bluuebunny/crossref_metadata_2025 huggingface dataset:
Accumulation of Virion Tegument and Envelope Proteins in a Stable Cytoplasmic Compartment during Human Cytomegalovirus Replication: Characterization of a Potential Site of Virus Assembly
Authors: Sanchez Veronica , Greis Kenneth D. , Sztul Elizabeth , Britt William J.
Published: July 2002

The assembly of human cytomegalovirus (HCMV) is thought to be similar to that which has been proposed for alphaherpesviruses and involve envelopment of tegumented subviral particles at the nuclear membrane followed by export from the cell by a poorly defined pathway. However, several studies have shown that at least two tegument virion proteins remain in the cytoplasm during the HCMV replicative cycle, thereby suggesting that HCMV cannot acquire its final envelope at the nuclear envelope. We investigated the assembly of HCMV by determining the intracellular trafficking of the abundant tegument protein pp150 (UL32) in productively infected human fibroblasts. Our results indicated that pp150 remained within the cytoplasm throughout the replicative cycle of HCMV and accumulated in a stable, juxtanuclear structure late in infection. Image analysis using a variety of cell protein-specific antibodies indicated that the pp150-containing structure was not a component of the endoplasmic reticulum, (ER), ER-Golgi intermediate compartment,
cis
or medial Golgi, or lysosomes. Partial colocalization of the structure was noted with the
trans
-Golgi network, and it appeared to lie in close proximity to the microtubule organizing center. Two additional tegument proteins (pp28 and pp65) and three envelope glycoproteins (gB, gH, and gp65) localized in this same structure late infection. This compartment appeared to be relatively stable since pp150, pp65, and the processed form of gB could be coisolated following cell fractionation. Our findings indicated that pp150 was expressed exclusively within the cytoplasm throughout the infectious cycle of HCMV and that the accumulation of the pp150 in this cytoplasmic structure was accompanied by at least five other virion proteins. These results suggested the possibility that this virus-induced structure represented a cytoplasmic site of virus assembly.
Human Herpesvirus 8 LANA Interacts with Proteins of the mSin3 Corepressor Complex and Negatively Regulates Epstein-Barr Virus Gene Expression in Dually Infected PEL Cells
Authors: Krithivas Anita , Young David B. , Liao Gangling , Greene Deborah , Hayward S. Diane
Published: July 2002

The human herpesvirus 8 (HHV-8) latency-associated nuclear antigen (LANA) is expressed in all latently HHV-8 infected cells and in HHV-8-associated tumors, including primary effusion lymphoma (PEL). To better understand the contribution of LANA to tumorigenesis and to the PEL phenotype, we performed a yeast two-hybrid screen which identified the corepressor protein SAP30 as a LANA binding protein. SAP30 is a constituent of a large multicomponent complex that brings histone deacetylases to the promoter. Glutathione
S
-transferase affinity assays confirmed interaction between LANA and SAP30 and also demonstrated interactions between LANA and two other members of the corepressor complex, mSin3A and CIR. The corepressors bound to the amino-terminal 340-amino-acid domain of LANA. In transient expression assays, this same domain of LANA mediated repression when targeted to a 5×Gal4tk-CAT reporter as a GAL4-LANA fusion. PEL cells have the unusual feature that they are frequently dually infected with both HHV-8 and Epstein-Barr virus (EBV). We found that EBV EBNA-1 expression is downregulated in PEL cells at both the RNA and protein levels. In transient expression assays, LANA repressed activated expression from the EBV Qp and Cp latency promoters. Reduction of endogenous Qp activity could also be demonstrated in EBV-infected Rael cells transfected with a LANA expression plasmid. In contrast to the effect of LANA on EBV latency promoters, LANA activated expression from its own promoter. The data indicate that LANA can mediate transcriptional repression through recruitment of an mSin3 corepressor complex and further that LANA-mediated repression is likely to contribute to the low level of EBV latency gene expression seen in dually infected PEL cells.
Genotypic, Phenotypic, and Modeling Studies of a Deletion in the β3-β4 Region of the Human Immunodeficiency Virus Type 1 Reverse Transcriptase Gene That Is Associated with Resistance to Nucleoside Reverse Transcriptase Inhibitors
Authors: Winters Mark A. , Coolley Kristi L. , Cheng Peng , Girard Yvette A. , Hamdan Hasnah , Kovari Ladislau C. , Merigan Thomas C.
Published: July 2002

Point mutations and inserts in the β3-β4 region of human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) are associated with resistance to nucleoside analog inhibitors. This report describes HIV-1 strains from seven patients that were found to have a 3-bp deletion in the β3-β4 region of the RT gene. These patient strains also had a mean of 6.2 drug resistance-associated mutations in their RT genes (range, 3 to 10 mutations). The deletion was most frequently found in strains with the Q151M mutation. Nonnucleoside RT inhibitor mutations were found in six of seven strains. Culture-based drug sensitivity assays showed that deletion-containing isolates had reduced susceptibility to four to eight RT inhibitors. Site-directed mutagenesis experiments showed that the deletion alone conferred reduced susceptibility to nucleoside analogs. Changes in the three-dimensional models of the RT deletion mutants were consistently observed at the β3-β4 loop and at helices C and E in both the presence and the absence of dTTP. Loss of hydrogen bonds between the RT and dTTP were also observed in the RT deletion mutant. These results suggest that the deletion in the RT gene contributes to resistance to several nucleoside analogs through a complex interaction with other mutations in the RT gene.
Deletions of Structural Glycoprotein E2 of Classical Swine Fever Virus Strain Alfort/187 Resolve a Linear Epitope of Monoclonal Antibody WH303 and the Minimal N-Terminal Domain Essential for Binding Immunoglobulin G Antibodies of a Pig Hyperimmune Serum
Authors: Lin Min , Lin Fang , Mallory Maria , Clavijo Alfonso
Published: July 2002

The major structural glycoprotein E2 of classical swine fever virus (CSFV) is responsible for eliciting neutralizing antibodies and conferring protective immunity. The current structural model of this protein predicts its surface-exposed region at the N terminus with a short stretch of the C-terminal residues spanning the membrane envelope. In this study, the N-terminal region of 221 amino acids (aa) covering aa 690 to 910 of the CSFV strain Alfort/187 E2, expressed as a fusion product in
Escherichia coli
, was shown to contain the epitope recognized by a monoclonal antibody (WH303) with affinity for various CSFV strains but not for the other members of the
Pestivirus
genus, bovine viral diarrhea virus (BVDV) and border disease virus (BDV). This region also contains the sites recognized by polyclonal immunoglobulin G (IgG) antibodies of a pig hyperimmune serum. Serial deletions of this region precisely defined the epitope recognized by WH303 to be TAVSPTTLR (aa 829 to 837) of E2. Comparison of the sequences around the WH303-binding site among the E2 proteins of pestiviruses indicated that the sequence TAVSPTTLR is strongly conserved in CSFV strains but highly divergent among BVDV and BDV strains. These results provided a structural basis for the reactivity patterns of WH303 and also useful information for the design of a peptide containing this epitope for potential use in the detection and identification of CSFV. By deletion analysis, an antigenic domain capable of reacting with pig polyclonal IgG was found 17 aa from the WH303 epitope within the N-terminal 123 residues (aa 690 to 812). Small N- or C-terminal deletions introduced into the domain disrupt its reactivity with pig polyclonal IgG, suggesting that this is the minimal antigenic domain required for binding to pig antibodies. This domain could have eliminated or reduced the cross-reactivity with other pestiviruses and may thus have an application for the serological detection of CSFV infection; evaluation of this is now possible, since the domain has been expressed in
E. coli
in large amounts and purified to homogeneity by chromatographic methods.
Binding of Rabbit Hemorrhagic Disease Virus to Antigens of the ABH Histo-Blood Group Family
Authors: Ruvoën-Clouet Nathalie , Ganière Jean Pierre , André-Fontaine Geneviève , Blanchard Dominique , Le Pendu Jacques
Published: July 2002

The ability of rabbit hemorrhagic disease virus to agglutinate human erythrocytes and to attach to rabbit epithelial cells of the upper respiratory and digestive tracts was shown to depend on the presence of ABH blood group antigens. Indeed, agglutination was inhibited by saliva from secretor individuals but not from nonsecretors, the latter being devoid of H antigen. In addition, erythrocytes of the rare Bombay phenotype, which completely lack ABH antigens, were not agglutinated. Native viral particles from extracts of infected rabbit liver as well as virus-like particles from the recombinant virus capsid protein specifically bound to synthetic A and H type 2 blood group oligosaccharides. Both types of particles could attach to adult rabbit epithelial cells of the upper respiratory and digestive tracts. This binding paralleled that of anti-H type 2 blood group reagents and was inhibited by the H type 2-specific lectin UEA-I and polyacrylamide-conjugated H type 2 trisaccharide. Young rabbit tissues were almost devoid of A and H type 2 antigens, and only very weak binding of virus particles could be obtained on these tissues.
Active Residues and Viral Substrate Cleavage Sites of the Protease of the Birnavirus Infectious Pancreatic Necrosis Virus
Authors: Petit Stéphanie , Lejal Nathalie , Huet Jean-Claude , Delmas Bernard
Published: July 2002

The polyprotein of infectious pancreatic necrosis virus (IPNV), a birnavirus, is processed by the viral protease VP4 (also named NS) to generate three polypeptides: pVP2, VP4, and VP3. Site-directed mutagenesis at 42 positions of the IPNV VP4 protein was performed to determine the active site and the important residues for the protease activity. Two residues (serine 633 and lysine 674) were critical for cleavage activity at both the pVP2-VP4 and the VP4-VP3 junctions. Wild-type activity at the pVP2-VP4 junction and a partial block (with an alteration of the cleavage specificity) at the VP4-VP3 junction were observed when replacement occurred at histidines 547 and 679. A similar observation was made when aspartic acid 693 was replaced by leucine, but wild-type activity and specificity were found when substituted by glutamine or asparagine. Sequence comparison between IPNV and two birnavirus (infectious bursal disease virus and
Drosophila
X virus) VP4s revealed that serine 633 and lysine 674 are conserved in these viruses, in contrast to histidines 547 and 679. The importance of serine 633 and lysine 674 is reminiscent of the protease active site of bacterial leader peptidases and their mitochondrial homologs and of the bacterial LexA-like proteases. Self-cleavage sites of IPNV VP4 were determined at the pVP2-VP4 and VP4-VP3 junctions by N-terminal sequencing and mutagenesis. Two alternative cleavage sites were also identified in the carboxyl domain of pVP2 by cumulative mutagenesis. The results suggest that VP4 cleaves the (Ser/Thr)-X-Ala↓(Ser/Ala)-Gly motif, a target sequence with similarities to bacterial leader peptidases and herpesvirus protease cleavage sites.
Interactions of Viral Protein 3CD and Poly(rC) Binding Protein with the 5′ Untranslated Region of the Poliovirus Genome
Authors: Gamarnik Andrea V. , Andino Raul
Published: July 2002

The poly(rC) binding protein (PCBP) is a cellular protein required for poliovirus replication. PCBP specifically interacts with two domains of the poliovirus 5′ untranslated region (5′UTR), the 5′ cloverleaf structure, and the stem-loop IV of the internal ribosome entry site (IRES). Using footprinting analysis and site-directed mutagenesis, we have mapped the RNA binding site for this cellular protein within the stem-loop IV domain. A C-rich sequence in a loop at the top of this large domain is required for PCBP binding and is crucial for viral translation. PCBP binds to stem-loop IV RNA with six-times-higher affinity than to the 5′ cloverleaf structure. However, the binding of the viral protein 3CD (precursor of the viral protease 3C and the viral polymerase 3D) to the cloverleaf RNA dramatically increases the affinity of PCBP for this RNA element. The viral protein 3CD binds to the cloverleaf RNA but does not interact directly with stem-loop IV nor with other RNA elements of the viral IRES. Our results indicate that the interactions of PCBP with the poliovirus 5′UTR are modulated by the viral protein 3CD.
Vaccinia Virus Envelope H3L Protein Binds to Cell Surface Heparan Sulfate and Is Important for Intracellular Mature Virion Morphogenesis and Virus Infection In Vitro and In Vivo
Authors: Lin Chi-Long , Chung Che-Sheng , Heine Hans G. , Chang Wen
Published: July 2002

An immunodominant antigen, p35, is expressed on the envelope of intracellular mature virions (IMV) of vaccinia virus. p35 is encoded by the viral late gene H3L, but its role in the virus life cycle is not known. This report demonstrates that soluble H3L protein binds to heparan sulfate on the cell surface and competes with the binding of vaccinia virus, indicating a role for H3L protein in IMV adsorption to mammalian cells. A mutant virus defective in expression of H3L (H3L
−
) was constructed; the mutant virus has a small plaque phenotype and 10-fold lower IMV and extracellular enveloped virion titers than the wild-type virus. Virion morphogenesis is severely blocked and intermediate viral structures such as viral factories and crescents accumulate in cells infected with the H3L
−
mutant virus. IMV from the H3L
−
mutant virus are somewhat altered and less infectious than wild-type virions. However, cells infected by the mutant virus form multinucleated syncytia after low pH treatment, suggesting that H3L protein is not required for cell fusion. Mice inoculated intranasally with wild-type virus show high mortality and severe weight loss, whereas mice infected with H3L
−
mutant virus survive and recover faster, indicating that inactivation of the H3L gene attenuates virus virulence in vivo. In summary, these data indicate that H3L protein mediates vaccinia virus adsorption to cell surface heparan sulfate and is important for vaccinia virus infection in vitro and in vivo. In addition, H3L protein plays a role in virion assembly.
Protection of the Villus Epithelial Cells of the Small Intestine from Rotavirus Infection Does Not Require Immunoglobulin A
Authors: O'Neal Christine M. , Harriman Gregory R. , Conner Margaret E.
Published: July 2002

Immunoglobulin A (IgA) is the primary immune response induced in the intestine by rotavirus infection, but vaccination with virus-like particles induces predominantly IgG, not IgA. To definitively assess the role of IgA in protection from rotavirus infection, IgA knockout mice, which are devoid of serum and secretory IgA, were infected and then rechallenged with murine rotavirus at either 6 weeks or 10 months. Following primary rotavirus infection, IgA knockout mice cleared virus as effectively as IgA normal control mice. Rotavirus-infected IgA knockout mice produced no serum or fecal IgA but did have high levels of antirotavirus serum IgG and IgM and fecal IgG, whereas IgA normal control mice made both serum IgA and IgG and fecal IgA. Both IgA normal and IgA knockout mice were totally protected from rotavirus challenge at 42 days. Ten months following a primary infection, both IgA normal and knockout mice still had high levels of serum and fecal antirotavirus antibody and were totally protected from rotavirus challenge. To determine if compensatory mechanisms other than IgG were responsible for protection from rotavirus infection in IgA knockout mice, mice were depleted of CD4+T cells or CD8+T cells. No changes in the level of protection were seen in depleted mice. These data show that fecal or systemic IgA is not essential for protection from rotavirus infection and suggest that in the absence of IgA, IgG may play a significant role in protection from mucosal pathogens.
Allogeneic Transplantation Induces Expression of Cytomegalovirus Immediate-Early Genes In Vivo: a Model for Reactivation from Latency
Authors: Hummel Mary , Zhang Zheng , Yan Shixian , DePlaen Isabelle , Golia Piyush , Varghese Thomas , Thomas Gail , Abecassis Michael I.
Published: July 2002

Reactivation of cytomegalovirus (CMV) from latency is a frequent complication of organ transplantation, and the molecular mechanism by which this occurs is unknown. Previous studies have shown that allogeneic stimulation induces reactivation of human CMV (HCMV) in vitro (64). We find that transplantation of vascularized allogeneic kidneys induces murine CMV (MCMV) and HCMV immediate-early (ie) gene expression. This induction is accompanied by increased expression of transcripts encoding inflammatory cytokines, including tumor necrosis factor (TNF), interleukin-2, and gamma interferon, and by activation of NF-κB. TNF alone can substitute for allogeneic transplantation in inducing HCMV and MCMViegene expression in some tissues. Our studies suggest that reactivation is a multistep process which is initiated by factors that induceiegene expression, including TNF and NF-κB. Allogeneic transplantation combined with immunosuppression may be required to achieve complete reactivation in vivo.
Design and Use of an Inducibly Activated Human Immunodeficiency Virus Type 1 Nef To Study Immune Modulation
Authors: Walk Scott F. , Alexander Melissa , Maier Bernhard , Hammarskjold Marie-Louise , Rekosh David M. , Ravichandran Kodi S.
Published: July 2002

The Nef protein of the human immunodeficiency virus type 1 (HIV-1) has been shown to enhance the infectivity of virus particles, downmodulate cell surface proteins, and associate with many intracellular proteins that are thought to facilitate HIV infection. One of the challenges in defining the molecular events regulated by Nef has been obtaining good expression of Nef protein in T cells. This has been attributed to effects of Nef on cell proliferation and apoptosis. We have designed a Nef protein that is readily expressed in T-cell lines and whose function is inducibly activated. It is composed of a fusion between full-length Nef and the estrogen receptor hormone-binding domain (Nef-ER). The Nef-ER is kept in an inactive state due to steric hindrance, and addition of the membrane-permeable drug 4-hydroxytamoxifen (4-HT), which binds to the ER domain, leads to inducible activation of Nef-ER within cells. We demonstrate that Nef-ER inducibly associates with the 62-kDa Ser/Thr kinase and is localized to specific membrane microdomains (lipid rafts) only after activation. Using this inducible Nef, we also compared the specific requirements for CD4 and HLA-A2 downmodulation in a SupT1 T-cell line. Half-maximal downmodulation of cell surface CD4 required very little active Nef-ER and occurred as early as 4 h after addition of 4-HT. In contrast, 50% downmodulation of HLA-A2 by Nef required 16 to 24 h and about 50- to 100-fold-greater concentrations of 4-HT. These data suggest that HLA-A2 downmodulation may require certain threshold levels of active Nef. The differential timing of CD4 and HLA-A2 downmodulation may have implications for HIV pathogenesis and immune evasion.
Cellular COPII Proteins Are Involved in Production of the Vesicles That Form the Poliovirus Replication Complex
Authors: Rust René C. , Landmann Lukas , Gosert Rainer , Tang Bor Luen , Hong Wanjin , Hauri Hans-Peter , Egger Denise , Bienz Kurt
Published: July 2002

Poliovirus (PV) replicates its genome in association with membranous vesicles in the cytoplasm of infected cells. To elucidate the origin and mode of formation of PV vesicles, immunofluorescence labeling with antibodies against the viral vesicle marker proteins 2B and 2BC, as well as cellular markers of the endoplasmic reticulum (ER), anterograde transport vesicles, and the Golgi complex, was performed in BT7-H cells. Optical sections obtained by confocal laser scanning microscopy were subjected to a deconvolution process to enhance resolution and signal-to-noise ratio and to allow for a three-dimensional representation of labeled membrane structures. The mode of formation of the PV vesicles was, on morphological grounds, similar to the formation of anterograde membrane traffic vesicles in uninfected cells. ER-resident membrane markers were excluded from both types of vesicles, and the COPII components Sec13 and Sec31 were both found to be colocalized on the vesicular surface, indicating the presence of a functional COPII coat. PV vesicle formation during early time points of infection did not involve the Golgi complex. The expression of PV protein 2BC or the entire P2 and P3 genomic region led to the production of vesicles carrying a COPII coat and showing the same mode of formation as vesicles produced after PV infection. These results indicate that PV vesicles are formed at the ER by the cellular COPII budding mechanism and thus are homologous to the vesicles of the anterograde membrane transport pathway.
Rep-Dependent Initiation of Adeno-Associated Virus Type 2 DNA Replication by a Herpes Simplex Virus Type 1 Replication Complex in a Reconstituted System
Authors: Ward Peter , Falkenberg Maria , Elias Per , Weitzman Matthew , Linden R. Michael
Published: July 2002

Productive infection by adeno-associated virus type 2 (AAV) requires coinfection with a helper virus, e.g., adenovirus or herpesviruses. In the case of adenovirus coinfection, the replication machinery of the host cell performs AAV DNA replication. In contrast, it has been proposed that the herpesvirus replication machinery might replicate AAV DNA. To investigate this question, we have attempted to reconstitute AAV DNA replication in vitro using purified herpes simplex virus type 1 (HSV-1) replication proteins. We show that the HSV-1 UL5, UL8, UL29, UL30, UL42, and UL52 gene products along with the AAV Rep68 protein are sufficient to initiate replication on duplex DNA containing the AAV origins of replication, resulting in products several hundred nucleotides in length. Initiation can occur also on templates containing only a Rep binding site and a terminal resolution site. We further demonstrate that initiation of DNA synthesis can take place with a subset of these factors: Rep68 and the UL29, UL30, and UL42 gene products. Since the HSV polymerase and its accessory factor (the products of the UL30 and UL42 genes) are unable to efficiently perform synthesis by strand displacement, it is likely that in addition to creating a hairpin primer, the AAV Rep protein also acts as a helicase for DNA synthesis. The single-strand DNA binding protein (the UL29 gene product) presumably prevents reannealing of complementary strands. These results suggest that AAV can use the HSV replication apparatus to replicate its DNA. In addition, they may provide a first step for the development of a fully reconstituted AAV replication assay.
Recruitment Times, Proliferation, and Apoptosis Rates during the CD8+T-Cell Response to Lymphocytic Choriomeningitis Virus
Authors: De Boer Rob J. , Oprea Mihaela , Antia Rustom , Murali-Krishna Kaja , Ahmed Rafi , Perelson Alan S.
Published: July 2002

The specific CD8+T-cell response during acute lymphocytic choriomeningitis virus (LCMV) infection of mice is characterized by a rapid proliferation phase, followed by a rapid death phase and long-term memory. In BALB/c mice the immunodominant and subdominant CD8+responses are directed against the NP118 and GP283 epitopes. These responses differ mainly in the magnitude of the epitope-specific CD8+T-cell expansion. Using mathematical models together with a nonlinear parameter estimation procedure, we estimate the parameters describing the rates of change during the three phases and thereby establish the differences between the responses to the two epitopes. We find that CD8+cell proliferation begins 1 to 2 days after infection and occurs at an average rate of 3 day−1, reaching the maximum population size between days 5 and 6 after immunization. The 10-fold difference in expansion to the NP118 and GP283 epitopes can be accounted for in our model by a 3.5-fold difference in the antigen concentration of these epitopes at which T-cell stimulation is half-maximal. As a consequence of this 3.5-fold difference in the epitope concentration needed for T-cell stimulation, the rates of activation and proliferation of T cells specific for the two epitopes differ during the response and in combination can account for the large difference in the magnitude of the response. After the peak, during the death phase, the population declines at a rate of 0.5 day−1, i.e., cells have an average life time of 2 days. The model accounts for a memory cell population of 5% of the peak population size by a reversal to memory of 1 to 2% of the activated cells per day during the death phase.
Role of Immunoglobulin A in Protection against Reovirus Entry into Murine Peyer's Patches
Authors: Silvey Katherine J. , Hutchings Amy B. , Vajdy Michael , Petzke Mary M. , Neutra Marian R.
Published: July 2002

Reovirus type 1 Lang (T1L) infects the mouse intestinal mucosa by adhering specifically to epithelial M cells and exploiting M-cell transport to enter the Peyer's patches. Oral inoculation of adult mice has been shown to elicit cellular and humoral immune responses that clear the infection within 10 days. This study was designed to determine whether adult mice that have cleared a primary infection are protected against viral entry upon oral rechallenge and, if so, whether antireovirus secretory immunoglobulin A (S-IgA) is a necessary component of protection. Adult BALB/c mice that were orally inoculated on day 0 with reovirus T1L produced antiviral S-IgA in feces and IgG in serum directed primarily against the reovirus ς1 attachment protein. Eight hours after oral reovirus challenge on day 21, the Peyer's patches of previously exposed mice contained no detectable virus whereas Peyer's patches of naive controls contained up to 2,300 PFU of reovirus/mg of tissue. Orally inoculated IgA knockout (IgA−/−) mice cleared the initial infection as effectively as wild-type mice and produced higher levels of reovirus-specific serum IgG and secretory IgM than C57BL/6 wild-type mice. When IgA−/−mice were rechallenged on day 21, however, their Peyer's patches became infected. These results indicate that intestinal S-IgA is an essential component of immune protection against reovirus entry into Peyer's patch mucosa.
Identification, Phylogeny, and Evolution of Retroviral Elements Based on Their Envelope Genes
Authors: Bénit Laurence , Dessen Philippe , Heidmann Thierry
Published: July 2002

Phylogenetic analyses of retroviral elements, including endogenous retroviruses, have relied essentially on the retroviralpolgene expressing the highly conserved reverse transcriptase. This enzyme is essential for the life cycle of all retroid elements, but other genes are also endowed with conserved essential functions. Among them, the transmembrane (TM) subunit of the envelope gene is involved in virus entry through membrane fusion. It has also been reported to contain a domain, named the immunosuppressive domain, that has immunosuppressive properties most probably essential for virus spread within the host. This domain is conserved among a large series of retroviral elements, and we have therefore attempted to generate phylogenetic links between retroviral elements identified from databases following tentative alignments of the immunosuppressive domain and adjacent sequences. This allowed us to unravel a conserved organization among TM domains, also found in the Ebola and Marburg filoviruses, and to identify a large number of human endogenous retroviruses (HERVs) from sequence databases. The latter elements are part of previously identified families of HERVs, and some of them define new families. A general phylogenetic analysis based on the TM proteins of retroelements, and including those with no clearly identified immunosuppressive domain, could then be derived and compared withpol-based phylogenetic trees, providing a comprehensive survey of retroelements and definitive evidence for recombination events in the generation of both the endogenous and the present-day infectious retroviruses.
Simian Immunodeficiency Virus Replicates to High Levels in Naturally Infected African Green Monkeys without Inducing Immunologic or Neurologic Disease
Authors: Broussard Suzanne R. , Staprans Silvija I. , White Robert , Whitehead Evelyn M. , Feinberg Mark B. , Allan Jonathan S.
Published: July 2002

African green monkeys can maintain long-term persistent infection with simian immunodeficiency viruses (SIVagm) without developing AIDS and thus provide an important model for understanding mechanisms of natural host resistance to disease. This study assessed the levels and anatomic distribution of SIVagm in healthy, naturally infected monkeys. Quantitative competitive reverse transcriptase PCR assays developed to measure SIVagm from two African green monkey subspecies demonstrated high levels of SIV RNA in plasma (>6 × 106RNA copies/ml) in sabaeus and vervet monkeys. Infectious virus was readily recovered from plasma and peripheral blood mononuclear cells and shown to be highly cytopathic in human cell lines and macrophages. SIVagm DNA levels were highest in the gastrointestinal tract, suggesting that the gut is a major site for SIVagm replication in vivo. Appreciable levels of virus were also found within the brain parenchyma and the cerebrospinal fluid (CSF), with lower levels detected in peripheral blood cells and lymph nodes. Virus isolates from the CSF and brain parenchyma readily infected macrophages in culture, whereas lymph node isolates were more restricted to growth in human T-cell lines. Comparison ofenvV2-C4 sequences showed extensive amino acid diversity between SIVagm recovered from the central nervous system and that recovered from lymphoid tissues. Homology between brain and CSF viruses, macrophage tropism, and active replication suggest compartmentalization in the central nervous system without associated neuropathology in naturally infected monkeys. These studies provide evidence that the nonpathogenic nature of SIVagm in the natural host can be attributed neither to more effective host control over viral replication nor to differences in the tissue and cell tropism from those for human immunodeficiency virus type 1-infected humans or SIV-infected macaques.
Murine Coronavirus Spike Protein Determines the Ability of the Virus To Replicate in the Liver and Cause Hepatitis
Authors: Navas Sonia , Seo Su-Hun , Chua Ming Ming , Sarma Jayasri Das , Lavi Ehud , Hingley Susan T. , Weiss Susan R.
Published: July 2002

Recombinant mouse hepatitis viruses (MHV) differing only in the spike gene, containing A59, MHV-4, and MHV-2 spike genes in the background of the A59 genome, were compared for their ability to replicate in the liver and induce hepatitis in weanling C57BL/6 mice infected with 500 PFU of each virus by intrahepatic injection. Penn98-1, expressing the MHV-2 spike gene, replicated to high titer in the liver, similar to MHV-2, and induced severe hepatitis with extensive hepatocellular necrosis. S
A59
R13, expressing the A59 spike gene, replicated to a somewhat lower titer and induced moderate to severe hepatitis with zonal necrosis, similar to MHV-A59. S
4
R21, expressing the MHV-4 spike gene, replicated to a minimal extent and induced few if any pathological changes, similar to MHV-4. Thus, the extent of replication and the degree of hepatitis in the liver induced by these recombinant viruses were determined largely by the spike protein.
Kaposi's Sarcoma-Associated Herpesvirus Latency-Associated Nuclear Antigen 1 Mediates Episome Persistence through
cis
-Acting Terminal Repeat (TR) Sequence and Specifically Binds TR DNA
Authors: Ballestas Mary E. , Kaye Kenneth M.
Published: July 2002

Kaposi's sarcoma (KS)-associated herpesvirus (KSHV) (also known as human herpesvirus 8) latently infects KS tumors, primary effusion lymphomas (PELs), and PEL cell lines. In latently infected cells, KSHV DNA is maintained as circularized, extrachromosomal episomes. To persist in proliferating cells, KSHV episomes must replicate and efficiently segregate to progeny nuclei. In uninfected B-lymphoblastoid cells, KSHV latency-associated nuclear antigen (LANA1) is necessary and sufficient for persistence of artificial episomes containing specific KSHV DNA. In previous work, the
cis
-acting sequence required for episome persistence contained KSHV terminal-repeat (TR) DNA and unique KSHV sequence. We now show that
cis
-acting KSHV TR DNA is necessary and sufficient for LANA1-mediated episome persistence. Furthermore, LANA1 binds TR DNA in mobility shift assays and a 20-nucleotide LANA1 binding sequence has been identified. Since LANA1 colocalizes with KSHV episomes along metaphase chromosomes, these results are consistent with a model in which LANA1 may bridge TR DNA to chromosomes during mitosis to efficiently segregate KSHV episomes to progeny nuclei.
Branched Polyamines Cure Prion-Infected Neuroblastoma Cells
Authors: Supattapone Surachai , Wille Holger , Uyechi Lisa , Safar Jiri , Tremblay Patrick , Szoka Francis C. , Cohen Fred E. , Prusiner Stanley B. , Scott Michael R.
Published: July 2002

Branched polyamines, including polyamidoamine and polypropyleneimine (PPI) dendrimers, are able to purge PrP
Sc
, the disease-causing isoform of the prion protein, from scrapie-infected neuroblastoma (ScN2a) cells in culture (S. Supattapone, H.-O. B. Nguyen, F. E. Cohen, S. B. Prusiner, and M. R. Scott, Proc. Natl. Acad. Sci. USA 96:14529–14534, 1999). We now demonstrate that exposure of ScN2a cells to 3 μg of PPI generation 4.0/ml for 4 weeks not only reduced PrP
Sc
to a level undetectable by Western blot but also eradicated prion infectivity as determined by a bioassay in mice. Exposure of purified RML prions to branched polyamines in vitro disaggregated the prion rods, reduced the β-sheet content of PrP 27-30, and rendered PrP 27-30 susceptible to proteolysis. The susceptibility of PrP
Sc
to proteolytic digestion induced by branched polyamines in vitro was strain dependent. Notably, PrP
Sc
from bovine spongiform encephalopathy-infected brain was susceptible to PPI-mediated denaturation in vitro, whereas PrP
Sc
from natural sheep scrapie-infected brain was resistant. Fluorescein-labeled PPI accumulated specifically in lysosomes, suggesting that branched polyamines act within this acidic compartment to mediate PrP
Sc
clearance. Branched polyamines are the first class of compounds shown to cure prion infection in living cells and may prove useful as therapeutic, disinfecting, and strain-typing reagents for prion diseases.
Definitive Assignment of Proton Selectivity and Attoampere Unitary Current to the M2 Ion Channel Protein of Influenza A Virus
Authors: Lin Tse-I , Schroeder Cornelia
Published: July 2002

The viral ion channel protein M2 supports the transit of influenza virus and its glycoproteins through acidic compartments of the cell. M2 conducts endosomal protons into the virion to initiate uncoating and, by equilibrating the pH at
trans
-Golgi membranes, preserves the native conformation of acid-sensitive viral hemagglutinin. The exceptionally low conductance of the M2 channel thwarted resolution of single channels by electrophysiological techniques. Assays of liposome-reconstituted M2 yielded the average unitary channel current of the M2 tetramer—1.2 aA (1.2 × 10
−18
A) at neutral pH and 2.7 to 4.1 aA at pH 5.7—which activates the channel. Extrapolation to physiological temperature predicts 4.8 and 40 aA, respectively, and a unitary conductance of 0.03 versus 0.4 fS. This minute activity, below previous estimates, appears sufficient for virus reproduction, but low enough to avert abortive cytotoxicity. The unitary permeability of M2 was within the range reported for other proton channels. To address the ion selectivity of M2, we exploited the coupling of ionic influx and efflux in sealed liposomes. Metal ion fluxes were monitored by proton counterflow, employing a pH probe 1,000 times more sensitive than available Na
+
or K
+
probes. Even low-pH-activated M2 did not conduct Na
+
and K
+
. The proton selectivity of M2 was estimated to be at least 3 × 10
6
(over sodium or potassium ions), in agreement with electrophysiological studies. The stringent proton selectivity of M2 suggests that the cytopathology of influenza virus does not involve direct perturbation of cellular sodium or potassium gradients.
The Late Domain of Human Immunodeficiency Virus Type 1 p6 Promotes Virus Release in a Cell Type-Dependent Manner
Authors: Demirov Dimiter G. , Orenstein Jan M. , Freed Eric O.
Published: July 2002

The p6 domain of human immunodeficiency virus type 1 (HIV-1) is located at the C terminus of the Gag precursor protein Pr55
Gag
. Previous studies indicated that p6 plays a critical role in HIV-1 particle budding from virus-expressing HeLa cells. In this study, we performed a detailed mutational analysis of the N terminus of p6 to map the sequences required for efficient virus release. We observed that the highly conserved P-T/S-A-P motif located near the N terminus of p6 is remarkably sensitive to change; even conservative mutations in this sequence imposed profound virus release defects in HeLa cells. In contrast, single and double amino acid substitutions outside the P-T/S-A-P motif had no significant effect on particle release. The introduction of stop codons one or two residues beyond the P-T/S-A-P motif markedly impaired virion release, whereas truncation four residues beyond P-T/S-A-P had no effect on particle production in HeLa cells. By examining the effects of p6 mutation in biological and biochemical analyses and by electron microscopy, we defined the role of p6 in particle release and virus replication in a panel of T-cell and adherent cell lines and in primary lymphocytes and monocyte-derived macrophages. We demonstrated that the effects of p6 mutation on virus replication are markedly cell type dependent. Intriguingly, even in T-cell lines and primary lymphocytes in which p6 mutations block virus replication, these changes had little or no effect on particle release. However, p6-mutant particles produced in T-cell lines and primary lymphocytes exhibited a defect in virion-virion detachment, resulting in the production of tethered chains of virions. Virus release in monocyte-derived macrophages was markedly inhibited by p6 mutation. To examine further the cell type-specific virus release defect in HeLa versus T cells, transient heterokaryons were produced between HeLa cells and the Jurkat T-cell line. These heterokaryons display a T-cell-like phenotype with respect to the requirement for p6 in particle release. The results described here define the role of p6 in virus replication in a wide range of cell types and reveal a strong cell type-dependent requirement for p6 in virus particle budding.
Activation of Cytomegalovirus in Pig-to-Primate Organ Xenotransplantation
Authors: Mueller Nicolas J. , Barth Rolf N. , Yamamoto Shin , Kitamura Hiroshi , Patience Clive , Yamada Kazuhiko , Cooper David K. C. , Sachs David H. , Kaur Amitinder , Fishman Jay A.
Published: July 2002

Xenotransplantation of porcine organs carries the risk of reactivation of latent virus in donor and recipient tissues as well as transmission of viruses between species. We have investigated the activation of baboon cytomegalovirus (BCMV) and porcine CMV (PCMV) in a pig-to-primate model of xenotransplantation. Tissues originating from a series of six swine-to-baboon composite thymokidney xenotransplants were investigated. Four immunosuppressed baboons died (survival range, 7 to 27 days) with the graft in situ. Increases in BCMV DNA copy numbers occurred in three (75%) of these baboons and was thought to be responsible for pneumonitis and the death of one animal. In two baboons, disseminated intravascular coagulation was successfully treated by graftectomy and discontinuation of immunosuppression. PCMV was upregulated in five of six xenografts (83%). PCMV infection was associated with ureteric necrosis in one xenograft. Although significantly increased in native tissues, low levels of BCMV and PCMV were also detected in tissues other than that of the native viral host species. The cross-species presence of CMV did not appear to cause clinical or histological signs of invasive disease. Thus, viral infections with clinical disease were restricted to tissues of the native species of each virus. Intensive immune suppression currently required for xenotransplantation results in a significant risk of reactivation of latent infections by BCMV and PCMV. It is not yet known whether viral DNA detected across species lines represents cellular microchimerism, ongoing viral infection, or uptake of free virus. The observation of graft injury by PCMV demonstrates that CMV will be an important pathogen in immunosuppressed xenograft recipients. Strategies must be developed to exclude CMV from porcine organ donors.
Comparison of Two Aquatic Alphaviruses, Salmon Pancreas Disease Virus and Sleeping Disease Virus, by Using Genome Sequence Analysis, Monoclonal Reactivity, and Cross-Infection
Authors: Weston Jonathan , Villoing Stéphane , Brémont Michel , Castric Jeanette , Pfeffer Martin , Jewhurst Victoria , McLoughlin Marian , Rødseth OddMagne , Christie Karen Elina , Koumans Joseph , Todd Daniel
Published: July 2002

Cell culture isolates of salmon pancreas disease virus (SPDV) of farmed Atlantic salmon and sleeping disease virus (SDV) of rainbow trout were compared. Excluding the poly(A) tracts, the genomic nucleotide sequences of SPDV and SDV RNAs include 11,919 and 11,900 nucleotides, respectively. Phylogenetic analysis places SPDV and SDV between the New World viruses of Venezuelan equine encephalitis virus and Eastern equine encephalitis virus and the Old World viruses of Aura virus and Sindbis virus. When compared to each other, SPDV and SDV show 91.1% nucleotide sequence identity over their complete genomes, with 95 and 93.6% amino acid identities over their nonstructural and structural proteins, respectively. Notable differences between the two viruses include a 24-nucleotide insertion in the C terminus of nsP3 protein of SPDV and amino acid sequence variation at the C termini of the capsid and E1 proteins. Experimental infections of Atlantic salmon and rainbow trout with SPDV and SDV confirmed that the disease lesions induced by SPDV and SDV were similar in nature. Although infections with SPDV and SDV produced similar levels of histopathology in rainbow trout, SDV induced significantly less severe lesions in salmon than did SPDV. Virus neutralization tests performed with sera from experimentally infected salmon indicated that SPDV and SDV belonged to the same serotype; however, antigenic variation was detected among SDV and geographically different SPDV isolates by using monoclonal antibodies. Although SPDV and SDV exhibit minor biological differences, we conclude on the basis of the close genetic similarity that SPDV and SDV are closely related isolates of the same virus species for which the name
Salmonid alphavirus
is proposed.
Structural Requirements for the Assembly of Norwalk Virus-Like Particles
Authors: Bertolotti-Ciarlet Andrea , White Laura J. , Chen Rong , Prasad B. V. Venkataram , Estes Mary K.
Published: July 2002

Norwalk virus (NV) is the prototype strain of a group of human caliciviruses responsible for epidemic outbreaks of acute gastroenteritis. While these viruses do not grow in tissue culture cells or animal models, expression of the capsid protein in insect cells results in the self-assembly of recombinant NV virus-like particles (rNV VLPs) that are morphologically and antigenically similar to native NV. The X-ray structure of the rNV VLPs has revealed that the capsid protein folds into two principal domains: a shell (S) domain and a protruding (P) domain (B. V. V. Prasad, M. E. Hardy, T. Dokland, J. Bella, M. G. Rossmann, and M. K. Estes, Science 286:287-290, 1999). To investigate the structural requirements for the assembly of rNV VLPs, we performed mutational analyses of the capsid protein. We examined the ability of 10 deletion mutants of the capsid protein to assemble into VLPs in insect cell cultures. Deletion of the N-terminal 20 residues, suggested by the X-ray structure to be involved in a switching mechanism during assembly, did not affect the ability of the mutant capsid protein to self-assemble into 38-nm VLPs with a T=3 icosahedral symmetry. Further deletions in the N-terminal region affected particle assembly. Deletions in the C-terminal regions of the P domain, involved in the interactions between the P and S domains, did not block the assembly process, but they affected the size and stability of the particles. Mutants carrying three internal deletion mutations in the P domain, involved in maintaining dimeric interactions, produced significantly larger 45-nm particles, albeit in low yields. The complete removal of the protruding domain resulted in the formation of smooth particles with a diameter that is slightly smaller than the 30-nm diameter expected from the rNV structure. These studies indicate that the shell domain of the NV capsid protein contains everything required to initiate the assembly of the capsid, whereas the entire protruding domain contributes to the increased stability of the capsid by adding intermolecular contacts between the dimeric subunits and may control the size of the capsid.
Glucose Hydrogenation in a Trickle-Bed Reactor
Authors: Tukač Vratislav
Published: July 2002

Catalytic hydrogenation of 40% aqueous solutions of D-glucose to D-glucitol was studied in a high-pressure trickle-bed reactor. The reactions were performed on a supported nickel catalyst at temperatures ranging from 115 to 165 °C and in the pressure range 0.5 to 10 MPa. The order of the reaction with respect to hydrogen is 0.65 and apparent activation energy 23.8-48.5 kJ mol-1, the latter depending on initial molar glucose concentration and density and viscosity of the solution. The influence of external diffusion is necessary to take into account for scaling-up the process.
Homogeneous Precipitation of Siliceous MCM-41 and Bimodal Silica
Authors: Rathouský Jiří , Zukalová Markéta , Zukal Arnošt , Had Jiří
Published: July 2002

A new procedure for synthesis of MCM-41 siliceous molecular sieves with regular morphologies and bimodal mesoporous silica was developed. It is based on the precipitation from an isotropic reaction mixture. The decrease in pH, which causes the formation of solid particles, is achieved by the hydrolysis of ethyl acetate. The procedure is excellently reproducible and enables to obtain not only siliceous MCM-41 with the highest degree of pore ordering and phase purity but also a material of a new type, viz. bimodal silica containing both the MCM-41 mesopore system and the system of larger mesopores whose size ranges from 15 to 25 nm.