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PubMed2004

Expression of human immunodeficiency virus type 1 gag modulates ligand-induced downregulation of EGF receptor.

Many enveloped viruses use the ESCRT proteins of the cellular vacuolar protein sorting pathway for efficient egress from the cell. Recruitment of the ESCRT proteins by human immunodeficiency virus type 1 (HIV-1) Gag is required for HIV-1 particle budding and egress. ESCRT proteins normally function at endosomal membranes, where they facilitate the downregulation of mitogen-activated receptors such as EGF receptor (EGFR) through multivesicular body biogenesis. It is not known whether the Gag-mediated recruitment of ESCRT proteins functionally depletes the pool of these molecules that is available for the downregulation of EGFR. Here we show that the expression of HIV-1 Gag decreases the rate of EGFR downregulation, as assessed by decreases in the rates of (125)I-EGF and EGFR degradation. The effect of Gag was dependent on the presence of the TSG101 binding motif (PTAP) within the Gag C-terminal p6 domain. Cells expressing HIV-1 Gag retained more EGFR in late endosomes. This effect occurred when Gag was expressed alone from a heterologous promoter and when Gag expression was driven by the HIV-1 long terminal repeat within pHXB2DeltaBalD25S, a noninfectious lentiviral vector. Gag-expressing cells exhibited higher levels of activated mitogen-activated protein kinase for longer times after EGF addition than did cells that did not express HIV-1 Gag. These results indicate that HIV-1 Gag can impinge upon the functioning of the cellular vacuolar protein sorting pathway and reveal yet another facet of the intricate effects of HIV-1 infection on host cell physiology.

Animals↗
PubMed2005

Analysis of human immunodeficiency virus type 1 Gag ubiquitination.

Ubiquitin is important for the release of human immunodeficiency virus type 1 (HIV-1) and several other retroviruses, but the functional significance of Gag ubiquitination is unknown. To address this problem, we decided to analyze Gag ubiquitination in detail. A low percentage of the HIV-1 p6 protein has previously been shown to be ubiquitinated, and published mutagenesis data suggested that Gag ubiquitination is largely lost upon mutation of the two lysine residues in p6. In this study, we show that Gag proteins lacking the p6 domain or the two lysine residues within p6 are ubiquitinated at levels comparable to those of the wild-type Gag protein. We detected monoubiquitinated forms of the matrix (MA), capsid (CA), and nucleocapsid (NC) proteins in mature virus preparations. Protease digestion of Gag polyproteins extracted from immature virions indicated that ubiquitinated MA, CA, and possibly NC are as abundant as ubiquitinated p6. The HIV-1 late-domain motifs PTAP and LRSLF were not required for Gag ubiquitination, and mutation of the PTAP motif even resulted in an increase in the amount of Gag-Ub conjugates detected. Finally, at steady state, ubiquitinated Gag proteins were not enriched in either membrane-associated or virus-derived Gag fractions. In summary, these results indicate that HIV-1 Gag can be monoubiquitinated in all domains and that ubiquitination of lysine residues outside p6 may thus contribute to viral release and/or infectivity.

Amino Acid Motifs↗
PubMed2005

Mutation of the SP1 sequence impairs both multimerization and membrane-binding activities of human immunodeficiency virus type 1 Gag.

The Gag protein of human immunodeficiency virus type 1 contains a 14-amino-acid region, termed SP1, between the capsid and downstream nucleocapsid sequences. Although SP1 is known to be indispensable for virus production, the mechanisms involved are mostly unclear. In this study, we demonstrate that an M368A mutation within SP1 severely diminished the ability of Gag to associate with cellular membranes. Although wild-type levels of membrane binding were restored to the M368A Gag by a second-site L20K mutation within matrix, the resultant Gag mutant L20K-M368A remained defective in virus production. This latter deficit was partially consequent to the binding of L20K-M368A Gag to nonraft membranes as opposed to raft association seen for wild-type Gag. Further analysis revealed that the majority of membrane-bound M368A Gag proteins were small oligomers, indicating a multimerization defect. In support of this observation, purified recombinant Gag derivatives containing the M368A mutation formed much lower amounts of high-molecular-weight complexes that were pelletable at 21,000 x g than did wild-type Gag. Based on the myristyl switch model, we propose that the M368A mutation inhibits Gag multimerization and, as a result, restricts the binding of Gag to cellular membranes.

Animals↗
PubMed2005

Phosphorylation and proteolytic cleavage of gag proteins in budded simian immunodeficiency virus.

The lentiviral Gag polyprotein (Pr55(Gag)) is cleaved by the viral protease during the late stages of the virus life cycle. Proteolytic cleavage of Pr55(Gag) is necessary for virion maturation, a structural rearrangement required for infectivity that occurs in budded virions. In this study, we investigate the relationship between phosphorylation of capsid (CA) domains in Pr55(Gag) and its cleavage intermediates and their cleavage by the viral protease in simian immunodeficiency virus (SIV). First, we demonstrate that phosphorylated forms of Pr55(Gag), several CA-containing cleavage intermediates of Pr55(Gag), and the free CA protein are detectable in SIV virions but not in virus-producing cells, indicating that phosphorylation of these CA-containing Gag proteins may require an environment that is unique to the virion. Second, we show that the CA domain of Pr55(Gag) can be phosphorylated in budded virus and that this phosphorylation does not require the presence of an active viral protease. Further, we provide evidence that CA domains (i.e., incompletely cleaved CA) are phosphorylated to a greater extent than free (completely cleaved) CA and that CA-containing Gag proteins can be cleaved by the viral protease in SIV virions. Finally, we demonstrate that Pr55(Gag) and several of its intermediates, but not free CA, are actively phosphorylated in budded virus. Taken together, these data indicate that, in SIV virions, phosphorylation of CA domains in Pr55(Gag) and several of its cleavage intermediates likely precedes the cleavage of these domains by the viral protease.

Animals↗
PubMed1992

Cytotoxic T lymphocytes do not appear to select for mutations in an immunodominant epitope of simian immunodeficiency virus gag.

Studies to date assessing HIV escape from CTL in vivo have yielded conflicting results. Previous studies have demonstrated that simian immunodeficiency virus of macaques (SIVmac)-infected rhesus monkeys expressing the MHC class I allele Mamu-A*01 reproducibly develop a gag-specific CTL response limited to a 9-amino acid epitope of the SIVmac gag protein (residues 182-190 within peptide 11C). To determine whether CTL have a role in selecting for AIDS virus mutants, we examined mutations in SIVmac proviral DNA encoding this gag CTL epitope in PBL of infected rhesus monkeys. Three Mamu-A*01+ rhesus monkeys were infected with SIVmac and assessed for gag- and peptide 11C-specific CTL responses. This specific CTL response was maintained in two monkeys, but lost in the third animal 2 yr after infection. The generation of proviral gag mutations was then determined by sequencing 500-bp proviral fragments amplified from fresh PBL obtained from the monkeys more than 2.5 yr after infection. Although numerous point mutations were characterized in 131 polymerase chain reaction-generated clones of SIVmac gag, only four mutations within the gag CTL epitope-coding region of the genome were identified. Comparison of synonymous and nonsynonymous nucleotide substitutions in the regions encoding peptide 11C (p11C) and the flanking gag protein indicated a lack of selective pressure for viral mutations in the CTL epitope coding region. Interestingly, a predominant gag mutant encoding a single amino acid change in p11C was found in a monkey which lost its CTL activity. However, even in this setting there was no evidence for selection of mutations in the CTL epitope coding region when compared with the flanking region. Furthermore, synthetic peptides corresponding to all naturally occurring variants in the gag epitope-coding region were recognized by cloned and bulk cultured effector cells of the infected monkeys with persistent CTL. These results indicate that SIVmac gag- and p11C-specific CTL do not select for mutations in the immunodominant epitope-coding region and that the naturally occurring mutants do not appear to escape CTL recognition.

Amino Acid Sequence↗
PubMed1994

Drug-induced intralysosomal storage of sulfated glycosaminoglycans (GAGs): a methodical pitfall occurring with acridine derivatives.

The present communication deals with an adverse drug action which is exerted by a series of dicationic amphiphilic compounds such as the immunomodulatory drug tilorone and congeners. The drugs induce lysosomal storage of sulfated glycosaminoglycans (GAGs) in intact organisms and in cultured cells by impairing the lysosomal GAG degradation. This impairment was proposed to be due to the formation of non-degradable GAG-drug complexes. GAGs are highly water-soluble and not preservable by aldehyde fixatives. Therefore, usually the lysosomes appear optically empty in histological preparations, unless the fixative is supplemented with a GAG-precipitating agent. When acridine derivatives were used for the induction of GAG-storage, the lysosomal storage material displayed unexpected and unsystematic variability with regard to its preservability and ultrastructure. In the present study, evidence is presented that the acridine derivatives (a) remain bound to the stored GAGs for some time after glutaraldehyde fixation; and (b) they precipitate GAGs in vitro. Thus, apart from their unwanted action in the living cell, i.e., disturbing lysosomal GAG-degradation, the drugs function as precipitants and "fixatives" for the intralysosomal GAGs. The uncontrolled persistence of the drugs after tissue fixation leads to variable degree of GAG-preservation and thus to unpredictable variability of the ultrastructure of the storage lysosomes. If this pitfall is not realized, the resulting inconsistencies may rise confusion among toxicologic pathologists who deal with drug-induced lysosomal storage disorders.

Acridines↗
PubMed1992

Signal transduction in glycosaminoglycan (GAG) synthesis by cultured chondrocytes and its inhibition by inflammatory cell-derived hydrogen peroxide.

Signal transduction in glycosaminoglycan (GAG) synthesis by chondrocytes has been studied. The activity of various subspecies of protein kinase C (PKC) in chondrocytes derived from rodent costal cartilage and bovine articular cartilage has been determined and the role of PKC in GAG synthesis as well as the possible interactions of PKC with calcium- or cyclic AMP (cAMP)-dependent systems in the synthesis of GAG. To investigate GAG synthesis in inflammatory conditions, the effects of hydrogen peroxide on PKC activity of the chondrocytes and PKC-mediated GAG synthesis have been studied. 12-O-Tetradecanoylphorbol-13-acetate (TPA), an activator of PKC, increased GAG synthesis in a dose-dependent fashion. This suggests that PKC up-regulates the synthesis of GAG in cultured chondrocytes. This increase was not significantly affected by simultaneous addition of the calcium ionophore, ionomycin, or dibutyryl cAMP (db-cAMP), a cAMP analogue. Ionomycin and db-cAMP, when used alone, did not significantly alter GAG synthesis by chondrocytes. Thus there appears to be no interaction between PKC and calcium- or cAMP-mediated systems in GAG synthesis. The increase in GAG synthesis induced by TPA was significantly (P less than 0.01) reduced by simultaneous addition of hydrogen peroxide (10(-6) M), without affecting cell viability. The activity of PKC in chondrocytes pretreated with 10(-6) M hydrogen peroxide was also significantly inhibited. Thus hydrogen peroxide which is generated by inflammatory cells may be important in suppression of GAG synthesis in inflammatory conditions.

Animals↗
PubMed2001

Further development of a recombinant feline herpesvirus type 1 expressing the Gag protein of feline immunodeficiency virus.

We have previously reported the construction of a recombinant feline herpesvirus type 1 (FHV-1), designated C7301ddlTK-gag, expressing the Gag precursor protein of feline immunodeficiency virus (FIV). In this study, we report the construction of a further recombinant FHV-1 (ddlTK(gBp)-gag) which carries an FHV-1 gB promoter sequence upstream of the FIV gag gene of C7301ddlTK-gag. Strong expression of the FIV Gag protein by ddlTK(gBp)-gag was confirmed by immunoblot analyses and enzyme-linked immunosorbent assays. Although C7301ddlTK-gag and ddlTK(gBp)-gag failed to induce anti-FIV Gag antibodies in cats, we confirmed the infectivity and stability of these recombinants in cats.

Animals↗
PubMed2004

Blocking of human immunodeficiency virus type-1 virion autolysis by autologous p2(gag) peptide.

Our previous study suggested that the p2(gag) peptide, AEAMSQVTNTATIM, inhibits human immunodeficiency virus type 1 (HIV-1) protease (PR) activity in vitro. In this study, Ala substitutions (Met4Ala and Thr8Ala) and deletion of amino acid Asn9 within the nona p2(gag) peptide (AEAMSQVTN) were found to decrease the inhibitory effect on HIV-1 PR activity. Furthermore, treatment of PMA-activated latently infected T lymphocytes, ACH-2 cells, with the p2(gag) peptide (100 and 250 micro M) resulted in a decrease in the amount of p24(gag )in the resultant viral lysates derived from the cell-free supernatant. In addition, the HIV-1-Tat-p2(gag) fusion peptide was synthesized to effectively deliver the p2(gag) peptide into the cells. The fusion peptide was incorporated into chronically infected T lymphocytes, CEM/LAV-1 cells, as detected on indirect immunofluorescence analysis using anti-p2(gag) peptide monoclonal antibodies, which recognize the nona peptide (AEAMSQVTN) derived from the N-terminus of the p2(gag) peptide, and cleaved by HIV-1 PR in vitro. Treatment of CEM/LAV-1 cells with the fusion peptide also resulted in a decrease in the amount of p24(gag )in the resultant viral lysate derived from the cell-free supernatant. Taken together, these data suggest that the p2(gag) peptide consequently blocks the autolysis of HIV-1 virions for the conservation of viral species.

Amino Acid Sequence↗
PubMed2001

Extended nucleocapsid protein is cleaved from the Gag-Pol precursor of human immunodeficiency virus type 1.

Human immunodeficiency virus type 1 Gag and Gag-Pol precursors are translated from an mRNA which is indistinguishable from the full-length genomic RNA. The ratio of Gag to Gag-Pol polyproteins is approximately 20:1 and is controlled by a frameshift of the reading frame, which takes place downstream of the p7 nucleocapsid (NC) in the N terminus of the p1 peptide. The viral precursors Gag and Gag-Pol are cleaved by the virus-encoded protease (PR) into the structural proteins, and into p6(Pol), PR, reverse transcriptase and integrase. Due to the frameshift event, the cleavage site at the C terminus of NC coded in the Gag frame (ERQAN-FLGKI) changes either to ERQANFLRED or ERQANFFRED. The results presented in this report demonstrate that the NC released from the Gag-Pol precursor is 8 amino acid residues longer than the NC cleaved from the Gag polyprotein. Our results also show that truncated Gag-Pol precursors bearing cleavage site mutation at the NC/p6(Pol), and/or p6(Pol)/PR junctions, undergo autoprocessing in bacterial and eukaryotic cells, indicating that PR is active when part of the precursor.

Animals↗
PubMed2007

Host ABCE1 is at plasma membrane HIV assembly sites and its dissociation from Gag is linked to subsequent events of virus production.

In primate cells, assembly of a single HIV-1 capsid involves multimerization of thousands of Gag polypeptides, typically at the plasma membrane. Although studies support a model in which HIV-1 assembly proceeds through complexes containing Gag and the cellular adenosine triphosphatase ABCE1 (also termed HP68 or ribonuclease L inhibitor), whether these complexes constitute true assembly intermediates remains controversial. Here we demonstrate by pulse labeling in primate cells that a population of Gag associates with endogenous ABCE1 within minutes of translation. In the next approximately 2 h, Gag-ABCE1 complexes increase in size to approximately that of immature capsids. Dissociation of ABCE1 from Gag correlates closely with Gag processing during virion maturation and occurs much less efficiently when the HIV-1 protease is inactivated. Finally, quantitative double-label immunogold electron microscopy reveals that ABCE1 is recruited to sites of assembling wild-type Gag at the plasma membrane but not to sites of an assembly-defective Gag mutant at the plasma membrane. Together these findings demonstrate that a population of Gag present at plasma membrane sites of assembly associates with ABCE1 throughout capsid formation until the onset of virus maturation, which is then followed by virus release. Moreover, the data suggest a linkage between Gag-ABCE1 dissociation and subsequent events of virion production.

ATP-Binding Cassette Transporters↗
PubMed1991

Assembly and processing of avian retroviral gag polyproteins containing linked protease dimers.

Assembly and maturation of retroviral particles requires the aggregation and controlled proteolytic cleavage of polyprotein core precursors by a precursor-encoded protease (PR). Active, mature retroviral PR is a dimer, and the accumulation of precursors at sites of assembly may facilitate subunit interaction and subsequent activation of this enzyme. In addition, it has been suggested that cellular cytoplasmic components act as inhibitors of PR activity, so that processing is delayed until the nascent virions leave this compartment and separate from the surface of host cells. To investigate the mechanisms that control PR activity during virus assembly, we studied the in vivo processing of retroviral gag precursors that contain tandemly linked PR subunits in which dimerization is concentration independent. Sequences encoding four different linked protease dimers were independently joined to the end of the Rous sarcoma virus (RSV) gag gene in a simian virus 40-based plasmid vector which expresses a myristoylated gag precursor upon transfection of COS-1 cells. Three of these plasmids produced gag precursors that were incorporated into viruslike particles and proteolytically cleaved by the dimers to mature core proteins that were indistinguishable from the processed products of wild-type gag. The amount of viral gag protein that was assembled and packaged in these transfections was inversely related to the relative proteolytic activities of the linked PR dimers. The fourth gag precursor, which contained the most active linked PR dimer, underwent rapid intracellular processing and did not form viruslike particles. In the absence of the plasma membrane targeting signal, processing of all four linked PR dimer-containing gag precursors was completed entirely within the cell. From these results, we conclude that the delay in polyprotein core precursor processing that occurs during normal virion assembly does not depend on a cytoplasmic inhibitor of PR activity. We suggest that dimer formation is not only necessary but may be sufficient for the initiation of PR-directed maturation of gag and gag-pol precursors.

Animals↗
PubMed1993

Characterization of a small (25-kilodalton) derivative of the Rous sarcoma virus Gag protein competent for particle release.

Retroviral Gag proteins have the ability to induce budding and particle release from the plasma membrane when expressed in the absence of all of the other virus-encoded components; however, the locations of the functional domains within the Gag protein that are important for this process are poorly understood. It was shown previously that the protease sequence of the Rous sarcoma virus (RSV) Gag protein can be replaced with a foreign polypeptide, iso-1-cytochrome c from a yeast, without disrupting particle assembly (R. A. Weldon, Jr., C. R. Erdie, M. G. Oliver, and J. W. Wills, J. Virol. 64:4169-4179, 1990). An unexpected product of the chimeric gag gene is a small, Gag-related protein named p25C. This product was of interest because of its high efficiency of packaging into particles. The goal of the experiments described here was to determine the mechanism by which p25C is synthesized and packaged into particles. The results demonstrate that it is not the product of proteolytic processing of the Gag-cytochrome precursor but is derived from an unusual spliced mRNA. cDNA clones of the spliced mRNA were obtained, and each expressed a product of approximately 25 kDa, designated p25M1, which was released into the growth medium in membrane-enclosed particles that were much lighter than authentic retrovirions as measured in sucrose density gradients. DNA sequencing revealed that the clones encode the first 180 of the 701 amino acids of the RSV Gag protein and no residues from iso-1-cytochrome c. This suggested that a domain in the carboxy-terminal half of Gag is important for the packaging of Gag proteins into dense arrays within the particles. In support of this hypothesis, particles of the correct density were obtained when a small segment from the carboxy terminus of the RSV Gag protein (residues 417 to 584) was included on the end of p25.

Alleles↗
PubMed1997

Effect of mutations in the nucleocapsid protein (NCp7) upon Pr160(gag-pol) and tRNA(Lys) incorporation into human immunodeficiency virus type 1.

COS-7 cells were transfected with DNAs containing mutations in the NCp7 sequences of human immunodeficiency virus. Selective incorporation into the virus of tRNA(Lys) was measured by two-dimensional polyacrylamide gel electrophoresis, and Pr160(gag-pol) incorporation into the virus was detected in Western blots of viral protein. Mutations tested included cysteine and histidine mutations in either of the Cys-His boxes, as well as mutations in the N- and C-terminal flanking regions and in the linker region between the two Cys-His boxes. Of 10 mutations tested, only 2 inhibited tRNA(Lys) incorporation: a P31L mutation in the linker region and a deletion which removed both Cys-His boxes and the linker region (deltaK14-T50). The P31L mutation prevents the incorporation of Pr160(gag-pol) into the virus. Cotransfection of COS cells with both P31L DNA and a plasmid coding only for unprocessed Pr160(gag-pol) resulted in the viral incorporation of Pr160(gag-pol) and the rescue of selective packaging of tRNA(Lys) into the virion. In the deltaK14-T50 mutant, Pr160(gag-pol) is incorporated into the virus. Selective tRNA(Lys) packaging is not rescued by cotransfection with a plasmid coding for Pr160(gag-pol) but is rescued by cotransfection with DNA coding for wild-type Pr55(gag). Since Pr55(gag) does not by itself selectively package tRNA(Lys), the deltaK14-T50 mutation may be affecting tRNA(Lys) binding to a cytoplasmic Pr55(gag)/Pr160(gag-pol) complex.

Amino Acid Sequence↗
PubMed2000

Differential narrow focusing of immunodominant human immunodeficiency virus gag-specific cytotoxic T-lymphocyte responses in infected African and caucasoid adults and children.

Cytotoxic T-lymphocyte (CTL) activity plays a central role in control of viral replication and in determining outcome in cases of human immunodeficiency virus type 1 (HIV-1) infection. Incorporation of important CTL epitope sequences into candidate vaccines is, therefore, vital. Most CTL studies have focused upon small numbers of adult Caucasoid subjects infected with clade-B virus, whereas the global epidemic is most severe in sub-Saharan African populations and predominantly involves clade-C infection in both adults and children. In this study, sensitive enzyme-linked immunospot (elispot) assays have been utilized to identify the dominant Gag-specific CTL epitopes targeted by adults and children infected with clade-B or -C virus. Cohorts evaluated included 44 B-clade-infected Caucasoid American and African American adults and children and 37 C-clade-infected African adults and children from Durban, South Africa. The results show that 3 out of 46 peptides spanning p17(Gag) and p24(Gag) sequences tested contain two-thirds of the dominant Gag-specific epitopes, irrespective of the clade, ethnicity, or age group studied. However, there were distinctive differences between the dominant responses made by Caucasoids and Africans. Dominant responses in Caucasoids were more often within p17(Gag) peptide residues 16 to 30 (38 versus 12%; P < 0.01), while p24(Gag) peptide residues 41 to 60 contained the dominant Gag epitope more often in the African subjects tested (39 versus 4%; P < 0.005). Within this 20-mer p24(Gag), an epitope presented by both B42 and B81 is defined which represents the dominant Gag response in >30% of the total infected population in Durban. This epitope is closely homologous with dominant HIV-2 and simian immunodeficiency virus Gag-specific CTL epitopes. The fine focusing of dominant CTL responses to these few regions of high immunogenicity is of significance to vaccine design.

Adult↗
PubMed2005

Characterization of human immunodeficiency virus Gag-specific gamma interferon-expressing cells following protective mucosal immunization with alphavirus replicon particles.

A safe, replication-defective viral vector that can induce mucosal and systemic immune responses and confer protection against many infectious pathogens, such as human immunodeficiency virus type 1 (HIV-1), may be an ideal vaccine platform. Accordingly, we have generated and tested alphavirus replicon particles encoding HIV-1 Gag from Sindbis virus (SIN-Gag) and Venezuelan equine encephalitis virus (VEE-Gag), as well as chimeras between the two (VEE/SIN-Gag). Following intramuscular (i.m.), intranasal (i.n.), or intravaginal (IVAG) immunization with VEE/SIN-Gag and an IVAG challenge with vaccinia virus encoding HIV Gag (VV-Gag), a larger number of Gag-specific CD8+ intracellular gamma interferon-expressing cells (iIFNEC) were detected in iliac lymph nodes (ILN), which drain the vaginal/uterine mucosa (VUM), than were observed after immunizations with SIN-Gag. Moreover, a single i.n. or IVAG immunization with VEE/SIN-Gag induced a larger number of cells expressing HIV Gag in ILN, and immunizations with VEE/SIN-Gag through any route induced better protective responses than immunizations with SIN-Gag. In VUM, a larger percentage of iIFNEC expressed alpha4beta7 or alpha(Ebeta)7 integrin than expressed CD62L integrin. However, in spleens (SP), a larger percentage of iIFNEC expressed alpha4beta7 or CD62L than expressed alpha(Ebeta)7. Moreover, a larger percentage of iIFNEC expressed the chemokine receptor CCR5 in VUM and ILN than in SP. These results demonstrate a better induction of cellular and protective responses following immunizations with VEE/SIN-Gag than that following immunizations with SIN-Gag and also indicate a differential expression of homing and chemokine receptors on iIFNEC in mucosal effector and inductive sites versus systemic lymphoid tissues.

Animals↗
PubMed1999

Tagging the human immunodeficiency virus gag protein with green fluorescent protein. Minimal evidence for colocalisation with actin.

The assembly and budding of human immunodeficiency virus type 1, encoded solely in the Gag protein precursor Pr55Gag, occur at the plasma membrane of infected cells. However, little is known about the routing of the Gag molecule from its site of synthesis in the cytoplasm to the site of budding, with past studies suggesting that the cytoskeleton, particularly actin, may be involved in the translocation. We have constructed a T7 promoter-driven gag gene fusion with green fluorescent protein (GFP) that expresses Gag-GFP in both cells and supernatant. The distribution of Gag-GFP was the same as Gag only, suggesting that cellular routing was not affected by fusion to GFP, and using colabelling techniques, Gag-GFP was shown to have no particular colocalisation with actin. After detergent extraction of expressing cells, Gag and Gag-GFP remained cell associated, whereas GFP only was wholly released. These data suggest that Gag may associate with other cytoskeletal components or, perhaps more likely, that a partial assembly to a large-molecular-weight intermediate occurs before localisation at the plasma membrane.

Actins↗
PubMed2005

Is the gag reflex useful in the management of swallowing problems in acute stroke?

The goal of this study was to compare the diagnostic value of an absent gag reflex in acute stroke patients with the bedside swallowing assessment (BSA) and assess its relationship to outcomes. Two hundred forty-two acute stroke patients had their gag reflex tested and a BSA performed. Numbers needing nasogastric or gastrostomy tube insertion were noted, also their discharge destination, discharge Barthel Index, and mortality. The mean age of the subjects was 76.5+/-10.2 years; 37.6% were male; 41.7% of the patients were dysphagic on BSA; 18.2% had an absent gag. Dysphagia was present in 88.6% of the patients with an absent gag and in 31.3% of those with an intact gag. The gag reflex was absent in 38.6% of dysphagic and 3.5% of nondysphagic patients. Comparing an absent gag against the criterion of the BSA, its specificity was 0.96, sensitivity 0.39, positive predictive value 0.89, and negative predictive value 0.69. Regression analyses found that an intact gag gave an Odds Ratio [CI] of 0.23 [0.06-0.91] for gastrostomy feeding but did not predict other outcomes. We conclude that the gag reflex is as specific as but less sensitive than the BSA in detecting dysphagia in acute stroke patients. An intact gag may be protective against longer-term swallowing problems and the need for enteral feeding.

Adult↗