBMCs. By phylogenetic analysis of NS5B, evidence of possible PBMC compartmentalization was observed for one woman, while statistical methods were consistent with PBMC compartmentalization for six women. Evidence of compartmentalization within a non‐structural genomic region may suggest that viral adaptation to a unique extracellular microenvironment(s) may be required for efficient replication and could contribute to HCV persistence. J. Med. Virol. 84:242–252, 2012. © 2011 Wiley Periodicals, Inc.     

Development of Transgenic Sheep That Express the Visna Virus Envelope Gene

The ovine lentiviruses cause encephalitis, pneumonia, and arthritis in sheep worldwide. Visna virus is a prototype of this family and the pathogenesis and molecular biology of the virus has been well characterized. The envelope proteins of visna virus are responsible for binding of virus to host cells and for causing cell fusion. The surface glycoprotein also elicits cellular and humoral immune responses to the virus, the former being thought to be responsible for eliminating infected cells as well as causing inflammatory lesions. In this study, transgenic sheep were constructed that expressed the envelope genes of visna virus under the control of the visna LTR to investigate the role of the env gene in the pathogenesis of lentiviral disease in its natural host. Three transgenic lambs were identified that contain the env transgene and express the envelope glycoproteins. These transgenic animals have remained healthy and expression of the viral gene has had no obvious deleterious effect. Expression of the visna envelope protein was demonstrated by cell fusion mediated by the envelope gene as well as by immunoprecipitation of the envelope proteins with monoclonal antibodies and immunofluorescence analyses of Env protein in cells. The target cell for visna virus replication in infected animals is the monocyte/macrophage. In natural infection, the level of viral gene expression in these cells increases with cell maturation. In the transgenic sheep, monocytes did not express the envelope glycoproteins until they differentiated into macrophages in vitro. Expression of the env mRNA in macrophages was quantitated by an RNase protection assay. In addition to expression in macrophages, the transgene was expressed by fibroblasts isolated from skin of the transgenic sheep. Expression of both the Env and Rev proteins was detected by immunoprecipitation and immunofluorescence. Two of the three lambs responded immunologically to the expression of the transgene by producing binding antibodies to the envelope glycoproteins. Thus, these transgenic sheep provide a model to study whether a lentivirus glycoprotein will prevent infection or modulate disease in its natural host after virus challenge.     

Expression of HTLV-specific polypeptides in various human T-cell lines

The adult T-cell leukemia(ATL)-associated antigen complex (ATLA) was first discovered with indirect immunofluorescence by Hinuma et al. (1981). Biochemical analysis with MT-2 cells revealed that ATLA consisted mainly of human T-cell leukemia virus (HTLV) structural polypeptides and their precursors (Yamamoto and Hinuma 1982a; Schneider et al. 1984). In this study, we have investigated the molecular nature of the ATLA antigen complex in various HTLV-positive human cell lines established by different methods including independently established HTLV-infected HUT 102 cells. We found that HTLVs infecting these cell lines have similar core polypeptides, p24 and p19, as well as an envelope glycopolypeptide, gp46, in all these cells. The intracellular gp61 and p53 appear to be precursors of the viral envelope and core polypeptides, respectively. Interestingly, MT-2 and MT-2 related T-cell lines contain two different species of envelope proteins, gp68 and gp61, whereas cell lines not related to MT-2 express only gp61.     

Expression of type-specific virion structural antigen(s) in L cells transformed by herpes simplex virus types 1 and 2.

Antisera prepared against thymidine kinaseless (tk^?) L cells (Ltk^?) transformed to the tk⁺ phenotype by herpes simplex virus type 1 (HSV-1) or type 2 (HSV-2) preferentially agglutinated purified homologous virions. Transformed cell antisera were much less effective in agglutinating nucleocapsids than they were in agglutinating envelope material from the homologous virus. Antiserum prepared against untransformed Ltk^? cells agglutinated about twice as much virus as did preimmune serum but only 13 to 19% as much as was agglutinated by transformed cell antisera. These results indicate that both HSV-1- and HSV-2-transformed L cells express an HSV type-specific structural antigen(s) and that this antigen is present in the envelope of the virion. Since the transformed cell antisera also agglutinated the heterologous virus but to a lesser extent than the homologous virus, these antisera were capable of detecting type-common antigenic determinants as well.     

Detection of flavivirus antigens in purified infected Vero cell plasma membranes.

The types of Kunjin virus-specified proteins present in purified Vero cell plasma membrane were studied. Immunofluorescence of unfixed Kunjin virus-infected whole cell monolayers, indicated that two structural proteins (envelope and prM) and three non-structural proteins (NS1, 3 and 5) were found at the plasma membrane. There was no obvious progressive accumulation of the observed antigens over the time periods between 8 to 24 h p.i. Thus SDS-PAGE analysis was performed using purified radiolabelled Vero cell plasma membranes. From the protein profiles, all five antigens detected by immunofluorescent staining were also present. In addition, two smaller molecular weight non-structural proteins NS4B and NS2B were also observed. Generally, all the non-structural proteins found in the purified plasma membranes were of the same molecular weights as those found in infected whole cell lysate. Interestingly, both the structural proteins, i.e., envelope (E) and prM proteins in the plasma membrane sample were of higher molecular weights as compared to the counterparts in the infected whole cell lysate. The envelope protein of purified extracellular Kunjin virus was also lower in molecular weight compared to the same protein in the plasma membrane.     

Electron paramagnetic resonance study of the structural characteristics of the envelope of the Venezuelan equine encephalitis virus

The method of electron paramagnetic resonance and spin labels was used to study the structural characteristics of Venezuelan equine encephalomyelitis virus envelope and cell membranes of chick embryo fibroblasts. The virus envelope lipids form a bilayer structural typical of biological membranes. The lipid bilayer of virions is more rigid than the cytoplasmic membrane of chick embryo fibroblasts. Investigation of interaction of a small hydrophilic molecule with Venezuelan equine encephalomyelitis virus particles showed free water to be absent in the virions. A structural translation was detected at 25 degrees-30 degrees C in the virion membrane. The role of membrane structures in virus reproduction is discussed.     

Protein synthesis in Bunyamwera virus-infected cells.

In Vero cells infected with Bunyamwera virus there is a rapid inhibition of cell RNA and protein synthesis to levels of 30 and 3% respectively of the control rate, both the rate of inhibition and the time lag before its initiation being multiplicity dependent. Using u.v.-irradiated virus, investigation of the mechanism of inhibition of host cell protein synthesis indicates that synthesis of new virus components is required for inhibition to occur. Quantitative comparison of the proteins synthesized in infected cells shows that at higher m.o.i. synthesis of virus, as well as cellular proteins, is inhibited. Bunyamwera virus-infected Vero cells synthesized three virus-specific proteins identified as the structural virion proteins. Nucleoprotein is synthesized predominantly early in infection while the major envelope glycoprotein and the minor glycoprotein are synthesized predominantly late in the infection cycle.     

Surface display of IgG Fc on baculovirus vectors enhances binding to antigen-presenting cells and cell lines expressing Fc receptors

Recombinant baculoviruses (recBV) were constructed with dual cassettes for constitutive expression of human IgG Fc following infection of insect cells and the structural proteins of hepatitis C virus (core, E1 and E2) following transduction of mammalian cells. The IgG Fc was expressed in insect cells as a fusion protein with the signal sequence and transmembrane region of either the native baculovirus envelope protein gp64 or the human transferrin receptor as a type I or type II integral membrane protein, respectively. The IgG Fc fusion proteins formed functional homodimers on the surface of recBV-infected insect cells and were incorporated into the envelope of recBV particles during egress from the infected cell. Both pseudotyped recBV bound specifically to recombinant soluble FcγRIIα receptor and to cell lines and antigen-presenting cells expressing Fc receptors (FcRs). These novel baculoviral vectors, which target cells of the immune system that express FcRs, have potential applications for vaccination or gene therapy.     

Cell entry by enveloped viruses: redox considerations for HIV and SARS-coronavirus

For enveloped viruses, genome entry into the target cell involves two major steps: virion binding to the cell-surface receptor and fusion of the virion and cell membranes. Virus-cell membrane fusion is mediated by the virus envelope complex, and its fusogenicity is the result of an active virus-cell interaction process that induces conformation changes within the envelope. For some viruses, such as influenza, exposure to an acidic milieu within the cell during the early steps of infection triggers the necessary structural changes. However, for other pathogens which are not exposed to such environmental stress, activation of fusogenicity can result from precise thiol/disulfide rearrangements mediated by either an endogenous redox autocatalytic isomerase or a cell-associated oxidoreductase. Study of the activation of HIV envelope fusogenicity has revealed new knowledge about how redox changes within a viral envelope trigger fusion. We discuss these findings and their implication for anti-HIV therapy. In addition, to compare and contrast the situation outlined for HIV with an enveloped virus that can fuse with the cell plasma membrane independent of the redox status of its envelope protein, we review parallel data obtained on SARS coronavirus entry.     

The C-terminal tail of the gp41 transmembrane envelope glycoprotein of HIV-1 clades A, B, C, and D may exist in two conformations: an analysis of sequence, structure, and function

In addition to the major ectodomain, the gp41 transmembrane glycoprotein of HIV-1 is now known to have a minor ectodomain that is part of the long C-terminal tail. Both ectodomains are highly antigenic, carry neutralizing and non-neutralizing epitopes, and are involved in virus-mediated fusion activity. However, data have so far been biologically based, and derived solely from T cell line-adapted (TCLA), B clade viruses. Here we have carried out sequence and theoretically based structural analyses of 357 gp41 C-terminal sequences of mainly primary isolates of HIV-1 clades A, B, C, and D. Data show that all these viruses have the potential to form a tail loop structure (the minor ectodomain) supported by three, beta-sheet, membrane-spanning domains (MSDs). This means that the first (N-terminal) tyrosine-based sorting signal of the gp41 tail is situated outside the cell membrane and is non-functional, and that gp41 that reaches the cell surface may be recycled back into the cytoplasm through the activity of the second tyrosine-sorting signal. However, we suggest that only a minority of cell-associated gp41 molecules - those destined for incorporation into virions - has 3 MSDs and the minor ectodomain. Most intracellular gp41 has the conventional single MSD, no minor ectodomain, a functional first tyrosine-based sorting signal, and in line with current thinking is degraded intracellularly. The gp41 structural diversity suggested here can be viewed as an evolutionary strategy to minimize HIV-1 envelope glycoprotein expression on the cell surface, and hence possible cytotoxicity and immune attack on the infected cell.     

Electron microscopic studies on the role of the envelope antigens of R-MuLV-ts29 in budding

The role of the envelope antigens of R-MuLV-ts29 in budding was studied with various agents known to interfere with the viral envelope antigens. Other agents, inhibiting the synthesis of certain cell constituents, were also used to investigate the requirements for budding. The temperature sensitive R-MuLV mutant ts29 was used to synchronize budding. Preincubating cells at the nonpermissive temperature (39°) with anti-gp70 or trypsin did lead to normal numbers of budding particles after shiftdown to the permissive temperature (31°). These particles did not contain gp70. Preincubating cells with antip15E led to a complete inhibition of budding. Large submembranous patches were observed. The effects on budding of tunicamycin, an inhibitor of glycosylation, and a few other agents interfering with cell metabolism, such as cytochalasin B and interferon, were investigated.     

Viral sequence diversity: challenges for AIDS vaccine designs

Among the greatest challenges facing AIDS vaccine development is the intrinsic diversity among circulating populations of HIV-1 in various geographical locations and the need to develop vaccines that can elicit enduring protective immunity to variant HIV-1 strains. While variation is observed in all of the viral proteins, the greatest diversity is localized to the viral envelope glycoproteins, evidently reflecting the predominant role of these proteins in eliciting host immune recognition and responses that result in progressive evolution of the envelope proteins during persistent infection. Interestingly, while envelope glycoprotein variation is widely assumed to be a major obstacle to AIDS vaccine development, there is very little experimental data in animal or human lentivirus systems addressing this critical issue. In this review, the state of vaccine development to address envelope diversity will be presented, focusing on the use of centralized and polyvalent sequence design as mechanisms to elicit broadly reactive immune responses.