bluuebunny/crossref_metadata_2025_split huggingface dataset:
Characterization and Structural Analysis of Novel Mutations in Human Immunodeficiency Virus Type 1 Reverse Transcriptase Involved in the Regulation of Resistance to Nonnucleoside Inhibitors
Authors: Ceccherini-Silberstein Francesca , Svicher Valentina , Sing Tobias , Artese Anna , Santoro Maria Mercedes , Forbici Federica , Bertoli Ada , Alcaro Stefano , Palamara Guido , d'Arminio Monforte Antonella , Balzarini Jan , Antinori Andrea , Lengauer Thomas , Perno Carlo Federico 
Published: August 2007

ABSTRACTResistance to antivirals is a complex and dynamic phenomenon that involves more mutations than are currently known. Here, we characterize 10 additional mutations (L74V, K101Q, I135M/T, V179I, H221Y, K223E/Q, and L228H/R) in human immunodeficiency virus type 1 (HIV-1) reverse transcriptase which are involved in the regulation of resistance to nonnucleoside reverse transcriptase inhibitors (NNRTIs). These mutations are strongly associated with NNRTI failure and strongly correlate with the classical NNRTI resistance mutations in a data set of 1,904 HIV-1 B-subtypepolsequences from 758 drug-naïve patients, 592 nucleoside reverse transcriptase inhibitor (NRTI)-treated but NNRTI-naïve patients, and 554 patients treated with both NRTIs and NNRTIs. In particular, L74V and H221Y, positively correlated with Y181C, were associated with an increase in Y181C-mediated resistance to nevirapine, while I135M/T mutations, positively correlated with K103N, were associated with an increase in K103N-mediated resistance to efavirenz. In addition, the presence of the I135T polymorphism in NNRTI-naïve patients significantly correlated with the appearance of K103N in cases of NNRTI failure, suggesting that I135T may represent a crucial determinant of NNRTI resistance evolution. Molecular dynamics simulations show that I135T can contribute to the stabilization of the K103N-induced closure of the NNRTI binding pocket by reducing the distance and increasing the number of hydrogen bonds between 103N and 188Y. H221Y also showed negative correlations with type 2 thymidine analogue mutations (TAM2s); its copresence with the TAM2s was associated with a higher level of zidovudine susceptibility. Our study reinforces the complexity of NNRTI resistance and the significant interplay between NRTI- and NNRTI-selected mutations. Mutations beyond those currently known to confer resistance should be considered for a better prediction of clinical response to reverse transcriptase inhibitors and for the development of more efficient new-generation NNRTIs.
Modulation of the Brd4/P-TEFb Interaction by the Human T-Lymphotropic Virus Type 1 Tax Protein
Authors: Cho Won-Kyung , Zhou Meisheng , Jang Moon Kyoo , Huang Keven , Jeong Soo-Jin , Ozato Keiko , Brady John N. 
Published: August 2007

ABSTRACT
Positive transcription elongation factor (P-TEFb), which is composed of CDK9 and cyclin T1, plays an important role in cellular and viral gene expression. Our lab has recently demonstrated that P-TEFb is required for Tax transactivation of the viral long terminal repeat (LTR). P-TEFb is found in two major complexes: the inactive form, which is associated with inhibitory subunits 7SK snRNA and HEXIM1, and the active form, which is associated with, at least in part, Brd4. In this study, we analyzed the effect of Brd4 on human T-lymphotropic virus type 1 (HTLV-1) transcription. Overexpression of Brd4 repressed Tax transactivation of the HTLV-1 LTR in a dose-dependent manner. In vitro binding studies suggest that Tax and Brd4 compete for binding to P-TEFb through direct interaction with cyclin T1. Tax interacts with cyclin T1 amino acids 426 to 533, which overlaps the region responsible for Brd4 binding. In vivo, overexpression of Tax decreased the amount of 7SK snRNA associated with P-TEFb and stimulates serine 2 phosphorylation of the RNA polymerase II carboxyl-terminal domain, suggesting that Tax regulates the functionality of P-TEFb. Our results suggest the possibility that Tax may compete and functionally substitute for Brd4 in P-TEFb regulation.
Rabbitpox Virus and Vaccinia Virus Infection of Rabbits as a Model for Human Smallpox
Authors: Adams Mathew M. , Rice Amanda D. , Moyer R. W. 
Published: August 2007

ABSTRACT

The threat of smallpox release and use as a bioweapon has encouraged the search for new vaccines and antiviral drugs, as well as development of new small-animal models in which their efficacy can be determined. Here, we reinvestigate a rabbit model in which the intradermal infection of rabbits with very low doses of either rabbitpox virus (RPV) or vaccinia virus Western Reserve (VV-WR) recapitulates many of the clinical features of human smallpox. Following intradermal inoculation with RPV, rabbits develop systemic disease characterized by extensive viremia, numerous secondary lesions on the skin and mucocutaneous tissues, severe respiratory disease, death by 9 days postinfection, and, importantly, natural aerosol transmission between animals. Contrary to previous reports, intradermal infection with VV-WR also resulted in a very similar lethal systemic disease in rabbits, again with natural aerosol transmission between animals. When sentinel and index animals were cohoused, transmission rates approached 100% with either virus, with sentinel animals exhibiting a similar, severe disease. Lower rates of transmission were observed when index and sentinel animals were housed in separate cages. Sentinel animals infected with RPV with one exception succumbed to the disease. However, the majority of VV-WR-infected sentinel animals, while becoming seriously ill, survived. Finally, we tested the efficacy of the drug 1-
O
-hexadecyloxypropyl-cidofovir in the RPV/rabbit model and found that an oral dose of 5 mg/kg twice a day for 5 days beginning 1 day before infection was able to completely protect rabbits from lethal disease.
Distinct Intracellular Trafficking of Equine Infectious Anemia Virus and Human Immunodeficiency Virus Type 1 Gag during Viral Assembly and Budding Revealed by Bimolecular Fluorescence Complementation Assays
Authors: Jin Jing , Sturgeon Timothy , Chen Chaoping , Watkins Simon C. , Weisz Ora A. , Montelaro Ronald C. 
Published: August 2007

ABSTRACT
Retroviral Gag polyproteins are necessary and sufficient for virus budding. Numerous studies of human immunodeficiency virus type 1 (HIV-1) Gag assembly and budding mechanisms have been reported, but relatively little is known about these fundamental pathways among animal lentiviruses. While there may be a general assumption that lentiviruses share common assembly mechanisms, studies of equine infectious anemia virus (EIAV) have indicated alternative cellular pathways and cofactors employed among lentiviruses for assembly and budding. In the current study, we used bimolecular fluorescence complementation to characterize and compare assembly sites and budding efficiencies of EIAV and HIV-1 Gag in both human and rodent cells. The results of these studies demonstrated that replacing the natural RNA nuclear export element (Rev-response element [RRE]) used by HIV-1 and EIAV with the hepatitis B virus posttranscriptional regulatory element (PRE) altered HIV-1, but not EIAV, Gag assembly sites and budding efficiency in human cells. Consistent with this novel observation, different assembly sites were revealed in human cells for Rev-dependent EIAV and HIV-1 Gag polyproteins. In rodent cells, Rev-dependent HIV-1 Gag assembly and budding were blocked, but changing RRE to PRE rescued HIV-1 Gag assembly and budding. In contrast, EIAV Gag polyproteins synthesized from mRNA exported via either Rev-dependent or PRE-dependent mechanisms were able to assemble and bud efficiently in rodent cells. Taken together, our results suggest that lentivirus assembly and budding are regulated by the RNA nuclear export pathway and that alternative cellular pathways can be adapted for lentiviral Gag assembly and budding.
Latent Infection of a New Alphanodavirus in an Insect Cell Line
Authors: Li Tian-Cheng , Scotti Paul D. , Miyamura Tatsuo , Takeda Naokazu 
Published: August 2007

ABSTRACT

Insect BTI-TN-5B1-4 (Tn5) cells have been used extensively with recombinant baculoviruses to express foreign genes. When a recombinant baculovirus containing the hepatitis E virus capsid protein gene was used to infect Tn5 cells, unknown virus particles in addition to the anticipated hepatitis E virus-like particles were produced in the infected cells. The unknown virus particles were 35 nm in diameter and contained RNA that was highly homologous to full-length RNA1 (3,107 bp) and RNA2 (1,383 bp) genomic RNAs of flock house virus. Surprisingly, both RNAs seen in these induced nodavirus particles could be amplified from commercially available Tn5 cells without infection with or induction by a baculovirus. The nucleotide sequences from the purified nodavirus particles and the normal Tn5 cells were identical, demonstrating that the Tn5 cells themselves were latently infected with a nodavirus. However, the generation of nodavirus particles was significantly stimulated by infection with recombinant baculoviruses. Phylogenetic analysis suggested that this new nodavirus belongs to the genus
Alphanodavirus
in the family
Nodaviridae
.
Infectivity and Neutralization of Simian Immunodeficiency Virus with FLAG Epitope Insertion in gp120 Variable Loops
Authors: Laird Melissa E. , Desrosiers Ronald C. 
Published: August 2007

ABSTRACT
A FLAG epitope tag was substituted within variable loop 1 (V1), 2 (V2), or 4 (V4) of the gp120 envelope glycoprotein of simian immunodeficiency virus strain 239 (SIV239) to evaluate the extent to which each variable loop may serve as a target for antibody-mediated neutralization. Two sites within each variable loop of SIV239 were chosen for individual epitope tag insertions. FLAG epitope substitutions were also made in the V1, V2, and V4 loops of a neutralization-sensitive derivative of SIV239, SIV316. Of the 10 FLAG-tagged recombinant viruses analyzed, three (SIV239FV1b, SIV239FV2b, and SIV239FV4a) replicated with kinetics similar to those of the parental strain, SIV239, in both CEMx174 cells and the immortalized rhesus monkey T-cell line 221. The SIV316FV1b and SIV316FV4a FLAG variants replicated with a substantial lag, and the five remaining recombinants did not replicate detectably. Both gp160 and gp120 from replication-competent FLAG variants could be immunoprecipitated from transfected 293T cells by the anti-gp120 rhesus monoclonal antibody (RhMAb) 3.11H, the anti-FLAG MAb M2, and CD4-immunoglobulin, whereas only unprocessed gp160 was detected in 293T cells transfected with replication-defective variants. Furthermore, gp120 was detectably incorporated only into virions that were infectious. SIV239FV1b was sensitive to neutralization by MAb M2, with a 50% inhibitory concentration of 1 μg/ml. Neither SIV239FV2b nor SIV239FV4a was sensitive to M2 neutralization. The ability of the M2 antibody to neutralize SIV239FV1b infectivity was associated with an increased ability of the M2 antibody to detect native, oligomeric SIV239FV1b envelope protein on the surfaces of cells relative to that for the other SIV FLAG variants. Furthermore, SIV239FV1b was globally more sensitive to antibody-mediated neutralization than was parental SIV239 when these strains were screened with a panel of anti-SIV MAbs of various specificities. These results indicate that the V1 loop can serve as an effective target for neutralization on SIV239FV1b. However, antibody-mediated neutralization of this variant, similar to that of other SIV239 variants that have been studied previously, was associated with a global increase in neutralization sensitivity. These results suggest that the variable loops on the neutralization-resistant SIV239 strain are difficult for antibodies to access effectively and that mutations that allow neutralization have global effects on the trimeric envelope glycoprotein structure and accessibility.
DNA Palindromes with a Modest Arm Length of ≳20 Base Pairs Are a Significant Target for Recombinant Adeno-Associated Virus Vector Integration in the Liver, Muscles, and Heart in Mice
Authors: Inagaki Katsuya , Lewis Susanna M. , Wu Xiaolin , Ma Congrong , Munroe David J. , Fuess Sally , Storm Theresa A. , Kay Mark A. , Nakai Hiroyuki 
Published: August 2007

ABSTRACT
Our previous study has shown that recombinant adeno-associated virus (rAAV) vector integrates preferentially in genes, near transcription start sites and CpG islands in mouse liver (H. Nakai, X. Wu, S. Fuess, T. A. Storm, D. Munroe, E. Montini, S. M. Burgess, M. Grompe, and M. A. Kay, J. Virol. 79:3606-3614, 2005). However, the previous method relied on in vivo selection of rAAV integrants and could be employed for the liver but not for other tissues. Here, we describe a novel method for high-throughput rAAV integration site analysis that does not rely on marker gene expression, selection, or cell division, and therefore it can identify rAAV integration sites in nondividing cells without cell manipulations. Using this new method, we identified and characterized a total of 997 rAAV integration sites in mouse liver, skeletal muscle, and heart, transduced with rAAV2 or rAAV8 vector. The results support our previous observations, but notably they have revealed that DNA palindromes with an arm length of ≳20 bp (total length, ≳40 bp) are a significant target for rAAV integration. Up to ∼30% of total integration events occurred in the vicinity of DNA palindromes with an arm length of ≳20 bp. Considering that DNA palindromes may constitute fragile genomic sites, our results support the notion that rAAV integrates at chromosomal sites susceptible to breakage or preexisting breakage sites. The use of rAAV to label fragile genomic sites may provide an important new tool for probing the intrinsic source of ongoing genomic instability in various tissues in animals, studying DNA palindrome metabolism in vivo, and understanding their possible contributions to carcinogenesis and aging.
Innate Recognition Network Driving Herpes Simplex Virus-Induced Corneal Immunopathology: Role of the Toll Pathway in Early Inflammatory Events in Stromal Keratitis
Authors: Sarangi Pranita P. , Kim Bumseok , Kurt-Jones Evelyn , Rouse Barry T. 
Published: August 2007

ABSTRACTOcular infection with herpes simplex virus (HSV) sets off an array of events that succeed in clearing virus from the cornea but leaves the tissue with a CD4+T-cell-orchestrated chronic inflammatory lesion that impairs vision. We demonstrate that Toll-like receptor (TLR) signaling forms a part of the recognition system that induces the syndrome that eventually culminates in immunopathology. Accordingly, in a comparison of the outcomes of infection in wild-type (WT) mice and those lacking TLR function, it was apparent that the absence of TLR2 and, to a lesser extent, TLR9 resulted in significantly diminished lesions. Similarly, mice lacking the adapter molecule MyD88 were resistant to lesion development, but such animals were also unable to control infection, with most succumbing to lethal encephalitis. The susceptibility of TLR4−/−animals was also evaluated. These animals developed lesions, which were more severe, more rapidly than did WT animals. We discuss the possible mechanisms by which early recognition of HSV constituents impacts the subsequent development of immunopathological lesions.
Vesicular Stomatitis Virus mRNA Capping Machinery Requires Specific
cis
-Acting Signals in the RNA
Authors: Wang Jennifer T. , McElvain Lauren E. , Whelan Sean P. J. 
Published: August 2007

ABSTRACT

Many viruses of eukaryotes that use mRNA cap-dependent translation strategies have evolved alternate mechanisms to generate the mRNA cap compared to their hosts. The most divergent of these mechanisms are those used by nonsegmented negative-sense (NNS) RNA viruses, which evolved a capping enzyme that transfers RNA onto GDP, rather than GMP onto the 5′ end of the RNA. Working with vesicular stomatitis virus (VSV), a prototype of the NNS RNA viruses, we show that mRNA cap formation is further distinct, requiring a specific
cis
-acting signal in the RNA. Using recombinant VSV, we determined the function of the eight conserved positions of the gene-start sequence in mRNA initiation and cap formation. Alterations to this sequence compromised mRNA initiation and separately formation of the GpppA cap structure. These studies provide genetic and biochemical evidence that the mRNA capping apparatus of VSV evolved an RNA capping machinery that functions in a sequence-specific manner.
Characterization of Human Immunodeficiency Virus Type 1 Replication in Immature and Mature Dendritic Cells Reveals Dissociablecis- andtrans-Infection
Authors: Dong Chunsheng , Janas Alicia M. , Wang Jian-Hua , Olson Wendy J. , Wu Li 
Published: August 2007

ABSTRACTDendritic cells (DCs) transmit human immunodeficiency virus type 1 (HIV-1) to CD4+T cells through thetrans- andcis-infection pathways; however, little is known about the relative efficiencies of these pathways and whether they are interdependent. Here we comparecis- andtrans-infections of HIV-1 mediated by immature DCs (iDCs) and mature DCs (mDCs), using replication-competent and single-cycle HIV-1. Monocyte-derived iDCs were differentiated into various types of mDCs by lipopolysaccharide (LPS), tumor necrosis factor alpha (TNF-α), and CD40 ligand (CD40L). iDCs and CD40L-induced mDCs were susceptible to HIV-1 infection and mediated efficient viral transmission to CD4+T cells. Although HIV-1cis-infection was partially restricted in TNF-α-induced mDCs and profoundly blocked in LPS-induced mDCs, these cells efficiently promoted HIV-1trans-infection of CD4+T cells. The postentry restriction of HIV-1 infection in LPS-induced mDCs was identified at the levels of reverse transcription and postintegration, using real-time PCR quantification of viral DNA and integration. Furthermore, nucleofection of DCs with HIV-1 proviral DNA confirmed that impaired gene expression of LPS-induced mDCs was responsible for the postentry restriction of HIV-1 infection. Our results suggest that various DC subsets in vivo may differentially contribute to HIV-1 dissemination via dissociablecis- andtrans-infections.
West Nile Virus Infection Activates the Unfolded Protein Response, Leading to CHOP Induction and Apoptosis
Authors: Medigeshi Guruprasad R. , Lancaster Alissa M. , Hirsch Alec J. , Briese Thomas , Lipkin W. Ian , DeFilippis Victor , Früh Klaus , Mason Peter W. , Nikolich-Zugich Janko , Nelson Jay A. 
Published: August 2007

ABSTRACT

West Nile virus (WNV)-mediated neuronal death is a hallmark of WNV meningitis and encephalitis. However, the mechanisms of WNV-induced neuronal damage are not well understood. We investigated WNV neuropathogenesis by using human neuroblastoma cells and primary rat hippocampal neurons. We observed that WNV activates multiple unfolded protein response (UPR) pathways, leading to transcriptional and translational induction of UPR target genes. We evaluated the role of the three major UPR pathways, namely, inositol-requiring enzyme 1-dependent splicing of X box binding protein 1 (XBP1) mRNA, activation of activating transcription factor 6 (ATF6), and protein kinase R-like endoplasmic reticulum (ER) kinase-dependent eukaryotic initiation factor 2α (eIF2α) phosphorylation, in WNV-infected cells. We show that XBP1 is nonessential or can be replaced by other UPR pathways in WNV replication. ATF6 was rapidly degraded by proteasomes, consistent with induction of ER stress by WNV. We further observed a transient phosphorylation of eIF2α and induction of the proapoptotic cyclic AMP response element-binding transcription factor homologous protein (CHOP). WNV-infected cells exhibited a number of apoptotic phenotypes, such as (i) induction of growth arrest and DNA damage-inducible gene 34, (ii) activation of caspase-3, and (iii) cleavage of poly(ADP-ribose) polymerase. The expression of WNV nonstructural proteins alone was sufficient to induce CHOP expression. Importantly, WNV grew to significantly higher viral titers in
chop
−
/
−
mouse embryonic fibroblasts (MEFs) than in wild-type MEFs, suggesting that CHOP-dependent premature cell death represents a host defense mechanism to limit viral replication that might also be responsible for the widespread neuronal loss observed in WNV-infected neuronal tissue.
A Rab-GAP TBC Domain Protein Binds Hepatitis C Virus NS5A and Mediates Viral Replication
Authors: Sklan Ella H. , Staschke Kirk , Oakes Tina M. , Elazar Menashe , Winters Mark , Aroeti Benjamin , Danieli Tsafi , Glenn Jeffrey S. 
Published: August 2007

ABSTRACT
Hepatitis C virus (HCV) is an important cause of liver disease worldwide. Current therapies are inadequate for most patients. Using a two-hybrid screen, we isolated a novel cellular binding partner interacting with the N terminus of HCV nonstructural protein NS5A. This partner contains a TBC Rab-GAP (GTPase-activating protein) homology domain found in all known Rab-activating proteins. As the first described interaction between such a Rab-GAP and a viral protein, this finding suggests a new mechanism whereby viruses may subvert host cell machinery for mediating the endocytosis, trafficking, and sorting of their own proteins. Moreover, depleting the expression of this partner severely impairs HCV RNA replication with no obvious effect on cell viability. These results suggest that pharmacologic disruption of this NS5A-interacting partner can be contemplated as a potential new antiviral strategy against a pathogen affecting nearly 3% of the world's population.
Signal Peptide and Helical Bundle Domains of Virulent Canine Distemper Virus Fusion Protein Restrict Fusogenicity
Authors: Plattet Philippe , Cherpillod Pascal , Wiener Dominique , Zipperle Ljerka , Vandevelde Marc , Wittek Riccardo , Zurbriggen Andreas 
Published: August 2007

ABSTRACTPersistence in canine distemper virus (CDV) infection is correlated with very limited cell-cell fusion and lack of cytolysis induced by the neurovirulent A75/17-CDV compared to that of the cytolytic Onderstepoort vaccine strain. We have previously shown that this difference was at least in part due to the amino acid sequence of the fusion (F) protein (P. Plattet, J. P. Rivals, B. Zuber, J. M. Brunner, A. Zurbriggen, and R. Wittek, Virology 337:312-326, 2005). Here, we investigated the molecular mechanisms of the neurovirulent CDV F protein underlying limited membrane fusion activity. By exchanging the signal peptide between both F CDV strains or replacing it with an exogenous signal peptide, we demonstrated that this domain controlled intracellular and consequently cell surface protein expression, thus indirectly modulating fusogenicity. In addition, by serially passaging a poorly fusogenic virus and selecting a syncytium-forming variant, we identified the mutation L372W as being responsible for this change of phenotype. Intriguingly, residue L372 potentially is located in the helical bundle domain of the F1subunit. We showed that this mutation drastically increased fusion activity of F proteins of both CDV strains in a signal peptide-independent manner. Due to its unique structure even among morbilliviruses, our findings with respect to the signal peptide are likely to be specifically relevant to CDV, whereas the results related to the helical bundle add new insights to our growing understanding of this class of F proteins. We conclude that different mechanisms involving multiple domains of the neurovirulent A75/17-CDV F protein act in concert to limit fusion activity, preventing lysis of infected cells, which ultimately may favor viral persistence.