bluuebunny/crossref_metadata_2025 huggingface dataset:
PKR-Dependent Mechanisms of Gene Expression from a Subgenomic Hepatitis C Virus Clone
Authors: Rivas-Estilla Ana Maria , Svitkin Yuri , Lopez Lastra Marcelo , Hatzoglou Maria , Sherker Averell , Koromilas Antonis E.
Published: October 2002

Studies on hepatitis C virus (HCV) replication have been greatly advanced by the development of cell culture models for HCV known as replicon systems. The prototype replicon consists of a subgenomic HCV RNA in which the HCV structural region is replaced by the neomycin phosphotransferase II (NPTII) gene, and translation of the HCV proteins NS3 to NS5 is directed by the encephalomyocarditis virus (EMCV) internal ribosome entry site (IRES). The interferon (IFN)-inducible protein kinase PKR plays an important role in cell defense against virus infection by impairing protein synthesis as a result of eIF-2α phosphorylation. Here, we show that expression of the viral nonstructural (NS) and PKR proteins and eIF-2α phosphorylation are all variably regulated in proliferating replicon Huh7 cells. In proliferating cells, induction of PKR protein by IFN-α is inversely proportional to viral RNA replication and NS protein expression, whereas eIF-2α phosphorylation is induced by IFN-α in proliferating but not in serum-starved replicon cells. The role of PKR and eIF-2α phosphorylation was further addressed in transient-expression assays in Huh7 cells. These experiments demonstrated that activation of PKR results in the inhibition of EMCV IRES-driven NS protein synthesis from the subgenomic viral clone through mechanisms that are independent of eIF-2α phosphorylation. Unlike NS proteins, HCV IRES-driven NPTII protein synthesis from the subgenomic clone was resistant to PKR activation. Interestingly, activation of PKR could induce HCV IRES-dependent mRNA translation from dicistronic constructs, but this stimulatory effect was mitigated by the presence of the viral 3′ untranslated region. Thus, PKR may assume multiple roles in modulating HCV replication and protein synthesis, and tight control of PKR activity may play an important role in maintaining virus replication and allowing infection to evade the host's IFN system.
In Situ Localization and Tissue Distribution of the Replication-Associated Proteins of
Cucumber Mosaic Virus
in Tobacco and Cucumber
Authors: Cillo Fabrizio , Roberts Ian M. , Palukaitis Peter
Published: October 2002

The replication-associated proteins encoded by
Cucumber mosaic virus
(CMV), the 1a and 2a proteins, were detected by immunogold labeling in two host species of this virus, tobacco (
Nicotiana tabacum
) and cucumber (
Cucumis sativus
). In both hosts, the 1a and 2a proteins colocalized predominantly to the vacuolar membranes, the tonoplast. While plus-strand CMV RNAs were found distributed throughout the cytoplasm by in situ hybridization, minus-strand CMV RNAs were barely detectable but were found associated with the tonoplast. In both cucumber and tobacco, 2a protein was detected at higher densities than 1a protein. The 1a and 2a proteins also showed quantitative differences with regard to tissue distributions in tobacco and cucumber. About three times as much 2a protein was detected in CMV-infected cucumber tissues as in CMV-infected tobacco tissues. In tobacco, high densities of these proteins were observed only in vascular bundle cells of minor veins. In contrast, in cucumber, high densities of 1a and 2a proteins were observed in mesophyll cells, followed by epidermis cells, with only low levels being observed in vascular bundle cells. Differences were also observed in the distributions of 2a protein and capsid protein in vascular bundle cells of the two host species. These observations may represent differences in the relative rates of tissue infection in different hosts or differences in the extent of virus replication in vascular tissues of different hosts.
Non-AUG-Initiated Internal Translation of the L* Protein of Theiler's Virus and Importance of This Protein for Viral Persistence
Authors: van Eyll Olivier , Michiels Thomas
Published: October 2002

Theiler's virus is a neurotropic murine picornavirus which, depending on the strain, causes either acute encephalitis or persistent demyelinating disease. Persistent strains of Theiler's virus (such as DA) produce an 18-kDa protein called L* from an open reading frame overlapping that encoding the viral polyprotein. Neurovirulent strains (such as GDVII) are thought not to produce the L* protein, as the alternative open reading frame of these strains starts with an ACG codon instead of an AUG codon. However, we observed that both persistent and neurovirulent strain derivatives can produce two forms of the L* protein through unusual type II internal ribosome entry site-mediated translation. A full-length 18-kDa protein can be expressed from an ACG or an AUG initiation codon, whereas an N-terminally truncated 15-kDa product can be translated from a downstream AUG initiation codon. The expression of the 18-kDa form is required for efficient persistence of DA virus derivatives in the central nervous system.
Selection for Human Immunodeficiency Virus Type 1 Recombinants in a Patient with Rapid Progression to AIDS
Authors: Liu Shan-Lu , Mittler John E. , Nickle David C. , Mulvania Thera M. , Shriner Daniel , Rodrigo Allen G. , Kosloff Barry , He Xi , Corey Lawrence , Mullins James I.
Published: October 2002

Although human immunodeficiency virus type 1 (HIV-1) recombinants have been found with high frequency, little is known about the forces that select for these viruses or their importance to pathogenesis. Here we document the emergence and dynamics of 11 distinct HIV-1 recombinants in a man who was infected with two subtype B HIV-1 strains and progressed rapidly to AIDS without developing substantial cellular or humoral immune responses. Although numerous frequency oscillations were observed, a single recombinant lineage eventually came to dominate the population. Numerical simulations indicate that the successive recombinant forms displaced each other too rapidly to be explained by any simple model of random genetic drift or sampling variation. All of the recombinants, including several resulting from independent recombination events, possessed the same sequence motif in the V3 loop, suggesting intense selection on this segment of the viral envelope protein. The outgrowth of the predominant V3 loop recombinants was not, however, associated with changes in coreceptor utilization. The final variant was instead notable for having lost 3 of 14 potential glycosylation sites. We also observed high ratios of synonymous-to-nonsynonymous nucleotide changes—suggestive of purifying selection—in all viral populations, with particularly high ratios in newly arising recombinants. Our study, therefore, illustrates the unusual and important patterns of viral adaptation that can occur in a patient with weak immune responses. Although it is hard to tease apart cause and effect in a single patient, the correlation with disease progression in this patient suggests that recombination between divergent viruses, with its ability to create chimeras with increased fitness, can accelerate progression to AIDS.
Simian Virus 40 Small Tumor Antigen Activates AKT and Telomerase and Induces Anchorage-Independent Growth of Human Epithelial Cells
Authors: Yuan Hang , Veldman Tim , Rundell Kathleen , Schlegel Richard
Published: October 2002

Human keratinocytes immortalized by full-length or early-region simian virus 40 (SV40) DNA grow in agarose and form tumors in nude mice, in contrast to keratinocytes immortalized by the E6/E7 genes of human papillomaviruses. To determine the molecular basis for this biological difference in growth, we have used the individual SV40 oncogenes (large T antigen [LT] and small t antigen [st]) and human papillomavirus oncogenes (E6/E7) to study the progression of human epithelial cells from the nonimmortal to the immortal state as well as from the immortal to the anchorage-independent state. Transfection of primary human foreskin keratinocytes with LT did not immortalize cells but did extend the in vitro life span and produced cells that were resistant to calcium- and serum-induced terminal differentiation. Cells transfected with st alone did not passage beyond vector-transfected keratinocytes. The simultaneous expression of LT- and st-immortalized keratinocytes occurred without evidence of crisis and, as anticipated, these immortal cells were anchorage- independent for growth. Interestingly, we found that keratinocytes expressing both LT and st, but not keratinocytes with LT alone, exhibited increased phosphorylation of the protein kinase AKT. In addition, AKT activation was paralleled by an increase in telomerase activity. Addition of st to anchorage-dependent keratinocytes, expressing either LT (nonimmortal) or E6/E7 (immortal), converted the cells to anchorage independence, with similar accompanying increases in AKT phosphorylation and telomerase activity. However, it was not possible to induce keratinocyte growth in agarose with activated AKT and/or overexpressed hTERT, indicating that these newly defined st-induced activities are not sufficient for progression to the anchorage-independent state.
Dendritic Cell-Mediated Viral Transfer to T Cells Is Required for Human Immunodeficiency Virus Type 1 Persistence in the Face of Rapid Cell Turnover
Authors: Gummuluru Suryaram , KewalRamani Vineet N. , Emerman Michael
Published: October 2002

Human immunodeficiency virus type 1 (HIV-1)-infected and activated CD4+T cells have short half-lives in vivo (<2 days). We have established an in vitro culture system in which infected T cells are turned over frequently to provide a model system that examines this important facet of in vivo HIV-1 replication. We observed that virus replication in T cells under rapid-turnover conditions was possible only when immature dendritic cells or DC-SIGN-expressing cells mediated HIV-1 transmission to T cells. Virus replication was initiated more rapidly in T cells infected with the cell-associated form of virus compared to infection by the cell-free route. This accelerated transfer of virus required adhesion molecule-mediated interactions between the virus-presenting cell and T cell, but surprisingly, HIV-1 transfer could occur independently of DC-SIGN (DC-specific intracellular adhesion molecule 3 [ICAM-3]-grabbing nonintegrin)in the dendritic-cell-T-cell cocultures. These results suggest that dendritic cell-mediated transmission of HIV-1 enables virus replication under conditions of rapid cell turnover in vivo.
Interleukin-18 Inhibits Hepatitis B Virus Replication in the Livers of Transgenic Mice
Authors: Kimura Kiminori , Kakimi Kazuhiro , Wieland Stefan , Guidotti Luca G. , Chisari Francis V.
Published: October 2002

Interleukin-18 (IL-18) produced by activated antigen-presenting cells stimulates natural killer (NK) cells, natural killer T (NKT) cells, and T cells to secrete gamma interferon (IFN-γ). In this study, injection of a single 10-μg dose of recombinant murine IL-18 rapidly, reversibly, and noncytopathically inhibited hepatitis B virus (HBV) replication in the livers of HBV transgenic mice. Furthermore, HBV replication was inhibited by as little as 1 μg of IL-18 injected repetitively, and also by a single 0.1-μg dose of IL-18 injected together with 1 ng of IL-12, neither of which inhibited HBV replication individually, demonstrating synergy between these cytokines in this system. The antiviral effect of IL-18 was mediated by its ability to activate resident intrahepatic NK cells and NKT cells to produce IFN-γ and by its ability to induce IFN-α/β production in the liver. These results suggest that IL-18 has the potential to contribute to the control of HBV replication during self-limited infection and that it may have therapeutic value for the treatment of patients with chronic hepatitis.
The Transmembrane Domain and Cytoplasmic Tail of Herpes Simplex Virus Type 1 Glycoprotein H Play a Role in Membrane Fusion
Authors: Harman Andrew , Browne Helena , Minson Tony
Published: October 2002

Herpes simplex virus glycoprotein H (gH) is one of the four virion envelope proteins which are required for virus entry and for cell-cell fusion in a transient system. In this report, the role of the transmembrane and cytoplasmic tail domains of gH in membrane fusion was investigated by generating chimeric constructs in which these regions were replaced with analogous domains from other molecules and by introducing amino acid substitutions within the membrane-spanning sequence. gH molecules which lack the authentic transmembrane domain or cytoplasmic tail were unable to mediate cell-cell fusion when coexpressed with gB, gD, and gL and were unable to rescue the infectivity of a gH-null virus as efficiently as a wild-type gH molecule. Many amino acid substitutions of specific amino acid residues within the transmembrane domain also affected cell-cell fusion, in particular, those introduced at a conserved glycine residue. Some gH mutants that were impaired in cell-cell fusion were nevertheless able to rescue the infectivity of a gH-negative virus, but these pseudotyped virions entered cells more slowly than wild-type virions. These results indicate that the fusion event mediated by the coexpression of gHL, gB, and gD in cells shares common features with the fusion of the virus envelope with the plasma membrane, they point to a likely role for the membrane-spanning and cytoplasmic tail domains of gH in both processes, and they suggest that a conserved glycine residue in the membrane-spanning sequence is crucial for efficient fusion.
Characterization of the 1918 “Spanish” Influenza Virus Matrix Gene Segment
Authors: Reid Ann H. , Fanning Thomas G. , Janczewski Thomas A. , McCall Sherman , Taubenberger Jeffery K.
Published: October 2002

The coding region of influenza A virus RNA segment 7 from the 1918 pandemic virus, consisting of the open reading frames of the two matrix genes M1 and M2, has been sequenced. While this segment is highly conserved among influenza virus strains, the 1918 sequence does not match any previously sequenced influenza virus strains. The 1918 sequence matches the consensus over the M1 RNA-binding domains and nuclear localization signal and the highly conserved transmembrane domain of M2. Amino acid changes that correlate with high yield and pathogenicity in animal models were not found in the 1918 strain. Phylogenetic analyses suggest that both genes were mammalian adapted and that the 1918 sequence is very similar to the common ancestor of all subsequent human and classical swine matrix segments. The 1918 sequence matches other mammalian strains at 4 amino acids in the extracellular domain of M2 that differ consistently between avian and mammalian strains, suggesting that the matrix segment may have been circulating in human strains for at least several years before 1918.
Hantavirus Infection of Dendritic Cells
Authors: Raftery Martin J. , Kraus Annette A. , Ulrich Rainer , Krüger Detlev H. , Schönrich Günther
Published: October 2002

Dendritic cells (DCs) play a pivotal role as antigen-presenting cells in the antiviral immune response. Here we show that Hantaan virus (HTNV), which belongs to theBunyaviridaefamily (genusHantavirus) and causes hemorrhagic fever with renal syndrome, productively infects human DCs in vitro. In the course of HTNV infection, DCs did not show any cytopathic effect and viral replication did not induce cell lysis or apoptosis. Furthermore, HTNV did not affect apoptosis-inducing signals that are important for the homeostatic control of mature DCs. In contrast to immunosuppressive viruses, e.g., human cytomegalovirus, HTNV activated immature DCs, resulting in upregulation of major histocompatibility complex (MHC), costimulatory, and adhesion molecules. Intriguingly, strong upregulation of MHC class I molecules and an increased intercellular cell adhesion molecule type 1 expression was also detected on HTNV-infected endothelial cells. In addition, antigen uptake by HTNV-infected DCs was reduced, another characteristic feature of DC maturation. Consistent with these findings, we observed that HTNV-infected DCs stimulated T cells as efficiently as did mature DCs. Finally, infection of DCs with HTNV induced the release of the proinflammatory cytokines tumor necrosis factor alpha and alpha interferon. Taken together, our findings indicate that hantavirus-infected DCs may significantly contribute to hantavirus-associated pathogenesis.
Efficient Generation and Amplification of High-Capacity Adeno-Associated Virus/Adenovirus Hybrid Vectors
Authors: Gonçalves Manuel A. F. V. , van der Velde Ietje , Janssen Josephine M. , Maassen Bram T. H. , Heemskerk Evert H. , Opstelten Dirk-Jan E. , Knaän-Shanzer Shoshan , Valerio Dinko , de Vries Antoine A. F.
Published: October 2002

Effective gene therapy is dependent on safe gene delivery vehicles that can achieve efficient transduction and sustained transgene expression. We are developing a hybrid viral vector system that combines in a single particle the large cloning capacity and efficient cell cycle-independent nuclear gene delivery of adenovirus (Ad) vectors with the long-term transgene expression and lack of viral genes of adeno-associated virus (AAV) vectors. The strategy being pursued relies on coupling the AAV DNA replication mechanism to the Ad encapsidation process through packaging of AAV-dependent replicative intermediates provided with Ad packaging elements into Ad capsids. The generation of these high-capacity AAV/Ad hybrid vectors takes place in Ad early region 1 (E1)-expressing cells and requires an Ad vector with E1 deleted to complement in
trans
both AAV helper functions and Ad structural proteins. The dependence on a replicating helper Ad vector leads to the contamination of AAV/Ad hybrid vector preparations with a large excess of helper Ad particles. This renders the further propagation and ultimate use of these gene delivery vehicles very difficult. Here, we show that Cre/
loxP
-mediated genetic selection against the packaging of helper Ad DNA can reduce helper Ad vector contamination by 99.98% without compromising hybrid vector rescue. This allowed amplification of high-capacity AAV/Ad hybrid vectors to high titers in a single round of propagation.
Intrapatient Diversity and Its Correlation with Viral Setpoint in Human Immunodeficiency Virus Type 1 CRF02_A/G-IbNG Infection
Authors: Mani Indu , Gilbert Peter , Sankalé Jean-Louis , Eisen Geoffrey , Mboup Souleymane , Kanki Phyllis J.
Published: October 2002

The human immunodeficiency virus type 1 (HIV-1) viral setpoint during the disease-free interval has been strongly associated with future risk of disease progression. An awareness of the correlation between viral setpoint and HIV-1 genetic evolution over time is important in the understanding of viral dynamics and infection. We examined genetic diversity in HIV-1 CRF02_A/G-IbNG-infected seroincident women in Dakar, Senegal; determined whether a viral setpoint kinetic pattern existed for CRF02_A/G-IbNG during the disease-free interval; and correlated viral load level and diversity. Samples were drawn during the disease-free interval from consenting CRF02_A/G-IbNG-infected, antiretroviral therapy-naïve female commercial sex workers in Dakar, Senegal. Based on sequential plasma RNA values, low and high viral setpoint groups were established. Intrapatient diversity and divergence over time was determined from earlier and later time point DNA samples from each person. Most individuals followed the viral setpoint paradigm. For each 1|−|log
10
copy/ml of plasma increase in viral load, intrapatient diversity increased by 1.4% (
P
= 0.028). A greater diversification rate was observed in the high viral setpoint group than in the low viral setpoint group (
P
= 0.01). Greater nucleotide (
P
= 0.015) and amino acid (
P
= 0.048) divergences and a greater nucleotide divergence rate (
P
= 0.03) were found in the high viral setpoint group. There was no difference between the groups in the ratio of the number of nonsynonymous substitutions per nonsynonymous site to the number of synonymous substitutions per synonymous site. The greater intrapatient diversity, divergence, and diversification rates observed in the high viral setpoint group supports the notion that diversity is driven by cycles of viral replication resulting in accumulated mutations. Recognizing diversity potential based on viral load levels in individuals may inform the design of vaccines and therapies.
Mutations Conferring Resistance to Neutralization by a Soluble Form of the Neurotrophin Receptor (p75NTR) Map outside of the Known Antigenic Sites of the Rabies Virus Glycoprotein
Authors: Langevin Christelle , Tuffereau Christine
Published: October 2002

The neurotrophin receptor (p75NTR) serves as a receptor for rabies virus (RV). We expressed and purified a soluble chimera consisting of the p75NTR ectodomain fused to the human immunoglobulin G1 (IgG1) Fc fragment (p75-Fc). Although p75-Fc interacts with RV, the infectivity of RV did not decrease significantly when it was incubated in the presence of the soluble receptor alone. However, when it was subsequently incubated with an antihuman IgG directed against the Fc fragment of p75-Fc, the infectivity of RV was significantly lowered (>90%), whereas incubation with antihuman IgG alone had no effect. We then selected eight independent RV mutants that were not neutralized by p75-Fc and antihuman IgG (srr[soluble receptor resistant] mutants). Each mutant carried a single mutation in the glycoprotein gene leading to one amino acid substitution in the protein. A total of four different substitutions were found. Two of the mutations were located at position 318 (phenylalanine replaced by a serine or a valine residue), and two were located at position 352 (histidine replaced by a tyrosine or an arginine residue). All of the mutations prevented the interaction with p75NTR as either a soluble or a membrane-anchored form. Two mutants (F318S) and (H352R) resulted in the formation of small plaques on BSR cells, probably due to the slower maturation of the glycoprotein. Immunoprecipitation, immunofluorescence, and neutralization assays showed that the four mutated glycoproteins still interacted with representative anti-RV glycoprotein monoclonal antibodies (MAbs), indicating that p75NTR binds outside of the known RV glycoprotein antigenic sites.
Complementation Analysis of the Flavivirus Kunjin NS3 and NS5 Proteins Defines the Minimal Regions Essential for Formation of a Replication Complex and Shows a Requirement of NS3 in
cis
for Virus Assembly
Authors: Liu Wen Jun , Sedlak Petra L. , Kondratieva Natasha , Khromykh Alexander A.
Published: October 2002

We have previously reported successful
trans
-complementation of defective Kunjin virus genomic RNAs with a range of large lethal deletions in the nonstructural genes NS1, NS3, and NS5 (A. A. Khromykh et al., J. Virol.
74:
3253-3263, 2000). In this study we have mapped further the minimal region in the NS5 gene essential for efficient
trans
-complementation of genome-length RNAs in repBHK cells to the first 316 of the 905 codons. To allow amplification and easy detection of complemented defective RNAs with deletions apparently affecting virus assembly, we have developed a dual replicon complementation system. In this system defective replicon RNAs with a deletion(s) in the nonstructural genes also encoded the puromycin resistance gene (PAC gene) and the reporter gene for β-galactosidase (β-Gal). Complementation of these defective replicon RNAs in repBHK cells resulted in expression of PAC and β-Gal which allowed establishment of cell lines stably producing replicating defective RNAs by selection with puromycin and comparison of replication efficiencies of complemented defective RNAs by β-Gal assay. Using this system we demonstrated that deletions in the C-terminal 434 codons of NS3 (codons 178 to 611) were complemented for RNA replication, while any deletions in the first 178 codons were not. None of the genome-length RNAs containing deletions in NS3 shown to be complementable for RNA replication produced secreted defective viruses during complementation in repBHK cells. In contrast, structural proteins produced from these complemented defective RNAs were able to package helper replicon RNA. The results define minimal regions in the NS3 and NS5 genes essential for the formation of complementable replication complex and show a requirement of NS3 in
cis
for virus assembly.
The Major Phosphorylation Sites of the Respiratory Syncytial Virus Phosphoprotein Are Dispensable for Virus Replication In Vitro
Authors: Lu Bin , Ma Chien-Hui , Brazas Robert , Jin Hong
Published: October 2002

The phosphoprotein (P protein) of respiratory syncytial virus (RSV) is a key component of the viral RNA-dependent RNA polymerase complex. The protein is constitutively phosphorylated at the two clusters of serine residues (116, 117, and 119 [116/117/119] and 232 and 237 [232/237]). To examine the role of phosphorylation of the RSV P protein in virus replication, these five serine residues were altered to eliminate their phosphorylation potential, and the mutant proteins were analyzed for their functions with a minigenome assay. The reporter gene expression was reduced by 20% when all five phosphorylation sites were eliminated. Mutants with knockout mutations at two phosphorylation sites (S232A/S237A [PP2]) and at five phosphorylation sites (S116L/S117R/S119L/S232A/S237A [PP5]) were introduced into the infectious RSV A2 strain. Immunoprecipitation of
33
P
i
-labeled infected cells showed that P protein phosphorylation was reduced by 80% for rA2-PP2 and 95% for rA2-PP5. The interaction between the nucleocapsid (N) protein and P protein was reduced in rA2-PP2- and rA2-PP5-infected cells by 30 and 60%, respectively. Although the two recombinant viruses replicated well in Vero cells, rA2-PP2 and, to a greater extent, rA2-PP5, replicated poorly in HEp-2 cells. Virus budding from the infected HEp-2 cells was affected by dephosphorylation of P protein, because the majority of rA2-PP5 remained cell associated. In addition, rA2-PP5 was also more attenuated than rA2-PP2 in replication in the respiratory tracts of mice and cotton rats. Thus, our data suggest that although the major phosphorylation sites of RSV P protein are dispensable for virus replication in vitro, phosphorylation of P protein is required for efficient virus replication in vitro and in vivo.
Identification of Conserved and Variable Structures in the Human Immunodeficiency Virus gp120 Glycoprotein of Importance for CXCR4 Binding
Authors: Basmaciogullari Stéphane , Babcock Gregory J. , Van Ryk Donald , Wojtowicz Woj , Sodroski Joseph
Published: October 2002

CD4 and the chemokine receptors, CXCR4 and CCR5, serve as receptors for human immunodeficiency virus type 1 (HIV-1). Binding of the HIV-1 gp120 envelope glycoprotein to the chemokine receptors normally requires prior interaction with CD4. Mapping the determinants on gp120 for the low-affinity interaction with CXCR4 has been difficult due to the nonspecific binding of this viral glycoprotein to cell surfaces. Here we examine the binding of a panel of gp120 mutants to paramagnetic proteoliposomes displaying CXCR4 on their surfaces. We show that the gp120 β19 strand and third variable (V3) loop contain residues important for CXCR4 interaction. Basic residues from both elements, as well as a conserved hydrophobic residue at the V3 tip, contribute to CXCR4 binding. Removal of the gp120 V1/V2 variable loops allows the envelope glycoprotein to bind CXCR4 in a CD4-independent manner. These results indicate that although some variable gp120 residues contribute to the specific binding to CCR5 or CXCR4, gp120 elements common to CXCR4- or CCR5-using strains are involved in the interaction with both coreceptors.
Characterization of Moloney Murine Leukemia Virus p12 Mutants Blocked during Early Events of Infection
Authors: Yuan Bing , Fassati Ariberto , Yueh Andrew , Goff Stephen P.
Published: October 2002

Mutations affecting either the N- or C-terminal regions of the Gag protein p12 block replication of Moloney murine leukemia virus (M-MuLV). Viruses carrying mutations in this portion of
gag
can mediate the assembly and release of virions but are unable to successfully carry out the early phase of the M-MuLV life cycle. Wild-type and mutant viruses were found to synthesize similar levels of linear viral DNA in both cytoplasmic and nuclear fractions, and there were no significant differences in either the density or sedimentation of the viral protein-nucleic acid complexes. Analysis of the termini of the linear viral DNAs showed that the 3′ ends of the mutant viral DNA were processed normally by the integrase. Further, the preintegration complexes extracted from the cytoplasm of cells infected with the mutant viruses were competent for integration into target DNA in vitro. Nevertheless, no circular viral DNAs were detected in cells infected by the mutants, and functional proviruses were not formed. These results suggest that p12 has an unexpected role in the early phase of the life cycle and is needed after viral DNA synthesis to deliver the incoming DNA to the correct location and in the appropriate state to permit either circularization or integration of the viral DNA in vivo.
The Mason-Pfizer Monkey Virus Internal Scaffold Domain Enables In Vitro Assembly of Human Immunodeficiency Virus Type 1 Gag
Authors: Sakalian Michael , Dittmer Stephanie S. , Gandy A. Dustin , Rapp Nathan D. , Zábranský Aleš , Hunter Eric
Published: October 2002

The Mason-Pfizer monkey virus (M-PMV) Gag protein possesses the ability to assemble into an immature capsid when synthesized in a reticulocyte lysate translation system. In contrast, the human immunodeficiency virus (HIV) Gag protein is incapable of assembly in parallel assays. To enable the assembly of HIV Gag, we have combined or inserted regions of M-PMV Gag into HIV Gag. By both biochemical and morphological criteria, several of these chimeric Gag molecules are capable of assembly into immature capsid-like structures in this in vitro system. Chimeric species containing large regions of M-PMV Gag fused to HIV Gag sequences failed to assemble, while species consisting of only the M-PMV p12 region, and its internal scaffold domain (ISD), fused to HIV Gag were capable of assembly, albeit at reduced kinetics compared to M-PMV Gag. The ability of the ISD to induce assembly of HIV Gag, which normally assembles at the plasma membrane, suggests a common requirement for a concentrating factor in retrovirus assembly. Despite the dramatic effect of the ISD on chimera assembly, the function of HIV Gag domains in that process was found to remain essential, since an assembly-defective mutant of HIV CA, M185A, abolished assembly when introduced into the chimera. This continued requirement for HIV Gag domain function in the assembly of chimeric molecules will allow this in vitro system to be used for the analysis of potential inhibitors of HIV immature particle assembly.
Identification, Localization, and Regulation of Expression of the UL24 Protein of Herpes Simplex Virus Type 1
Authors: Pearson Angela , Coen Donald M.
Published: October 2002

The
UL24
gene of herpes simplex virus type 1 is conserved across many herpesviruses, but its protein product has not been identified. We expressed the
UL24
gene in insect cells from a recombinant baculovirus and used the resulting protein to raise a rat antiserum. In immunoblotting experiments, this antiserum recognized a 30-kDa protein in lysates from infected cells. The identity of this species as UL24 was confirmed by using a virus encoding a truncated form of UL24. On the basis of biochemical fractionation of infected cells, UL24 appeared to be predominantly nucleus associated, especially at later times in infection. Although certain
UL24
transcripts exhibit early kinetics, UL24 protein accumulated at later times in infection and levels were decreased sixfold in the presence of the viral DNA synthesis inhibitor phosphonoacetic acid; thus, UL24 was expressed with leaky-late kinetics. The entire
UL24
open reading frame is encoded by mRNAs with two different 5′ ends. A mutation that eliminates the more abundant transcripts that originate at the first transcription start site resulted in a 10-fold reduction in the level of UL24 expressed but did not eliminate expression. Thus, the less abundant transcripts originating at the second transcription start site can evidently be translated, although transcripts originating at the first start site appear to be the major contributors to the expression of UL24. We conclude that UL24 is a bona fide herpes simplex virus type 1 protein that associates primarily with nuclei and whose expression is regulated at multiple levels.
Characterization of Two New Structural Glycoproteins, GP
3
and GP
4
, of Equine Arteritis Virus
Authors: Wieringa Roeland , de Vries Antoine A. F. , Raamsman Martin J. B. , Rottier Peter J. M.
Published: October 2002

Equine arteritis virus (EAV) is an enveloped, positive-stranded RNA virus belonging to the family
Arteriviridae
of the order
Nidovirales
. Four envelope proteins have hitherto been identified in EAV particles: the predominant membrane proteins M and G
L
, the unglycosylated small envelope protein E, and the nonabundant membrane glycoprotein G
S
. In this study, we established that the products of EAV open reading frame 3 (ORF3) and ORF4 (designated GP
3
and GP
4
, respectively) are also minor structural glycoproteins. The proteins were first characterized by various analyses after in vitro translation of RNA transcripts in a rabbit reticulocyte lysate in the presence and absence of microsomal membranes. We subsequently expressed ORF3 and -4 in baby hamster kidney cells by using the vaccinia virus expression system and, finally, analyzed the GP
3
and GP
4
proteins synthesized in EAV-infected cells. The results showed that GP
4
is a class I integral membrane protein of 28 kDa with three functional N-glycosylation sites and with little, if any, of its carboxy terminus exposed. Both after independent expression and in EAV-infected cells, the protein localizes in the endoplasmic reticulum (ER), as demonstrated biochemically by analysis of its oligosaccharide side chains and as visualized directly by immunofluorescence studies. GP
3
, on the other hand, is a heavily glycosylated protein whose hydrophobic amino terminus is not cleaved off. It is an integral membrane protein anchored by either or both of its hydrophobic terminal domains and with no parts detectably exposed cytoplasmically. Also, GP
3
localizes in the ER when expressed independently and in the context of an EAV infection. Only a small fraction of the GP
3
and GP
4
proteins synthesized in infected cells ends up in virions. Most, but not all, of the oligosaccharides of these virion glycoproteins are biochemically mature. Our results bring the number of EAV envelope proteins to six.
The Genes Encoding the gCIII Complex of Human Cytomegalovirus Exist in Highly Diverse Combinations in Clinical Isolates
Authors: Rasmussen Lucy , Geissler Aimee , Cowan Catherine , Chase Amanda , Winters Mark
Published: October 2002

The UL74 (glycoprotein O [gO])-UL75 (gH)-UL115 (gL) complex of human cytomegalovirus (CMV), known as the gCIII complex, is likely to play an important role in the life cycle of the virus. The gH and gL proteins have been associated with biological activities, such as the induction of virus-neutralizing antibody, cell-virus fusion, and cell-to-cell spread of the virus. The sequences of the two gH gene variants, readily recognizable by restriction endonuclease polymorphism, are well conserved among clinical isolates, but nothing is known about the sequence variability of the gL and gO genes. Sequencing of the full-length gL and gO genes was performed with 22 to 39 clinical isolates, as well as with laboratory strains AD169, Towne, and Toledo, to determine phylogenetically based variants of the genes. The sequence information provided the basis for identifying gL and gO variants by restriction endonuclease polymorphism. The predicted gL amino acid sequences varied less than 2% among the isolates, but the variability of gO among the isolates approached 45%. The variants of the genes coding for gCIII in laboratory strains Towne, AD169, and Toledo were different from those in most clinical isolates. When clinical isolates from different patient populations with various degrees of symptomatic CMV disease were surveyed, the gO1 variant occurred almost exclusively with the gH1 variant. The gL2 variant occurred with a significantly lower frequency in the gH1 variant group. There were no configurations of the gCIII complex that were specifically associated with symptomatic CMV disease or human immunodeficiency virus serologic status. The potential for the gCIII complex to exist in diverse genetic combinations in clinical isolates points to a new aspect that must be considered in studies of the significance of CMV strain variability.
Mutational Analysis of the Uukuniemi Virus (
Bunyaviridae
Family) Promoter Reveals Two Elements of Functional Importance
Authors: Flick Ramon , Elgh Fredrik , Pettersson Ralf F.
Published: October 2002

We have performed an extensive mutational analysis of the proposed promoter region of the phlebovirus Uukuniemi (UUK), a member of the
Bunyaviridae
family. This was achieved by using a recently developed RNA polymerase I (Pol I)-driven reverse genetics system (R. Flick and R. F. Pettersson, J. Virol.
75:
1643-1655, 2001). Chimeric cDNAs containing the coding region for the reporter chloramphenicol acetyltransferase (CAT) in an antisense orientation were flanked by the 5′- and 3′-terminal nontranslated regions of the UUK virus-sense RNA (vRNA) derived from the medium-sized (M) RNA segment. The chimeric cDNAs (Pol I expression cassettes) were cloned between the murine Pol I promoter and terminator, and the plasmids were transfected into BHK-21 cells. CAT activity was determined after cotransfection with viral expression plasmids encoding the RNA-dependent RNA polymerase (L) and the nucleoprotein (N) or, alternatively, after superinfection with UUK virus helper virus. Using oligonucleotide-directed mutagenesis, single point mutations (substitutions, deletions, and insertions) were introduced into the viral promoter region. Differences in CAT activities were interpreted to reflect the efficiency of mRNA transcription from the mutated promoter and the influence on RNA replication. Analysis of 109 mutants allowed us to define two important regulatory regions within the proximal promoter region (site A, positions 3 to 5 and
2
to
4
; site B, positions 8 and
8
, where underlined nucleotides refer to positions in the vRNA 3′ end). Complementary double nucleotide exchanges in the proximal promoter region, which maintained the possibility for base pairing between the 5′ and 3′ ends, demonstrated that nucleotides in the two described regions are essential for viral polymerase recognition in a base-specific manner. Thus, mere preservation of panhandle base pairing between the 5′ and 3′ ends is not sufficient for promoter activity. In conclusion, we have been able to demonstrate that both ends of the M RNA segment build up the promoter region and are involved in the specific recognition by the viral polymerase.
Envelope Proteins Containing Single Amino Acid Substitutions Support a Structural Model of the Receptor-Binding Domain of Bovine Leukemia Virus Surface Protein
Authors: Johnston Elizabeth R. , Albritton Lorraine M. , Radke Kathryn
Published: October 2002

Functional domains of the strikingly conserved envelope (Env) glycoproteins of bovine leukemia virus (BLV) and its close relative, human T-cell leukemia virus type 1 (HTLV-1), are still being defined. We have used BLV Env protein variants to gain insights into the structure and function of this important determinant of viral infectivity. Each of 23 different single amino acid variants found in cDNA clones of
env
transcripts present after short-term culture of peripheral blood mononuclear cells from BLV-infected sheep was expressed in COS-1 cells and tested for the ability to mediate cell fusion and to be cleaved to surface (SU) and transmembrane (TM) protein subunits. Of 11 Env variants that failed to induce syncytia or did so poorly, 7 contained changes in amino acids identical or chemically conserved in the HTLV-1 Env protein. These seven included the four variants that showed aberrant proteolytic cleavage and poor cell surface expression, underscoring their importance for Env structure. Ten of 12 variants that retained wild-type syncytium-inducing ability clustered in the N-terminal half of BLV SU, which forms the putative receptor-binding domain (RBD). Several variants in the RBD showed evidence of subtle misfolding, as judged by reduced binding to monoclonal antibodies recognizing conformational epitopes F, G, and H formed by the N terminus of SU. We modeled the BLV RBD by aligning putative structural elements with known elements of the ecotropic Friend murine leukemia virus RBD monomer. All the variant RBD residues but one are exposed on the surface of this BLV model. These variants as well as function-altering, antibody-reactive residues defined by other investigators group on one face of the molecular model. They are strikingly absent from the opposite face, implying that it is likely to face inward in Env complexes. This surface might interact with the C-terminal domain of SU or with an adjacent monomer in the Env oligomer. This location suggests an orientation for the monomer of ecotropic Friend murine leukemia virus RBD.
Optimized Chimeras between Kinase-Inactive Mutant Cdk9 and Truncated Cyclin T1 Proteins Efficiently Inhibit Tat Transactivation and Human Immunodeficiency Virus Gene Expression
Authors: Fujinaga Koh , Irwin Dan , Geyer Matthias , Peterlin B. Matija
Published: October 2002

The human cyclin T1 (hCycT1) protein from the positive transcription elongation factor b (P-TEFb) binds the transactivator Tat and the transactivation response (TAR) RNA stem loop from human immunodeficiency virus type 1 (HIV). This complex activates the elongation of viral transcription. To create effective inhibitors of Tat and thus HIV replication, we constructed mutant hCycT1 proteins that are defective in binding its kinase partner, Cdk9, or TAR. Although these mutant hCycT1 proteins did not increase Tat transactivation in murine cells, their dominant-negative effects were small in human cells. Higher inhibitory effects were obtained when hCycT1 was fused with the mutant Cdk9 protein. Since the autophosphorylation of the C terminus of Cdk9 is required for the formation of the stable complex between P-TEFb, Tat, and TAR, these serines and threonines were changed to glutamate in a kinase-inactive Cdk9 protein. This chimera inhibited Tat transactivation and HIV gene expression in human cells. Therefore, this dominant-negative kinase-inactive mutant Cdk9.hCycT1 chimera could be used for antiviral gene therapy.
Structure-Based Analysis of the Herpes Simplex Virus Glycoprotein D Binding Site Present on Herpesvirus Entry Mediator HveA (HVEM)
Authors: Connolly Sarah A. , Landsburg Daniel J. , Carfi Andrea , Wiley Don C. , Eisenberg Roselyn J. , Cohen Gary H.
Published: October 2002

Binding of herpes simplex virus (HSV) envelope glycoprotein D (gD) to a cell surface receptor is an essential step of virus entry. We recently determined the crystal structure of gD bound to one receptor, HveA. HveA is a member of the tumor necrosis factor receptor family and contains four characteristic cysteine-rich domains (CRDs). The first two CRDs of HveA are necessary and sufficient for gD binding. The structure of the gD-HveA complex reveals that 17 amino acids in HveA CRD1 and 4 amino acids in HveA CRD2 directly contact gD. To determine the contribution of these 21 HveA residues to virus entry, we constructed forms of HveA mutated in each of these contact residues. We determined the ability of the mutant proteins to bind gD, facilitate virus entry, and form HveA oligomers. Our results point to a binding hot spot centered around HveA-Y23, a residue that protrudes into a crevice on the surface of gD. Both the hydroxyl group and phenyl group of HveA-Y23 contribute to HSV entry. Our results also suggest that an intermolecular β-sheet formed between gD and HveA residues 35 to 37 contributes to binding and that a C37-C19 disulfide bond in CRD1 is a critical component of HveA structure necessary for gD binding. The results argue that CRD2 is required for gD binding mainly to provide structural support for a gD binding site in CRD1. Only one mutant, HveA-R75A, exhibited enhanced gD binding. While some mutations influenced complex formation, the majority did not affect HSV entry, suggesting that most contact residues contribute to HveA receptor function collectively rather than individually. This structure-based dissection of the gD-HveA binding site highlights the contribution of key residues within HveA to gD binding and HSV entry and defines a target region for the design of small-molecule inhibitors.
Microglia from Creutzfeldt-Jakob Disease-Infected Brains Are Infectious and Show Specific mRNA Activation Profiles
Authors: Baker Christopher A. , Martin Daniel , Manuelidis Laura
Published: October 2002

Neurons are often assumed to be the principal sites for replication of the infectious agents causing Creutzfeldt-Jakob disease (CJD), scrapie, and bovine spongiform encephalopathy because they express high levels of normal and pathological prion protein (PrP). However, isolated brain cell types have not been evaluated for either infection or gene expression. Microglia purified from CJD-infected mice showed infectivity comparable to that of starting brain homogenate but expressed ∼50-fold less PrP. CJD-infected microglia also displayed morphological changes indicative of cellular activation. To determine the molecular pathways of activation, we evaluated pertinent transcripts, including those linked to inflammation. Semiquantitative reverse transcription-PCR showed a >4-fold increase in cathepsin S, an enzyme important in antigen presentation, the cytokine interleukin-1β, and the chemokine B-lymphocyte chemoattractant. The profile of microglial changes induced by the CJD agent differed substantially from activation induced by bacterial lipopolysaccharide or by β-amyloid, a structure comparable to pathological PrP. These microglial studies emphasize migratory hematopoietic cells in the dispersion, and possibly replication, of the CJD agent. The low PrP levels in these highly infectious and activated cells further support the concept that pathological PrP is the result of infection rather than the infectious agent itself. Because microglia develop a specific pattern of responses to the CJD agent, microglial markers may be exploited in the diagnosis of these spongiform encephalopathies.
Modulation of the Cell Division Cycle by Human Papillomavirus Type 18 E4
Authors: Nakahara Tomomi , Nishimura Akiko , Tanaka Masakazu , Ueno Takaharu , Ishimoto Akinori , Sakai Hiroyuki
Published: October 2002

The life cycle of human papillomaviruses (HPVs) is tightly coupled to the differentiation program of their host epithelial cells. HPV E4 gene expression is first observed in the parabasal layers of squamous epithelia, suggesting that the E4 gene product contributes to the mechanism of differentiation-dependent virus replication, although its biological function remains unclear. We analyzed the effect of HPV type 18 E4 on cell proliferation and found that E4 expression induced cell cycle arrest at the G
2
/M boundary. The functional region of E4 necessary for the growth arrest activity was located in the central portion of the molecule, and this activity was independent of the E4-mediated collapse of cytokeratin intermediate filament structures.