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Biomedical subjects

K Suryanarayana

Publications and source records attributed to K Suryanarayana.

18 recordsLinked to original sources
PubMed2002

The central nervous system as a reservoir for simian immunodeficiency virus (SIV): steady-state levels of SIV DNA in brain from acute through asymptomatic infection.

Latent reservoirs of human immunodeficiency virus (HIV) present significant challenges for eradicating HIV from infected persons, particularly reservoirs in the brain established during acute infection. A simian immunodeficiency virus (SIV)/macaque model of HIV dementia was used to show that viral DNA levels in the brain remained at constant levels from acute through asymptomatic infection, despite significant down-regulation of viral RNA in the brain after the acute phase of infection. Viral replication in the brain coincided with activation of macrophages and microglia in the central nervous system; down-regulation of viral replication coincided with increased infiltration of cytotoxic lymphocytes and reduced activation of macrophages and microglia in the brain. Comparison of viral genotypes in the central nervous system and peripheral blood mononuclear cells suggests that recrudescence of viral replication in brain occurs by reactivation of latent viral DNA. Latent virus in the brain must be considered in therapeutic strategies to eliminate HIV from infected persons.

Acute Disease↗
PubMed2001

Partial activation and induction of apoptosis in CD4(+) and CD8(+) T lymphocytes by conformationally authentic noninfectious human immunodeficiency virus type 1.

Increased levels of apoptosis are seen in human immunodeficiency virus (HIV) infection, and this has been proposed as an important mechanism contributing to HIV pathogenesis. However, interpretation of in vitro studies aimed at understanding HIV-related apoptosis has been complicated by the use of high concentrations of recombinant proteins or by direct cytopathic effects of replicating virus. We have developed an inactivation procedure that destroys retroviral infectivity while preserving the structural and functional integrity of the HIV surface proteins. These noninfectious virions interact authentically with target cells, providing a powerful tool to dissect mechanisms of HIV pathogenesis that do or do not require viral replication. Noninfectious CXCR4-tropic HIV-1 virions, but not microvesicles, partially activated freshly isolated CD4(+) and CD8(+) peripheral blood mononuclear cell T lymphocytes to express FasL and Fas, but not CD69 or CD25 (interleukin-2 receptor alpha) and eventually die via apoptosis starting 4 to 6 days postexposure. These effects required conformationally intact virions, as heat-denatured virions or equivalent amounts of recombinant gp120 did not induce apoptosis. The maximal apoptotic effect was dependent on major histocompatibility complex (MHC) class II proteins being present on the virion, but was not MHC restricted. The results suggest that the immunopathogenesis of HIV infection may not depend solely on direct cytopathic effects of HIV replication, but that effects due to noninfectious HIV-1 virions may also contribute importantly.

Apoptosis↗
PubMed2000

Real-time polymerase chain reaction assay for cell-associated HTLV type I DNA viral load.

We have developed a quantitative real-time PCR assay for HTLV-I DNA. This assay approach uses real-time monitoring of fluorescent signal generation as a consequence of Taq-mediated amplification of specific target sequences to allow real-time kinetic analysis of amplicon production. This kinetic approach yields excellent sensitivity and an extremely broad linear dynamic range, and ensures that quantitation is based on analysis during the exponential phase of amplification, regardless of the input template copy number. The HTLV-I DNA assay has a nominal threshold sensitivity of 10 copy Eq/reaction, although single-copy plasmid template can be detected at frequencies consistent with statistical prediction. The linear dynamic range is in excess of 5 logs. Interassay reproducibility averages 14% (coefficient of variation) for control templates over a range of 10(1) to 10(6) copy Eq/reaction and 25%, based on studies of extraction and analysis of replicate aliquots of PBMC specimens from HTLV-I-infected subjects. The primer/probe combination targets tax sequences conserved across described HTLV-I and HTLV-II isolates. Parallel quantitation in the same samples of an endogenous sequence present at a known copy number per cell allows normalization of results for potential variation in DNA recovery. Availability of this assay should facilitate studies of basic pathogenesis and clinical evaluation of HTLV-I and HTLV-II infection, as well as assessment of therapeutic approaches.

DNA Primers↗
PubMed1999

High viral load in the cerebrospinal fluid and brain correlates with severity of simian immunodeficiency virus encephalitis.

AIDS dementia and encephalitis are complications of AIDS occurring most frequently in patients who are immunosuppressed. The simian immunodeficiency virus (SIV) model used in this study was designed to reproducibly induce AIDS in macaques in order to examine the effects of a neurovirulent virus in this context. Pigtailed macaques (Macaca nemestrina) were coinoculated with an immunosuppressive virus (SIV/DeltaB670) and a neurovirulent molecularly cloned virus (SIV/17E-Fr), and more than 90% of the animals developed moderate to severe encephalitis within 6 months of inoculation. Viral load in plasma and cerebrospinal fluid (CSF) was examined longitudinally to onset of AIDS, and viral load was measured in brain tissue at necropsy to examine the relationship of systemic and central nervous system (CNS) viral replication to the development of encephalitis. In all animals, plasma viral load peaked at 10 to 14 days postinfection and remained high throughout infection with no correlation found between plasma viremia and SIV encephalitis. In contrast, persistent high levels of CSF viral RNA after the acute phase of infection correlated with the development of encephalitis. Although high levels of viral RNA were found in the CSF of all macaques (six of six) during the acute phase, this high level was maintained only in macaques developing SIV encephalitis (five of six). Furthermore, the level of both viral RNA and antigen in the brain correlated with the severity of the CNS lesions. The single animal in this group that did not have CNS lesions had no detectable viral RNA in any of the regions of the brain. The results substantiate the use of CSF viral load measurements in the postacute phase of SIV infection as a marker for encephalitis and CNS viral replication.

Animals↗
PubMed1999

Nef enhances human immunodeficiency virus replication and responsiveness to interleukin-2 in human lymphoid tissue ex vivo.

The nef gene is important for the pathogenicity associated with simian immunodeficiency virus infection in rhesus monkeys and with human immunodeficiency virus type 1 (HIV-1) infection in humans. The mechanisms by which nef contributes to pathogenesis in vivo remain unclear. We investigated the contribution of nef to HIV-1 replication in human lymphoid tissue ex vivo by studying infection with parental HIV-1 strain NL4-3 and with a nef mutant (DeltanefNL4-3). In human tonsillar histocultures, NL4-3 replicated to higher levels than DeltanefNL4-3 did. Increased virus production with NL4-3 infection was associated with increased numbers of productively infected cells and greater loss of CD4(+) T cells over time. While the numbers of productively infected T cells were increased in the presence of nef, the levels of viral expression and production per infected T cell were similar whether the nef gene was present or not. Exogenous interleukin-2 (IL-2) increased HIV-1 production in NL4-3-infected tissue in a dose-dependent manner. In contrast, DeltanefNL4-3 production was enhanced only marginally by IL-2. Thus, Nef can facilitate HIV-1 replication in human lymphoid tissue ex vivo by increasing the numbers of productively infected cells and by increasing the responsiveness to IL-2 stimulation.

Animals↗
PubMed1998

Plasma SIV RNA viral load determination by real-time quantification of product generation in reverse transcriptase-polymerase chain reaction.

Internally controlled RT-PCR methods (QC-RT-PCR) for quantification of SIV RNA are effective, but are relatively cumbersome, expensive, and time and labor intensive. For greater throughput and efficiency, we have developed a method for quantification of plasma SIV RNA levels by real-time RT-PCR using the Applied Biosystems Prism 7700 sequence detection system. This assay format allows real-time kinetic analysis of PCR product generation, providing a broad linear dynamic range and ensuring that quantification is based on analysis during the exponential phase of amplification, regardless of the input template copy number. Simultaneous amplification and analysis eliminates any requirement for handling amplified products, increasing throughput and eliminating a potential source of assay contamination. The assay we have developed for quantification of SIV RNA has a nominal threshold sensitivity of 300 copy Eq/ml of plasma, although as little as 10 copy Eq/reaction of SIV RNA template can be detected. The linear dynamic range is in excess of 5 logs. Interassay reproducibility averages 25% (coefficient of variation), based on studies of extraction and analysis of replicate aliquots of the same plasma specimens. The combination of sensitivity, precision, and broad dynamic range allows reliable quantification of viral load even during dynamic phases of SIV infection, such as through the onset and resolution of primary infection, or during treatment with antiretroviral agents. The primer-probe combinations we have developed allow quantification of SIV isolates most commonly used for experimental studies. Availability of this assay should greatly facilitate studies of basic pathogenesis and evaluation of therapeutic and prophylactic approaches in the SIV-infected macaque.

Animals↗
PubMed1998

Inactivation of human immunodeficiency virus type 1 infectivity with preservation of conformational and functional integrity of virion surface proteins.

Whole inactivated viral particles have been successfully used as vaccines for some viruses, but procedures historically used for inactivation can denature virion proteins. Results have been inconsistent, with enhancement of disease rather than protection seen in some notable instances following vaccination. We used the compound 2,2'-dithiodipyridine (aldrithiol-2; AT-2) to covalently modify the essential zinc fingers in the nucleocapsid (NC) protein of human immunodeficiency virus type 1 (HIV-1) or simian immunodeficiency virus (SIV) virions, thereby inactivating infectivity. The inactivated virus was not detectably infectious in vitro (up to 5 log units of inactivation). However, in contrast to virions inactivated by conventional methods such as heat or formalin treatment, viral and host cell-derived proteins on virion surfaces retained conformational and functional integrity. Thus, immunoprecipitation of AT-2-treated virions was comparable to precipitation of matched untreated virus, even when using antibodies to conformational determinants on gp120. AT-2 inactivated virions bound to CD4(+) target cells and mediated virus-induced, CD4-dependent "fusion from without" comparably to native virions. However, viral entry assays demonstrated that the viral life cycle of AT-2-treated virions was arrested before initiation of reverse transcription. The major histocompatibility complex (MHC) class II molecules on the surface of AT-2-treated virions produced from MHC class II-expressing cells retained the ability to support class II-dependent, superantigen-triggered proliferative responses by resting T lymphocytes. These findings indicate that inactivation via this method results in elimination of infectivity with preservation of conformational and functional integrity of virion surface proteins, including both virally encoded determinants and proteins derived from the host cells in which the virus was produced. Such inactivated virions should provide a promising candidate vaccine antigen and a useful reagent for experimentally probing the postulated involvement of virion surface proteins in indirect mechanisms of HIV-1 pathogenesis.

HIV-1↗
PubMed1995

Replication of measles virus in human monocytes and T cells.

Replication of measles virus (MV) in populations of peripheral blood mononuclear cells enriched for T cells and monocytes was studied using a temperature-sensitive mutant, MV ts38, and the parent counterpart, MV Lec. Stimulation of the cells was required for a full cycle of virus replication in both cell types. More infectious virus was released after stimulation from MV-infected populations enriched for T cells, T cell-enriched than from monocyte-enriched populations. However, similar amounts of viral mRNA, genomic RNA, and viral proteins of the expected size were found in both cell populations. The results indicate that MV-specific macromolecular synthesis is similar in both T cells and monocytes, but the assembly and (or) release of infectious virus is greatly reduced in monocytes as compared with T cells.

Cell Division↗
PubMed1994

Membrane-binding domains and cytopathogenesis of the matrix protein of vesicular stomatitis virus.

The membrane-binding affinity of the matrix (M) protein of vesicular stomatitis virus (VSV) was examined by comparing the cellular distribution of wild-type (wt) virus M protein with that of temperature-sensitive (ts) and deletion mutants probed by indirect fluorescent-antibody staining and fractionation of infected or plasmid-transfected CV1 cells. The M-gene mutant tsO23 caused cytopathic rounding of cells infected at permissive temperature but not of cells at the nonpermissive temperature; wt VSV also causes rounding, which prohibits study of M protein distribution by fluorescent-antibody staining. Little or no M protein can be detected in the plasma membrane of cells infected with tsO23 at the nonpermissive temperature, whereas approximately 20% of the M protein colocalized with the membrane fraction of cells infected with tsO23 at the permissive temperature. Cells transfected with a plasmid expressing intact 229-amino-acid wt M protein (M1-229) exhibited cytopathic cell rounding and actin filament dissolution, whereas cells retained normal polygonal morphology and actin filaments when transfected with plasmids expressing M proteins truncated to the first 74 N-terminal amino acids (M1-74) or deleted of the first 50 amino acids (M51-229) or amino acids 1 to 50 and 75 to 106 (M51-74/107-229). Truncated proteins M1-74 and M51-229 were readily detectable in the plasma membrane and cytosol of transfected cells as determined by both fluorescent-antibody staining and cell fractionation, as was the plasmid-expressed intact wt M protein. However, the expressed doubly deleted protein M51-74/107-229 could not be detected in plasma membrane by fluorescent-antibody staining or by cell fractionation, suggesting the presence of two membrane-binding sites spanning the region of amino acids 1 to 50 and amino acids 75 to 106 of the VSV M protein. These in vivo data were confirmed by an in vitro binding assay in which intact M protein and its deletion mutants were reconstituted in high- or low-ionic-strength buffers with synthetic membranes in the form of sonicated unilammelar vesicles. The results of these experiments appear to confirm the presence of two membrane-binding sites on the VSV M protein, one binding peripherally by electrostatic forces at the highly charged NH2 terminus and the other stably binding membrane integration of hydrophobic amino acids and located by a hydropathy plot between amino acids 88 and 119.

1,2-Dipalmitoylphosphatidylcholine↗
PubMed1994

Transcription inhibition and other properties of matrix proteins expressed by M genes cloned from measles viruses and diseased human brain tissue.

Ribonucleoprotein (RNP) cores extracted from virions of wild-type (Edmonston strain) measles virus (MV) or obtained from MV-infected cells (cRNP) were shown to be capable of transcribing RNA in vitro but at relatively low efficiency. The tightly bound matrix (M) protein could be effectively removed from virion RNP (vRNP) and from cRNP by exposure to buffers of high ionic strength (0.5 to 1.0 M KCl) but only at pH 8.0 or higher. The vRNP and cRNP cores complexed with M protein exhibited markedly reduced transcriptional activity at increasing concentrations, whereas vRNP and cRNP cores free of M protein exhibited linear and substantially higher transcriptional activity; these data suggest that M protein is the endogenous inhibitor of MV RNP transcription. M-gene cDNA clones derived from three strains of wild-type (wt) MV and 10 clones from mRNAs isolated from the brain tissue of patients who had died from subacute sclerosing panencephalitis (SSPE) and from measles inclusion body encephalitis (MIBE) were recloned in the pTM-1 expression vector driven by the bacteriophage T7 RNA polymerase expressed by a coinfecting vaccinia virus recombinant. All 10 mutant SSPE and MIBE clones expressed in vitro and in vivo M proteins that reacted with monospecific anti-M polyclonal antibody and migrated on polyacrylamide gels to positions identical to or only slightly different from those of the M proteins expressed by wt MV clones. When reconstituted with cRNP cores, the three expressed wt M proteins and 6 of the 10 mutant-expressed M proteins showed equivalent capacity to down-regulate MV transcription. Three of the M proteins from SSPE clones and one from the MIBE clone showed little or no capacity to down-regulate transcription when reconstituted with cRNP cores. The only plausible explanations for loss of transcription inhibition activity by the four SSPE/MIBE M proteins were exceedingly high degrees of hypermutations leading to U-->C transitions and cloning-corrected mutations in the initiator codon (ATG-->ACG) of the four M genes. However, only the hypermutated M protein expressed by the MIBE cDNA clone exhibited virtually no capacity to bind cRNP cores in a reconstitution assay. These experiments provide some preliminary data to support the hypothesis that MV encephalitis may result from certain selective mutations in the M gene.

Animals↗
PubMed1988

DNA-mediated transformation in the aquatic filamentous fungus Achlya ambisexualis.

A DNA-mediated transformation system was developed for the aquatic filamentous fungus Achlya ambisexualis using the chimeric plasmid vector pSV2neo2 microns. Hyphal colonies resistant to the neomycin analogue G-418 sulphate were regenerated from transformed protoplasts on soft agar. Southern blot analyses of the transformed-cell DNA produced multiple hybridization bands, suggesting integration of vector DNA into the host genome at multiple sites. Northern blot analyses revealed the presence of three APHII-gene-specific transcripts in the transformant, indicating that the G-418-resistant phenotype was due to the expression of the APHII gene. The presence of multiple RNA transcripts of unexpectedly large size suggested that RNA initiation and/or termination is under the control of regulatory element(s) other than the SV40 promoter. Plasmid DNAs recovered by transformation of Escherichia coli cells with total DNA preparations from the fungal transformants showed considerable DNA rearrangements. However, at least a portion of the plasmid DNA recovered from each of the transformants carried a functional APHII gene, suggesting that the episomal vector DNA may have played a role in maintaining the G-418-resistant phenotype.

Chytridiomycota↗
PubMed1987

Expression of herpes simplex virus thymidine kinase gene in aquatic filamentous fungus Achlya ambisexualis.

We have constructed a plasmid vector pSV2neo-MK alpha G in which the structural tk gene for Herpes simplex virus thymidine kinase (HSV-TK) was placed downstream from the metallothionein-I promoter. The vector also contained the selection marker aminoglycoside 3'-phosphotransferase (Km). This vector was able to transform the filamentous fungus Achlya ambisexualis and G-418-resistant colonies were obtained. Southern blot analyses revealed that multiple bands hybridizing to the HSV tk gene probe were present in the genomic DNA of the transformants. Upon analysis by gel electrophoresis, one of the transformants exhibited TK activity bearing electrophoretic mobility similar to that of the HSV-TK. An increase of approx. 40% of [3H]thymidine uptake and incorporation into cellular DNA was also observed in this transformant. This study suggested that the HSV tk gene can be expressed in the fungus A. ambisexualis that can be considered as a candidate host cell for further gene-expression studies.

Chytridiomycota↗
PubMed1985

Calmodulin from the water mold Achlya ambisexualis: isolation and characterization.

A protein-activator of bovine cyclic nucleotide phosphodiesterase from the water mold Achlya ambisexualis has been affinity-purified to apparent electrophoretic homogeneity. The heat-stable protein is similar in amino acid content and electrophoretic mobility on SDS acrylamide gels, to bovine brain calmodulin. It also cross-reacts with antibodies raised to the bovine protein. Achlya calmodulin activates PDE increasing its activity up to 9-fold in a Ca2+-dependent manner. The mold protein appears unusual in that its tyrosine fluorescence is unaltered by Ca2+ or by EGTA.

Amino Acids↗