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At least 757 records · Page 42Linked to original sources
PubMed2006

Paramyxovirus fusion: real-time measurement of parainfluenza virus 5 virus-cell fusion.

Although cell-cell fusion assays are useful surrogate methods for studying virus fusion, differences between cell-cell and virus-cell fusion exist. To examine paramyxovirus fusion in real time, we labeled viruses with fluorescent lipid probes and monitored virus-cell fusion by fluorimetry. Two parainfluenza virus 5 (PIV5) isolates (W3A and SER) and PIV5 containing mutations within the fusion protein (F) were studied. Fusion was specific and temperature-dependent. Compared to many low pH-dependent viruses, the kinetics of PIV5 fusion was slow, approaching completion within several minutes. As predicted from cell-cell fusion assays, virus containing an F protein with an extended cytoplasmic tail (rSV5 F551) had reduced fusion compared to wild-type virus (W3A). In contrast, virus-cell fusion for SER occurred at near wild-type levels, despite the fact that this isolate exhibits a severely reduced cell-cell fusion phenotype. These results support the notion that virus-cell and cell-cell fusion have significant differences.

Animals↗
PubMed2006

Feline leukemia virus T entry is dependent on both expression levels and specific interactions between cofactor and receptor.

Feline leukemia virus (FeLV) subgroup T uses both a multiple membrane-spanning receptor, FePit1, and a soluble cofactor, FeLIX, to enter feline cells. FeLIX is expressed from endogenous FeLV-related sequence and resembles the receptor binding domain (RBD) of the viral envelope protein. It remains unclear whether FeLV-T receptor activity requires specific residues within FePit1 and FeLIX and/or a threshold level of receptor/cofactor expression. To address this, we examined FeLV-T infection of cells expressing variable levels of FePit1 and other gammaretroviral receptors in the presence of variable amounts of soluble cofactor, either RBD or the envelope surface subunit (SU). Cofactor-receptor pairs fall into three groups with regard to mediating FeLV-T infection: those that are efficient at all concentrations tested, such as FePit1 and FeLIX; those requiring high expression of both cofactor and receptor; and those that are non-functional as receptors even at high expression. This suggests that both expression levels and specific interactions with receptor and cofactor are critical for mediating entry of FeLV-T.

Animals↗
PubMed2006

Labeling HIV-1 virions with two fluorescent proteins allows identification of virions that have productively entered the target cell.

GFP-Vpr labeled HIV-1 virions have provided a method to visually examine the interactions between the virus and target cell during infection. However, existing methods to discriminate between virions that have been non-specifically endocytosed from those that have productively entered the host cell cytoplasm have remained problematic. Therefore, we examined the ability of a series of membrane-targeted fluorescent fusion protein constructs to be incorporated into virions. We find that a fluorescent protein fusion targeted to the plasma membrane by the addition of the N-terminal 15 amino acid sequence of c-Src (S15) is efficiently packaged into HIV virions. Using fluorescent proteins fused to this sequence, we have generated virions dually labeled with S15-mCherry and GFP-Vpr. Importantly, we can detect the loss of this S15-mCherry membrane signal following fusion. After infection with VSV-g pseudotyped HIV virions, we find a measurable, specific loss of membrane label during infection. This loss of fluorescence is not observed when fusion is prevented using bafilomycin A. This increased ability to discriminate between non-productively endocytosed virions and those actively undergoing steps in the infectious process will facilitate efforts to examine early steps in infection microscopically.

Cell Line↗
PubMed2006

Characterization of soluble glycoprotein D-mediated herpes simplex virus type 1 infection.

Herpes simplex virus type 1 (HSV-1) entry into permissive cells involves attachment to cell-surface glycosaminoglycans (GAGs) and fusion of the virus envelope with the cell membrane triggered by the binding of glycoprotein D (gD) to cognate receptors. In this study, we characterized the observation that soluble forms of the gD ectodomain (sgD) can mediate entry of gD-deficient HSV-1. We examined the efficiency and receptor specificity of this activity and used sequential incubation protocols to determine the order and stability of the initial interactions required for entry. Surprisingly, virus binding to GAGs did not increase the efficiency of sgD-mediated entry and gD-deficient virus was capable of attaching to GAG-deficient cells in the absence of sgD. These observations suggested a novel binding interaction that may play a role in normal HSV infection.

Animals↗
PubMed2004

An internal element of the measles virus antigenome promoter modulates replication efficiency.

The cis-acting sequence elements that direct measles virus (MV) genome synthesis reside in the 109 base non-coding region at the 5' trailer (3' antigenomic) end of MV genome that makes up the antigenomic promoter (AGP). The MV-AGP nucleotides 79-96, corresponding to nucleotide hexamers 14, 15 and 16 (the C' element), show sequence similarity with the equivalent region of many paramyxoviruses and are analogous to the three nucleotide hexamers that form the second replication control element in the Sendai virus AGP. In this study, results of two independent procedures demonstrate that the MV C' element also is a replication control sequence. Results of in vivo nucleotide selection experiments show that selection pressure for retaining the wild type nucleotides at the first position of each of the three hexamers, and for the fifth position of the 14th hexamer was relatively high. However, with continued replication, preference for the conservation of wild type nucleotides across the entire C' element was clearly evident. Results of mutational analysis of individual nucleotides in one or more hexamers in a measles-helper-virus driven reporter gene rescue system agreed with these results. Substitutions at the first position of the 14th, the 15th or the 16th hexamers reduced minireplicon activity dramatically. In contrast, changes at the other five positions of any one hexamer had little or no effect on minireplicon activity, even when all the five bases were changed at the same time. However, when minireplicons were analyzed which contained point mutations at equivalent positions in all three hexamers, it was evident that the nucleotides, particularly those at the 5th position, were also important components of the C' element. This pattern of sequence requirement in the C' element based on mutational analysis could be described as a distinct motif, 5'-(GNNNAN)2GNNNCN-3', that is important for MV replication.

Base Sequence↗
PubMed2006

Functional and antigenic analyses of the 1918 influenza virus haemagglutinin using a recombinant vaccinia virus expression system.

The influenza pandemic of 1918 caused unprecedented levels of morbidity and mortality in its 12-month period of circulation around the globe. The haemagglutinin molecule has been shown to affect the pathogenicity of some subtypes of influenza A viruses. Using a recombinant vaccinia system that allowed expression of the 1918 influenza haemagglutinin, we performed functional assays to assess the glycoprotein's involvement in determining the high pathogenicity of the 1918 virus. We show that in respect of expression levels, proteolytic processing, receptor-binding, membrane fusion and antigenic properties, the haemagglutinin of the 1918 virus is unremarkable when compared with the haemagglutinins of other 'early' H1 influenza viruses. This suggests that whilst the 1918 haemagglutinin, as a new/novel antigen in the human population, was responsible for the influenza pandemic its functions per se were not responsible for the high mortality and acute symptoms experienced by patients infected with the 1918 influenza virus.

Amino Acid Sequence↗
PubMed2006

HIV infection of mononuclear cells is calcium-dependent.

Strategies that prevent initial HIV infection of cells are greatly needed. In this study, we determined the requirement of divalent cations for HIV infection of and attachment to peripheral blood mononuclear cells (PBMC), which contain several types of HIV-infectable cells-CD4(+) T cells, monocytes and dendritic cells. EDTA, added only during PBMC exposure to HIV, reduced infection by an average of 92%. The reduction of infection by EDTA was accompanied by a reduction in HIV binding to PBMC; R5, X4 and dual-tropic HIV binding to PBMC were inhibited by >85%. EGTA similarly reduced HIV binding to PBMC, while addition of Ca(2+) or Mn(2+), but not Mg(2+), fully restored binding. Virus attachment was inhibited in a dose-dependent manner by trypsin treatment of PBMC, indicating protein involvement in HIV binding. In contrast, mannan or soluble ICAM-1 did not inhibit HIV binding to PBMC. These data indicate that a Ca(2+)-dependent cell-surface protein(s) is responsible for the majority of HIV attachment to and infection of PBMC. Further studies of this are likely to reveal novel strategies to prevent infection of PBMC.

Antiviral Agents↗
PubMed2006

Role of envelope processing and gp41 membrane spanning domain in the formation of human immunodeficiency virus type 1 (HIV-1) fusion-competent envelope glycoprotein complex.

Human immunodeficiency virus type 1 (HIV-1) entry into target cells is directed by the envelope (Env) glycoproteins, which are present on the surface of HIV-1 virion or infected cells in the form of trimers consisting of gp120/gp41 complexes. The surface subunit, gp120, initiates the entry process by interacting sequentially with the CD4 receptor and a co-receptor, thereby inducing a conformational change that allows the transmembrane (TM) gp41 subunit to mediate fusion between viral and target cell membranes. Cleavage of Env into its gp120 and gp41 components is necessary for activation of its fusogenic activity. Here, the gp41 TM glycoprotein was altered by either deleting an isoleucine residue (DeltaI642) in a critical region of its ectodomain or by substituting its membrane spanning domain (MSD) by that of the influenza hemagglutinin (HA) glycoprotein (TM-HA) to examine the contribution of these regions to Env functions. Characterization of these mutant forms of gp41 revealed that they both affected the infectivity of pseudotyped virions, however, through distinct defects in Env functions. While deletion of Ile 642 drastically altered processing of Env, replacement of gp41 MSD by that of HA led to a marked fusion defect even though the TM-HA Env was efficiently processed and incorporated into viral particles. Interestingly, both DeltaI642 and TM-HA Env were found to act as trans dominant-negative mutant of viral infectivity, presumably via their ability to form hetero-oligomers with wild type Env. Together, these results support a previously proposed model whereby all three gp120-gp41 monomers must be cleaved for the Env homo-trimer to function and suggest that the gp41 MSD plays a critical role in the formation of fusion-competent Env trimers.

Cell Line↗
PubMed2003

Studies on interaction between hTNF-alpha and its two receptors with expressed hsTR55-preS1/hsTR75-preS1 fusion soluble receptors.

The gene encoding the N-terminal 2-50 amino acids of HBsAg-preS1 was amplified by PCR and fused to the 3'-end of two human soluble TNF receptor genes to form the hsTR55-preS1/hsTR75-preS1 fusion genes. The recombinant bicistronic expression vectors were further constructed, which contained one of the human soluble TNF receptor fusion genes and the encephalomyocarditis virus (EMCV) internal ribosome entry site (IRES), followed by the neomycin phosphotransferases as the selectable marker. BHK-21 cells transfected with those vectors by electroporation were selected with G-418, and the positive colonies expressing the protein of interest were obtained. All of the culture media of those transfects could fairly neutralize hTNFalpha-induced cytotoxicity to L929 cells. The expression of hsTR55-preS1/hsTR75-preS1 in those cells has been further demonstrated by RT-PCR and indirect ELISA at RNA transcription and protein translation levels. Their K(d) (dissociation constant) value has also been assayed with BIOSENSOR method. The results showed that the fused HBsAg-preS1 peptide did not affect the dissociation constant of hsTR55 or hsTR75 with hTNFalpha and its muteins. Thus, a novel nonradioactive ELISA method was developed for studies on interaction between hTNFalpha and its two receptors using those expressed fusion receptors.

Animals↗
PubMed1999

Detection and typing of lymphotropic herpesviruses by multiplex polymerase chain reaction.

A multiplex nested-polymerase chain reaction (PCR) method was developed for the simultaneous detection and typing of all human lymphotropic herpesviruses described to date, including Ebstein Barr virus (EBV), cytomegalovirus (CMV), human herpesvirus 6, variants A and B (HHV6-A, HHV6-B), human herpesvirus 7 (HHV7) and human herpesvirus 8 (HHV8). Oligonucleotide primers were designed to amplify a highly conserved region within the DNA polymerase gene. Each reaction component and thermal cycling parameters were thoroughly standardized to achieve optimal specificity and sensitivity for the PCR assay, which was estimated at about 10-100 molecules for each virus. An internal control, consisting of 100 molecules of a cloned fragment of the porcine pseudorabies herpesvirus (PrV) genome, was included to detect false negative results. To assess suitability and clinical application of the multiplex PCR method, a total of 35 well-characterized specimens, including Kaposi's sarcoma skin lesions, serum, cerebrospinal fluid, saliva and urine samples, were tested. Results obtained suggest this technique could be applied as a sole diagnostic tool in several clinical settings in which herpesviral infection is suspected and differential diagnosis required, avoiding the need to test specimens by separate PCR methods.

Adult↗
PubMed1996

Inducible retroviral vectors regulated by lac repressor in mammalian cells.

We have developed lac repressor-regulated retroviral expression vectors that are induced by beta-galactosides upon stable transduction in mammalian cells. These vectors, derived from a Moloney-virus-based vector LNCX, contain an internal Rous sarcoma virus (RSV) or cytomegalovirus (CMV) promoter, coupled with 2 to 4 lac operator sequences and placed in anti orientation relative to the retroviral long terminal repeat (LTR). Three different vectors were tested in stably infected mass populations of mouse and human cells expressing the lac repressor, in parallel with the constitutively expressed LNCX vector. The highest expression levels from these vectors ranged from 1-4% to 25-33% of the LNCX level, and the induction by beta-galactosides ranged from 6-11-fold to 29-54-fold. These vectors should be suitable for studies requiring efficient gene transfer and regulated expression in mass populations of stably transduced mammalian cells.

3T3 Cells↗
PubMed1997

Development and characterization of a binary gene expression system based on bacteriophage T7 components in adenovirus vectors.

To explore the utility of the bacteriophage T7 binary system in adenovirus (Ad) vectors we constructed three Ad5-based vectors containing the T7 RNA polymerase (T7pol) gene in either early region 1 (E1) or E3. The recombinant Ad vectors were either deficient (AdT7pol1, AdT7pol2) or competent (AdT7pol3) for replication in human cells other than Ad5 transformed (293) cells. To test the ability of the T7 polymerase produced by these vectors to drive gene expression, a reporter vector was constructed with an E1 substitution comprising the bacterial beta-galactosidase (betaGal) (lacZ) gene under the control of the T7 gene 10 promoter (T7pro) and linked to the encephalomyocarditis virus (EMCV) internal ribosome entry site (IRES) (AdBHG10T7betaGal). Coinfections were performed with the various AdT7pol vectors and the reporter vector, and expression was analysed in three different human cell lines: 293, A549 and MRC-5. Depending on the AdT7pol vector used, different levels of expression were obtained from the reporter gene. In 293 cells, expression was detected following infection at very low multiplicities of infection (moi) with all of the T7pol vectors when coinfected with the reporter vector AdBHG10T7betaGal. In A549 and MRC-5 cells very little expression was detected using AdT7pol1 or pol2 and efficient expression was only obtained when relatively high moi values of the replication-competent vector were used in the coinfections. We also constructed a single vector containing both elements of the T7 system (T7pol in E3 and T7 promoter driving expression of the chloramphenicol acetyl transferase (cat) gene in E1). This vector proved difficult to rescue but was stable once isolated. Finally, experiments performed to evaluate the 'leakiness' of the Ad-T7 system detected very little expression from the T7pro in the absence of T7 polymerase suggesting this system may be useful for the cloning and expression of genes encoding cytotoxic proteins.

Adenoviridae↗
PubMed1998

Routine diagnosis of herpes simplex virus (HSV) encephalitis by an internal DNA controlled HSV PCR and an IgG-capture assay for intrathecal synthesis of HSV antibodies.

BACKGROUND: The development of antiviral therapy increases the need for rapid, sensitive and reliable methods or combination of methods for diagnosis and monitoring herpes simplex encephalitis, HSE. OBJECTIVES: Evaluation of diagnostic performance of three successively developed HSV PCR assays when combined with a new capture ELISA for HSV intrathecal antibody production (ITT). STUDY DESIGN: During a 3.6 year period a total of 4.206 CSF and serum samples from about 4.140 hospitalized patients with a tentative diagnosis of HSE were analyzed by a new ELISA for ITT. 1.962 CSF samples were examined also by PCR. Clinical signs and symptoms and additional tests were obtained on all ITT and/or PCR positive patients. In 1993 the PCR was a double PCR. In 1994 the PCR was a single PCR with internal inhibition control. Positive samples were confirmed by a different confirmative PCR to increase the specificity. From 1995 the PCR was as in 1994 but samples were no longer divided in the serology routine laboratory. RESULTS: A total of 33 HSE cases was found (incidence 1.8 HSE per million people). All patients were treated with aciclovir. Three patients died, 9 patients had primary infection, 2 patients had HSE previously, and 2 patients relapsed. Only 11 patients recovered satisfactory. Of all 37 positive ITT 7 were unlikely positive. False positive PCR was seen in 1993 and 1994, due to sample-to-sample contamination during division of samples, but was not seen since 1995 when this procedure was changed. The test results depended on the state of the disease. Thus, the sensitivity, specificity, PPV and NPV for ITT were highest when performed more than 1 week after debut of symptoms whereas these values were highest using PCR within the first week. CONCLUSION: Routine PCR diagnosis of HSE type 1 and 2 is a highly sensitive and specific method that should be performed together with serological ITT to cover the whole time span from debut of symptoms to several weeks after hospitalization.

Acyclovir↗
PubMed2003

Heparan sulfate proteoglycan as a plasma membrane carrier.

The plasma membrane defines the border of living cells and provides a barrier to extracellular components. Advances in molecular biology have resulted in the development of novel therapeutic strategies (e.g. gene therapy and cellular protein delivery) which rely on the entry of charged macromolecules into the intracellular compartment. Recent reports demonstrate an intriguing role for heparan sulfate proteoglycans in cellular internalization of viruses, basic peptides and polycation-nucleic-acid complexes and the possibility that they have important implications for gene transfer and protein delivery to mammalian cells. This review focuses on heparan sulfate proteoglycan as a plasma membrane carrier.

Amino Acid Sequence↗
PubMed1988

Internalization of the human immunodeficiency virus does not require the cytoplasmic domain of CD4.

Binding of the human immunodeficiency virus (HIV) to infectable host cells, such as B and T lymphocytes, monocytes and colorectal cells, is mediated by a high-affinity interaction between the gp120 component of the viral envelope glycoprotein and the CD4 receptor. Upon binding, it is thought that the second component of the envelope, gp41, mediates fusion between the viral envelope and host cell membranes. However, the early steps of HIV infection have not yet been thoroughly elucidated. Viral entry was first reported to be mediated by pH-dependent receptor-mediated endocytosis; subsequent studies have shown entry to be pH-independent. Although direct fusion of virus to plasma membranes of infected cells has been observed by electron microscopy, it is still formally possible that the infectious path of the virus involves receptor-mediated endocytosis. To gain a better understanding of receptor function in viral entry, we have analysed the ability of several altered or truncated forms of CD4 to serve as effective viral receptors. Our results indicate that domains beyond the HIV-binding region of CD4 are not required for viral infection. Some of the altered forms of CD4 that serve as effective HIV receptors are severely impaired in their ability to be endocytosed. These experiments therefore support the notion that viral fusion to the plasma membrane is sufficient for infection.

Antigens, Differentiation, T-Lymphocyte↗
PubMed2006

Dendritic-cell interactions with HIV: infection and viral dissemination.

Dendritic cells (DCs) are crucial for the generation and the regulation of adaptive immunity. Because DCs have a pivotal role in marshalling immune responses, HIV has evolved ways to exploit DCs, thereby facilitating viral dissemination and allowing evasion of antiviral immunity. Defining the mechanisms that underlie cell-cell transmission of HIV and understanding the role of DCs in this process should help us in the fight against HIV infection. This Review highlights the latest advances in our understanding of the interactions between DCs and HIV, focusing on the mechanisms of DC-mediated viral dissemination.

CD4-Positive T-Lymphocytes↗
PubMed2002

Crystal structure of an RNA tertiary domain essential to HCV IRES-mediated translation initiation.

The hepatitis C virus (HCV) internal ribosome entry site (IRES) RNA drives internal initiation of viral protein synthesis during host cell infection. In the tertiary structure of the IRES RNA, two helical junctions create recognition sites for direct binding of the 40S ribosomal subunit and eukaryotic initiation factor 3 (eIF3). The 2.8 A resolution structure of the IIIabc four-way junction, which is critical for binding eIF3, reveals how junction nucleotides interact with an adjacent helix to position regions directly involved in eIF3 recognition. Two of the emergent helices stack to form a nearly continuous A-form duplex, while stacking of the other two helices is interrupted by the insertion of junction residues into the helix minor groove. This distorted stack probably serves as an important recognition surface for the translational machinery.

Base Sequence↗