bluuebunny/crossref_metadata_2025 huggingface dataset:
In Vitro Activities of Doripenem and Comparator Agents against 364 Anaerobic Clinical Isolates
Authors: Wexler Hannah M. , Engel Adrian E. , Glass Daniel , Li Calida
Published: September 2005

The in vitro activities of doripenem against 364 anaerobic isolates were measured and compared to those of ertapenem, imipenem, meropenem, ceftriaxone, and levofloxacin. All of the carbapenems were active against nearly all
Bacteroides fragilis
group isolates. Doripenem was either comparable to or slightly less active than imipenem and meropenem against most isolates but more active than the other penems against
Clostridium difficile
. Doripenem appears to have excellent activity against a broad range of anaerobes.
Intratypic Recombination among Lineages of Type 1 Vaccine-Derived Poliovirus Emerging during Chronic Infection of an Immunodeficient Patient
Authors: Yang Chen-Fu , Chen Hour-Young , Jorba Jaume , Sun Hui-Chih , Yang Su-Ju , Lee Hsiang-Chi , Huang Yhu-Chering , Lin Tzou-Yien , Chen Pei-Jer , Shimizu Hiroyuki , Nishimura Yorihiro , Utama Andi , Pallansch Mark , Miyamura Tatsuo , Kew Olen , Yang Jyh-Yuan
Published: September 2005

We determined the complete genomic sequences of nine type 1 immunodeficient vaccine-derived poliovirus (iVDPV) isolates obtained over a 337-day period from a poliomyelitis patient from Taiwan with common variable immunodeficiency. The iVDPV isolates differed from the Sabin type 1 oral poliovirus vaccine (OPV) strain at 1.84% to 3.15% of total open reading frame positions and had diverged into at least five distinct lineages. Phylogenetic analysis suggested that the chronic infection was initiated by the fifth and last OPV dose, given 567 days before onset of paralysis, and that divergence of major lineages began very early in the chronic infection. Key determinants of attenuation in Sabin 1 had reverted in the iVDPV isolates, and representative isolates of each lineage showed increased neurovirulence for PVR-Tg21 transgenic mice. None of the isolates had retained the temperature-sensitive phenotype of Sabin 1. All isolates were antigenic variants of Sabin 1, having multiple amino acid substitutions within or near neutralizing antigenic sites 1, 2, and 3a. Antigenic divergence of the iVDPV variants from Sabin 1 followed two major independent evolutionary pathways. The emergence of distinct coreplicating lineages suggests that iVDPVs can replicate for many months at separate sites in the gastrointestinal tract. Some isolates had mosaic genome structures indicative of recombination across and within lineages. iVDPV excretion apparently ceased after 30 to 35 months of chronic infection. The appearance of a chronic VDPV excretor in a tropical, developing country has important implications for the strategy to stop OPV immunization after eradication of wild polioviruses.
Endocytosis Plays a Critical Role in Proteolytic Processing of the Hendra Virus Fusion Protein
Authors: Meulendyke Kelly Ann , Wurth Mark Allen , McCann Richard O. , Dutch Rebecca Ellis
Published: September 2005

The Hendra virus fusion (F) protein is synthesized as a precursor protein, F
0
, which is proteolytically processed to the mature form, F
1
+F
2
. Unlike the case for the majority of paramyxovirus F proteins, the processing event is furin independent, does not require the addition of exogenous proteases, is not affected by reductions in intracellular Ca
2+
, and is strongly affected by conditions that raise the intracellular pH (C. T. Pager, M. A. Wurth, and R. E. Dutch, J. Virol.
78:
9154-9163, 2004). The Hendra virus F protein cytoplasmic tail contains a consensus motif for endocytosis, YXXΦ. To analyze the potential role of endocytosis in the processing and membrane fusion promotion of the Hendra virus F protein, mutation of tyrosine 525 to alanine (Hendra virus F Y525A) or phenylalanine (Hendra virus F Y525F) was performed. The rate of endocytosis of Hendra virus F Y525A was significantly reduced compared to that of the wild-type (wt) F protein, confirming the functional importance of the endocytosis motif. An intermediate level of endocytosis was observed for Hendra virus F Y525F. Surprisingly, dramatic reductions in the rate of proteolytic processing were observed for Hendra virus F Y525A, although initial transport to the cell surface was not affected. The levels of surface expression for both Hendra virus F Y525A and Hendra virus F Y525F were higher than that of the wt protein, and these mutants displayed enhanced syncytium formation. These results suggest that endocytosis is critically important for Hendra virus F protein cleavage, representing a new paradigm for proteolytic processing of paramyxovirus F proteins.
A Sabin 3-Derived Poliovirus Recombinant Contained a Sequence Homologous with Indigenous Human Enterovirus Species C in the Viral Polymerase Coding Region
Authors: Arita Minetaro , Zhu Shuang-Li , Yoshida Hiromu , Yoneyama Tetsuo , Miyamura Tatsuo , Shimizu Hiroyuki
Published: September 2005

Outbreaks of poliomyelitis caused by circulating vaccine-derived polioviruses (cVDPVs) have been reported in areas where indigenous wild polioviruses (PVs) were eliminated by vaccination. Most of these cVDPVs contained unidentified sequences in the nonstructural protein coding region which were considered to be derived from human enterovirus species C (HEV-C) by recombination. In this study, we report isolation of a Sabin 3-derived PV recombinant (Cambodia-02) from an acute flaccid paralysis (AFP) case in Cambodia in 2002. We attempted to identify the putative recombination counterpart of Cambodia-02 by sequence analysis of nonpolio enterovirus isolates from AFP cases in Cambodia from 1999 to 2003. Based on the previously estimated evolution rates of PVs, the recombination event resulting in Cambodia-02 was estimated to have occurred within 6 months after the administration of oral PV vaccine (99.3% nucleotide identity in VP1 region). The 2BC and the 3D
pol
coding regions of Cambodia-02 were grouped into the genetic cluster of indigenous coxsackie A virus type 17 (CAV17) (the highest [87.1%] nucleotide identity) and the cluster of indigenous CAV13-CAV18 (the highest [94.9%] nucleotide identity) by the phylogenic analysis of the HEV-C isolates in 2002, respectively. CAV13-CAV18 and CAV17 were the dominant HEV-C serotypes in 2002 but not in 2001 and in 2003. We found a putative recombination between CAV13-CAV18 and CAV17 in the 3CD
pro
coding region of a CAV17 isolate. These results suggested that a part of the 3D
pol
coding region of PV3(Cambodia-02) was derived from a HEV-C strain genetically related to indigenous CAV13-CAV18 strains in 2002 in Cambodia.
Varicella-Zoster Virus Activates Inflammatory Cytokines in Human Monocytes and Macrophages via Toll-Like Receptor 2
Authors: Wang Jennifer P. , Kurt-Jones Evelyn A. , Shin Ok S. , Manchak Michael D. , Levin Myron J. , Finberg Robert W.
Published: September 2005

The pattern recognition receptor Toll-like receptor 2 (TLR2) has been implicated in the response to several human viruses, including herpes simplex viruses (types 1 and 2) and cytomegalovirus. We demonstrated that varicella-zoster virus (VZV) activates inflammatory cytokine responses via TLR2. VZV specifically induced interleukin-6 (IL-6) in human monocytes via TLR2-dependent activation of NF-κB, and small interfering RNA designed to suppress TLR2 mRNA reduced the IL-6 response to VZV in human monocyte-derived macrophages. Unlike other herpesviruses, the cytokine response to VZV was species specific. VZV did not induce cytokines in murine embryonic fibroblasts or in a mouse cell line, although VZV did activate NF-κB in a human cell line expressing a murine TLR2 construct. Together, these results suggest that TLR2 may play a role in the inflammatory response to VZV infection.
Distinct Functional Sites for Human Immunodeficiency Virus Type 1 and Stromal Cell-Derived Factor 1α on CXCR4 Transmembrane Helical Domains
Authors: Tian Shaomin , Choi Won-Tak , Liu Dongxiang , Pesavento James , Wang Youli , An Jing , Sodroski Joseph G. , Huang Ziwei
Published: September 2005

The entry of human immunodeficiency virus type 1 (HIV-1) into the cell is initiated by the interaction of the viral surface envelope protein with two cell surface components of the target cell, CD4 and a chemokine coreceptor, usually CXCR4 or CCR5. The natural ligand of CXCR4 is stromal cell-derived factor 1α (SDF-1α). Whereas the overlap between HIV-1 and SDF-1α functional sites on the extracellular domains of CXCR4 has been well documented, it has yet to be determined whether there are sites in the transmembrane (TM) helices of CXCR4 important for HIV-1 and/or SDF-1α functions, and if such sites do exist, whether they are overlapping or distinctive for the separate functions of CXCR4. For this study, by employing alanine-scanning mutagenesis,
125
I-SDF-1α competition binding, Ca
2+
mobilization, and cell-cell fusion assays, we found that the mutation of many CXCR4 TM residues, including Tyr
45
, His
79
, Asp
97
, Pro
163
, Trp
252
, Tyr
255
, Asp
262
, Glu
288
, His
294
, and Asn
298
, could selectively decrease HIV-1-mediated cell fusion but not the binding activity of SDF-1α. Phe
87
and Phe
292
, which were involved in SDF-1α binding, did not play a significant role in the coreceptor activity of CXCR4, further demonstrating the disconnection between physiological and pathological activities of CXCR4 TM domains. Our data also show that four mutations of the second extracellular loop, D182A, D187A, F189A, and P191A, could reduce HIV-1 entry without impairing either ligand binding or signaling. Taken together, our first detailed characterization of the different functional roles of CXCR4 TM domains may suggest a mechanistic basis for the discovery of new selective anti-HIV agents.
Use of a Combined Ex Vivo/In Vivo Population Approach for Screening of Human Genes Involved in the Human Immunodeficiency Virus Type 1 Life Cycle for Variants Influencing Disease Progression
Authors: Bleiber Gabriela , May Margaret , Martinez Raquel , Meylan Pascal , Ott Jürg , Beckmann Jacques S. , Telenti Amalio
Published: September 2005

Humans differ substantially with respect to susceptibility to human immunodeficiency virus type 1 (HIV-1). We evaluated variants of nine host genes participating in the viral life cycle for their role in modulating HIV-1 infection. Alleles were assessed ex vivo for their impact on viral replication in purified CD4 T cells from healthy blood donors (
n
= 128). Thereafter, candidate alleles were assessed in vivo in a cohort of HIV-1-infected individuals (
n
= 851) not receiving potent antiretroviral therapy. As a benchmark test, we tested 12 previously reported host genetic variants influencing HIV-1 infection as well as single nucleotide polymorphisms in the nine candidate genes. This led to the proposition of three alleles of
PML
,
TSG101
, and
PPIA
as potentially associated with differences in progression of HIV-1 disease. In a model considering the combined effects of new and previously reported gene variants, we estimated that their effect might be responsible for lengthening or shortening by up to 2.8 years the period from 500 CD4 T cells/μl to <200 CD4 T cells/μl.
The CD8+T-Cell Response to an Epstein-Barr Virus-Related Gammaherpesvirus Infecting Rhesus Macaques Provides Evidence for Immune Evasion by the EBNA-1 Homologue
Authors: Fogg Mark H. , Kaur Amitinder , Cho Young-Gyu , Wang Fred
Published: September 2005

Epstein-Barr virus (EBV) infection persists for life in humans, similar to other gammaherpesviruses in the same lymphocryptovirus (LCV) genus that naturally infect Old World nonhuman primates. The specific immune elements required for control of EBV infection and potential immune evasion strategies essential for persistent EBV infection are not well defined. We evaluated the cellular immune response to latent infection proteins in rhesus macaques with naturally and experimentally acquired rhesus LCV (rhLCV) infection. RhLCV EBNA-1 (rhEBNA-1) was the most frequently targeted latent infection protein and induced the most robust responses by peripheral blood mononuclear cells tested ex vivo using the gamma interferon ELISPOT assay. In contrast, although in vitro stimulation and expansion of rhLCV-specific T lymphocytes demonstrated cytotoxic T-lymphocyte (CTL) activity against autologous rhLCV-infected B cells, rhEBNA-1-specific CTL activity could not be detected. rhEBNA-1 CTL epitopes were identified and demonstrated that rhEBNA-1-specific CTL were stimulated and expanded in vitro but did not lyse targets expressing rhEBNA-1. Similarly, rhEBNA-1-specific CTL clones were able to lyse targets pulsed with rhEBNA-1 peptides or expressing rhEBNA-1 deleted for the glycine-alanine repeat (GAR) but not full-length rhEBNA-1 or rhLCV-infected B cells. These studies show that the rhLCV-specific immune response to latent infection proteins is similar to the EBV response in humans, and a potential immune evasion mechanism for EBNA-1 has been conserved in rhLCV. Thus, the rhLCV animal model can be used to analyze the immune responses important for control of persistent LCV infection and the role of the EBNA-1 GAR for immune evasion in vivo.
Heparan Sulfate Proteoglycans Mediate Attachment and Entry of Human T-Cell Leukemia Virus Type 1 Virions into CD4
+
T Cells
Authors: Jones Kathryn S. , Petrow-Sadowski Cari , Bertolette Daniel C. , Huang Ying , Ruscetti Francis W.
Published: September 2005

Heparan sulfate proteoglycans (HSPGs) are used by a number of viruses to facilitate entry into host cells. For the retrovirus human T-cell leukemia virus type 1 (HTLV-1), it has recently been reported that HSPGs are critical for efficient binding of soluble HTLV-1 SU and the entry of HTLV pseudotyped viruses into non-T cells. However, the primary in vivo targets of HTLV-1, CD4
+
T cells, have been reported to express low or undetectable levels of HSPGs. For this study, we reexamined the expression of HSPGs in CD4
+
T cells and examined their role in HTLV-1 attachment and entry. We observed that while quiescent primary CD4
+
T cells do not express detectable levels of HSPGs, HSPGs are expressed on primary CD4
+
T cells following immune activation. Enzymatic modification of HSPGs on the surfaces of either established CD4
+
T-cell lines or primary CD4
+
T cells dramatically reduced the binding of both soluble HTLV-1 SU and HTLV-1 virions. HSPGs also affected the efficiency of HTLV-1 entry, since blocking the interaction with HSPGs markedly reduced both the internalization of HTLV-1 virions and the titer of HTLV-1 pseudotyped viral infection in CD4
+
T cells. Thus, HSPGs play a critical role in the binding and entry of HTLV-1 into CD4
+
T cells.
Activation of the Jun N-Terminal Kinase Pathway by Friend Spleen Focus-Forming Virus and Its Role in the Growth and Survival of Friend Virus-Induced Erythroleukemia Cells
Authors: Nishigaki Kazuo , Hanson Charlotte , Thompson Delores , Yugawa Takashi , Ruscetti Sandra
Published: September 2005

Members of the mitogen-activated protein kinase (MAPK) family, including Jun amino-terminal kinase (JNK) and extracellular signal-related kinase (ERK), play an important role in the proliferation of erythroid cells in response to erythropoietin (Epo). Erythroid cells infected with the Friend spleen focus-forming virus (SFFV) proliferate in the absence of Epo and show constitutive activation of Epo signal transduction pathways. We previously demonstrated that the ERK pathway was constitutively activated in Friend SFFV-infected erythroid cells, and in this study JNK is also shown to be constitutively activated. Pharmacological inhibitors of both the ERK and JNK pathways stopped the proliferation of primary erythroleukemic cells from Friend SFFV-infected mice, with little induction of apoptosis, and furthermore blocked their ability to form Epo-independent colonies. However, only the JNK inhibitor blocked the proliferation of erythroleukemia cell lines derived from these mice. The JNK inhibitor caused significant apoptosis in these cell lines as well as an increase in the fraction of cells in G
2
/M and undergoing endoreduplication. In contrast, the growth of erythroleukemia cell lines derived from Friend murine leukemia virus (MuLV)-infected mice was inhibited by both the MEK and JNK inhibitors. JNK is important for AP1 activity, and we found that JNK inhibitor treatment reduced AP1 DNA-binding activity in primary erythroleukemic splenocytes from Friend SFFV-infected mice and in erythroleukemia cell lines from Friend MuLV-infected mice but did not alter AP1 DNA binding in erythroleukemia cell lines from Friend SFFV-infected mice. These data suggest that JNK plays an important role in cell proliferation and/or the survival of erythroleukemia cells.
Low TRBP Levels Support an Innate Human Immunodeficiency Virus Type 1 Resistance in Astrocytes by Enhancing the PKR Antiviral Response
Authors: Ong Chi L. , Thorpe Janine C. , Gorry Paul R. , Bannwarth Sylvie , Jaworowski Anthony , Howard Jane L. , Chung Sean , Campbell Shahan , Christensen Helen S. , Clerzius Guerline , Mouland Andrew J. , Gatignol Anne , Purcell Damian F. J.
Published: September 2005

Acute human immunodeficiency virus type 1 (HIV-1) replication in astrocytes produces minimal new virus particles due, in part, to inefficient translation of viral structural proteins despite high levels of cytoplasmic viral mRNA. We found that a highly reactive double-stranded (ds) RNA-binding protein kinase (PKR) response in astrocytes underlies this inefficient translation of HIV-1 mRNA. The dsRNA elements made during acute replication of HIV-1 in astrocytes triggers PKR activation and the specific inhibition of HIV-1 protein translation. The heightened PKR response results from relatively low levels of the cellular antagonist of PKR, the TAR RNA binding protein (TRBP). Efficient HIV-1 production was restored in astrocytes by inhibiting the innate PKR response to HIV-1 dsRNA with dominant negative PKR mutants, or PKR knockdown by siRNA gene silencing. Increasing the expression of TRBP in astrocytes restored acute virus production to levels comparable to those observed in permissive cells. Therefore, the robust innate PKR antiviral response in astrocytes results from relatively low levels of TRBP expression and contributes to their restricted infection. Our findings highlight TRBP as a novel cellular target for therapeutic interventions to block productive HIV-1 replication in cells that are fully permissive for HIV-1 infection.
Site-Directed Mutagenesis of Large DNA Palindromes: Construction and In Vitro Characterization of Herpes Simplex Virus Type 1 Mutants Containing Point Mutations That Eliminate the oriL or oriS Initiation Function
Authors: Balliet John W. , Min Jonathan C. , Cabatingan Mark S. , Schaffer Priscilla A.
Published: September 2005

Technical challenges associated with mutagenesis of the large oriL palindrome have hindered comparisons of the functional roles of the herpes simplex virus type 1 (HSV-1) origins of DNA replication, oriL and oriS, in viral replication and pathogenesis. To address this problem, we have developed a novel PCR-based strategy to introduce site-specific mutations into oriL and other large palindromes. Using this strategy, we generated three plasmids containing mutant forms of oriL, i.e., pDoriL-IL, pDoriL-IR, and pDoriL-ILR, containing point mutations in the left, right, and both copies, respectively, of theoriginbindingprotein (OBP) binding site (site I) which eliminate OBP binding. In in vitro DNA replication assays, plasmids with mutations in only one arm of the palindrome supported origin-dependent DNA replication, whereas plasmids with symmetrical mutations in both arms of the palindrome were replication incompetent. An analysis of the cloned mutant plasmids used in replication assays revealed that a fraction of each plasmid mutated in only one arm of the palindrome had lost the site I mutation. In contrast, plasmids containing symmetrical mutations in both copies of site I retained both mutations. These observations demonstrate that the single site I mutations in pDoriL-ILand pDoriL-IRare unstable upon propagation in bacteria and suggest that functional forms of both the left and right copies of site I are required to initiate DNA replication at oriL. To examine the role of oriL and oriS site I in virus replication, we introduced the two site I mutations in pDoriL-ILRinto HSV-1 DNA to yield the mutant virus DoriL-ILRand the same point mutations into the single site I sequence present in both copies of oriS to yield the mutant virus DoriS-I. In Vero cells andprimaryrat embryonic corticalneurons (PRN) infected with either mutant virus, viral DNA synthesis and viral replication were efficient, confirming that the two origins can substitute functionally for one another in vitro. Measurement of the levels of oriL and oriS flanking gene transcripts revealed a modest alteration in the kinetics of ICP8 transcript accumulation in DoriL-ILR-infected PRN, but not in Vero cells, implicating a cell-type-specific role for oriL in regulating ICP8 transcription.
Low-Dose Adenovirus Vaccine Encoding Chimeric Hepatitis B Virus Surface Antigen-Human Papillomavirus Type 16 E7 Proteins Induces Enhanced E7-Specific Antibody and Cytotoxic T-Cell Responses
Authors: Báez-Astúa Andrés , Herráez-Hernández Elsa , Garbi Natalio , Pasolli Hilda A. , Juárez Victoria , zur Hausen Harald , Cid-Arregui Angel
Published: September 2005

Induction of effective immune responses may help prevent cancer progression. Tumor-specific antigens, such as those of human papillomaviruses involved in cervical cancer, are targets with limited intrinsic immunogenicity. Here we show that immunization with low doses (10
6
infectious units/dose) of a recombinant human adenovirus type 5 encoding a fusion of the E7 oncoprotein of human papillomavirus type 16 to the carboxyl terminus of the surface antigen of hepatitis B virus (HBsAg) induces remarkable E7-specific humoral and cellular immune responses. The HBsAg/E7 fusion protein assembled efficiently into virus-like particles, which stimulated antibody responses against both carrier and foreign antigens, and evoked antigen-specific kill of an indicator cell population in vivo. Antibody and T-cell responses were significantly higher than those induced by a control adenovirus vector expressing wild-type E7. Such responses were not affected by preexisting immunity against either HBsAg or adenovirus. These data demonstrate that the presence of E7 on HBsAg particles does not interfere with particle secretion, as it occurs with bigger proteins fused to the C terminus of HBsAg, and results in enhancement of CD8
+
-mediated T-cell responses to E7. Thus, fusion to HBsAg is a convenient strategy for developing cervical cancer therapeutic vaccines, since it enhances the immunogenicity of E7 while turning it into an innocuous secreted fusion protein.
Persistent Gene Expression in Mouse Nasal Epithelia following Feline Immunodeficiency Virus-Based Vector Gene Transfer
Authors: Sinn Patrick L. , Burnight Erin R. , Hickey Melissa A. , Blissard Gary W. , McCray Paul B.
Published: September 2005

Gene transfer development for treatment or prevention of cystic fibrosis lung disease has been limited by the inability of vectors to efficiently and persistently transduce airway epithelia. Influenza A is an enveloped virus with natural lung tropism; however, pseudotyping feline immunodeficiency virus (FIV)-based lentiviral vector with the hemagglutinin envelope protein proved unsuccessful. Conversely, pseudotyping FIV with the envelope protein from influenza D (Thogoto virus GP75) resulted in titers of 10
6
transducing units (TU)/ml and conferred apical entry into well-differentiated human airway epithelial cells. Baculovirus GP64 envelope glycoproteins share sequence identity with influenza D GP75 envelope glycoproteins. Pseudotyping FIV with GP64 from three species of baculovirus resulted in titers of 10
7
to 10
9
TU/ml. Of note, GP64 from
Autographa californica
multicapsid nucleopolyhedrovirus resulted in high-titer FIV preparations (∼10
9
TU/ml) and conferred apical entry into polarized primary cultures of human airway epithelia. Using a luciferase reporter gene and bioluminescence imaging, we observed persistent gene expression from in vivo gene transfer in the mouse nose with
A. californica
GP64-pseudotyped FIV (AcGP64-FIV). Longitudinal bioluminescence analysis documented persistent expression in nasal epithelia for ∼1 year without significant decline. According to histological analysis using a LacZ reporter gene, olfactory and respiratory epithelial cells were transduced. In addition, methylcellulose-formulated AcGP64-FIV transduced mouse nasal epithelia with much greater efficiency than similarly formulated vesicular stomatitis virus glycoprotein-pseudotyped FIV. These data suggest that AcGP64-FIV efficiently transduces and persistently expresses a transgene in nasal epithelia in the absence of agents that disrupt the cellular tight junction integrity.
Identification and Functional Evaluation of Cellular and Viral Factors Involved in the Alteration of Nuclear Architecture during Herpes Simplex Virus 1 Infection
Authors: Simpson-Holley Martha , Colgrove Robert C. , Nalepa Grzegorz , Harper J. Wade , Knipe David M.
Published: September 2005

Herpes simplex virus 1 (HSV-1) replicates in the nucleus of host cells and radically alters nuclear architecture as part of its replication process. Replication compartments (RCs) form, and host chromatin is marginalized. Chromatin is later dispersed, and RCs spread past it to reach the nuclear edge. Using a lamin A-green fluorescent protein fusion, we provide direct evidence that the nuclear lamina is disrupted during HSV-1 infection and that the UL31 and UL34 proteins are required for this. We show nuclear expansion from 8 h to 24 h postinfection and place chromatin rearrangement and disruption of the lamina in the context of this global change in nuclear architecture. We show HSV-1-induced disruption of the localization of Cdc14B, a cellular protein and component of a putative nucleoskeleton. We also show that UL31 and UL34 are required for nuclear expansion. Studies with inhibitors of globular actin (G-actin) indicate that G-actin plays an essential role in nuclear expansion and chromatin dispersal but not in lamina alterations induced by HSV-1 infection. From analyses of HSV infections under various conditions, we conclude that nuclear expansion and chromatin dispersal are dispensable for optimal replication, while lamina rearrangement is associated with efficient replication.
Host Cell Nuclear Proteins Are Recruited to Cytoplasmic Vaccinia Virus Replication Complexes
Authors: Oh Jaewook , Broyles Steven S.
Published: September 2005

The initiation and termination of vaccinia virus postreplicative transcription have been reported to require cellular proteins, some of which are believed to be nuclear proteins. Vaccinia virus replicates in the cytoplasmic compartment of the cell, raising questions as to whether vaccinia virus has access to nuclear proteins. This was addressed here by following the fate of several nuclear proteins after infection of cells with vaccinia virus. The nuclear transcription factors YY1, SP1, and TATA binding protein were found to colocalize with virus replication complexes in the cytoplasm of infected cells. In addition, the nuclear proteins RNA polymerase II, TAFIIp32, and histone deacetylase 8, but not the structural protein lamin B, also were found in the cytoplasm of the cell. The association of YY1 with replication complexes was dependent on DNA replication and required only the DNA binding domain of the protein, indicating that DNA binding alone may be responsible for the association of nuclear transcription factors with viral replication complexes in the cytoplasm. The cytoplasmic localization of YY1 was resistant to the nuclear export inhibitor leptomycin B. Evidence is presented indicating that nuclear import and export pathways were not adversely affected by vaccinia virus infection. These observations indicate that vaccinia virus replication complexes have ready access to nuclear proteins by allowing leakage from the nucleus.
Mutational Analyses of the Nonconserved Sequences in the Bunyamwera Orthobunyavirus S Segment Untranslated Regions
Authors: Lowen Anice C. , Elliott Richard M.
Published: September 2005

Bunyamwera virus
(BUNV) is the prototype of the genus
Orthobunyavirus
and the family
Bunyaviridae
. BUNV has a tripartite genome of negative-sense RNA composed of small (S), medium (M), and large (L) segments. Partially complementary untranslated regions (UTRs) flank the coding region of each segment. The terminal 11 nucleotides of these UTRs are conserved between the three segments and throughout the genus, while the internal regions are unique to each segment and largely nonconserved between different viruses. To investigate the functions of the UTR sequences, we constructed a series of BUNV S segment cDNA clones with deletions in the 3′ and/or 5′ UTR and then attempted to rescue these segments into recombinant viruses. We found that the genomic 5′ UTR was much more sensitive to mutation than the 3′ UTR and, in general, sequences proximal to the termini were more important than those flanking the coding region. Northern blot analyses of infected-cell RNA showed that the internal, nonconserved sequences of the S segment 3′ UTR play a role in the regulation of transcription and replication and the balance between these two processes. In contrast, deletions in the 5′ UTR caused attenuation of the recombinant virus but did not specifically affect levels of S segment RNAs or the encoded nucleocapsid protein. Thus, the internal regions of both UTRs are functional: most of the 5′ UTR is essential to viral growth, and, while nonessential, the internal 3′ UTR is important to the regulation of viral RNA synthesis.
New Genes from Old: Redeployment of dUTPase by Herpesviruses
Authors: Davison Andrew J. , Stow Nigel D.
Published: September 2005

Published work (D. J. McGeoch, Nucleic Acids Res.
18:
4105-4110, 1990; J. E. McGeehan, N. W. Depledge, and D. J. McGeoch, Curr. Protein Peptide Sci.
2:
325-333, 2001) has indicated that evolution of dUTPase in the class of herpesviruses that infect mammals and birds involved capture of a host gene followed by a duplication event that resulted in a coding region comprising two fused dUTPase domains. Some of the conserved residues required for enzyme activity were then lost, resulting in a dUTPase containing a single active site with different elements contributed by each half of the protein. Further conserved residues were lost in one subfamily (the
Betaherpesvirinae
), yielding a protein that is related to herpesvirus dUTPases but has a different and as yet unrecognized function. Evidence from sequence similarities and structural predictions now indicates that several additional genes were derived from the herpesvirus dUTPase gene, probably by duplication. These are UL31, UL82, UL83, and UL84 in human cytomegalovirus (and counterparts in other members of the
Betaherpesvirinae
) and ORF10 and ORF11 in human herpesvirus 8 (and counterparts in other members of the
Gammaherpesvirinae
). The findings clarify the evolutionary history of these genes and provide novel insights for structural and functional studies.
Hepatitis B Virus Large and Middle Glycoproteins Are Degraded by a Proteasome Pathway in Glucosidase-Inhibited Cells but Not in Cells with Functional Glucosidase Enzyme
Authors: Simsek Ender , Mehta Anand , Zhou Tianlun , Dwek Raymond A. , Block Timothy
Published: September 2005

The secretion of hepatitis B virus (HBV) large (LHBs) and middle (MHBs) envelope polypeptides from tissue cultures requires proper protein folding and is prevented by inhibitors of the endoplasmic reticulum (ER) glucosidase. Using competitive inhibitors of the ER glucosidase, here it is shown that the amounts of glycosylated and unglycosylated forms of LHBs and MHBs proteins are all greatly reduced in tissue cultures producing HBV envelope glycoproteins. In contrast, the HBV small (SHBs) protein was not affected. The reduction in secretion of LHBs and MHBs proteins appears to be mediated by proteasomal degradation pathways, since it is prevented by either lactacystin or epoxomicin, two inhibitors of proteasomal degradation. Although there is no detectable proteasomal degradation of LHBs and MHBs in cells with functional glucosidase, the implications of the nearly quantitative sensitivity of glycosylated and unglycosylated forms of LHBs and MHBs proteins, with selective sparing of SHBs protein, in cells in which glucosidase is inhibited is surprising, and its implications are discussed.
T-Cell Tropism and the Role of ORF66 Protein in Pathogenesis of Varicella-Zoster Virus Infection
Authors: Schaap Anne , Fortin Jean-Francois , Sommer Marvin , Zerboni Leigh , Stamatis Shaye , Ku Chia-Chi , Nolan Garry P. , Arvin Ann M.
Published: September 2005

The pathogenesis of varicella-zoster virus (VZV) involves a cell-associated viremia during which infectious virus is carried from sites of respiratory mucosal inoculation to the skin. We now demonstrate that VZV infection of T cells is associated with robust virion production and modulation of the apoptosis and interferon pathways within these cells. The VZV serine/threonine protein kinase encoded by ORF66 is essential for the efficient replication of VZV in T cells. Preventing ORF66 protein expression by stop codon insertion (pOka66S) impaired the growth of the parent Oka (pOka) strain in T cells in SCID-hu T-cell xenografts in vivo and reduced formation of VZV virions. The lack of ORF66 protein also increased the susceptibility of infected T cells to apoptosis and reduced the capacity of the virus to interfere with induction of the interferon (IFN) signaling pathway following exposure to IFN-γ. However, preventing ORF66 protein expression only slightly reduced growth in melanoma cells in culture and did not diminish virion formation in these cells. The pOka66S virus showed only a slight defect in growth in SCID-hu skin implants compared with intact pOka. These observations suggest that the ORF66 kinase plays a unique role during infection of T cells and supports VZV T-cell tropism by contributing to immune evasion and enhancing survival of infected T cells.
De Novo Generation of Escape Variant-Specific CD8+T-Cell Responses following Cytotoxic T-Lymphocyte Escape in Chronic Human Immunodeficiency Virus Type 1 Infection
Authors: Allen Todd M. , Yu Xu G. , Kalife Elizabeth T. , Reyor Laura L. , Lichterfeld Mathias , John Mina , Cheng Michael , Allgaier Rachel L. , Mui Stanley , Frahm Nicole , Alter Galit , Brown Nancy V. , Johnston Mary N. , Rosenberg Eric S. , Mallal Simon A. , Brander Christian , Walker Bruce D. , Altfeld Marcus
Published: September 2005

Human immunodeficiency virus type 1 (HIV-1) evades CD8+T-cell responses through mutations within targeted epitopes, but little is known regarding its ability to generate de novo CD8+T-cell responses to such mutants. Here we examined gamma interferon-positive, HIV-1-specific CD8+T-cell responses and autologous viral sequences in an HIV-1-infected individual for more than 6 years following acute infection. Fourteen optimal HIV-1 T-cell epitopes were targeted by CD8+T cells, four of which underwent mutation associated with dramatic loss of the original CD8+response. However, following the G357S escape in the HLA-A11-restricted Gag349-359epitope and the decline of wild-type-specific CD8+T-cell responses, a novel CD8+T-cell response equal in magnitude to the original response was generated against the variant epitope. CD8+T cells targeting the variant epitope did not exhibit cross-reactivity against the wild-type epitope but rather utilized a distinct T-cell receptor Vβ repertoire. Additional studies of chronically HIV-1-infected individuals expressing HLA-A11 demonstrated that the majority of the subjects targeted the G357S escape variant of the Gag349-359epitope, while the wild-type consensus sequence was significantly less frequently recognized. These data demonstrate that de novo responses against escape variants of CD8+T-cell epitopes can be generated in chronic HIV-1 infection and provide the rationale for developing vaccines to induce CD8+T-cell responses directed against both the wild-type and variant forms of CD8 epitopes to prevent the emergence of cytotoxic T-lymphocyte escape variants.
Isolation of Cell Lines That Show Novel, Murine Leukemia Virus-Specific Blocks to Early Steps of Retroviral Replication
Authors: Bruce James W. , Bradley Kenneth A. , Ahlquist Paul , Young John A. T.
Published: September 2005

In order to identify cellular proteins required for early stages of retroviral replication, a high volume screening with mammalian somatic cells was performed. Ten pools of chemically mutagenized Chinese hamster ovary (CHO-K1) cells were challenged with a murine leukemia virus (MLV) vector pseudotyped with the vesicular stomatitis virus glycoprotein (VSV-G), and cells that failed to be transduced were enriched by cell sorting. Each pool yielded a clonally derived cell line with a 5-fold or greater resistance to virus infection, and five cell lines exhibited a >50-fold resistance. These five cell lines were efficiently infected by a human immunodeficiency virus vector pseudotyped with VSV-G. When engineered to express the TVA receptor for subgroup A avian sarcoma and leukosis virus (ASLV-A), the five cell lines were resistant to infection with a MLV vector pseudotyped with the ASLV-A envelope protein but were fully susceptible to infection with an ASLV-A vector. Thus, the defect in these cells resides after virus-cell membrane fusion and, unlike those in other mutant cell lines that have been described, is specific for the MLV core. To identify the specific stages of MLV infection that are impaired in the resistant cell lines, real-time quantitative PCR analyses were employed and two phenotypic groups were identified. Viral infection of three cell lines was restricted before reverse transcription; in the other two cell lines, it was blocked after reverse transcription, nuclear localization, and two-long terminal repeat circle formation but before integration. These data provide genetic evidence that at least two distinct intracellular gene products are required specifically for MLV infection. These cell lines are important tools for the biochemical and genetic analysis of early stages in retrovirus infection.
Full-Breadth Analysis of CD8+T-Cell Responses in Acute Hepatitis C Virus Infection and Early Therapy
Authors: Lauer Georg M. , Lucas Michaela , Timm Joerg , Ouchi Kei , Kim Arthur Y. , Day Cheryl L. , zur Wiesch Julian Schulze , Paranhos-Baccala Glaucia , Sheridan Isabelle , Casson Deborah R. , Reiser Markus , Gandhi Rajesh T. , Li Bin , Allen Todd M. , Chung Raymond T. , Klenerman Paul , Walker Bruce D.
Published: September 2005

Multispecific CD8+T-cell responses are thought to be important for the control of acute hepatitis C virus (HCV) infection, but to date little information is actually available on the breadth of responses at early time points. Additionally, the influence of early therapy on these responses and their relationships to outcome are controversial. To investigate this issue, we performed comprehensive analysis of the breadth and frequencies of virus-specific CD8+T-cell responses on the single epitope level in eight acutely infected individuals who were all started on early therapy. During the acute phase, responses against up to five peptides were identified. During therapy, CD8+T-cell responses decreased rather than increased as virus was controlled, and no new specificities emerged. A sustained virological response following completion of treatment was independent of CD8+T-cell responses, as well as CD4+T-cell responses. Rapid recrudescence also occurred despite broad CD8+T-cell responses. Importantly, in vivo suppression of CD3+T cells using OKT3 in one subject did not result in recurrence of viremia. These data suggest that broad CD8+T-cell responses alone may be insufficient to contain HCV replication, and also that early therapy is effective independent of such responses.
Analysis of a Foot-and-Mouth Disease Virus Type A
24
Isolate Containing an SGD Receptor Recognition Site In Vitro and Its Pathogenesis in Cattle
Authors: Rieder Elizabeth , Henry Tina , Duque Hernando , Baxt Barry
Published: September 2005

Foot-and-mouth disease virus (FMDV) initiates infection by binding to integrin receptors via an Arg-Gly-Asp (RGD) sequence found in the G-H loop of the structural protein VP1. Following serial passages of a type A
24
Cruzeiro virus (A
24
Cru) in bovine, via tongue inoculation, a virus was generated which contained an SGD sequence in the cell receptor-binding site and expressed a turbid plaque phenotype in BHK-21 cells. Propagation of this virus in these cells resulted in the rapid selection of viruses that grew to higher titers, produced clear plaques, and now contained an RGD sequence in place of the original SGD. To study the role of the SGD sequence in FMDV receptor recognition and bovine virulence, we assembled an infectious cDNA clone of an RGD-containing A
24
Cru and derived mutant clones containing either SGD with a single nucleotide substitution in the R
144
codon or double substitutions at this position to prevent mutation of the S to an R. The SGD viruses grew poorly in BHK-21 cells and stably maintained the sequence during propagation in BHK-21 cells expressing the bovine α
V
β
6
integrin (BHK3-α
V
β
6
), as well as in experimentally infected and contact steers. While all the SGD-containing viruses used only the bovine α
V
β
6
integrin as a cellular receptor with relatively high efficiency, the revertant RGD viruses utilized either the α
V
β
1
or α
V
β
3
bovine integrins with higher efficiency than α
V
β
6
and grew well in BHK-21 cells. Replacing the R at the −1 SGD position with either K or E showed that this residue did not contribute to integrin utilization in vitro. These results illustrate the rapid evolution of FMDV with alteration in receptor specificity and suggest that viruses with sequences other than RGD, but closely related to it, can still infect via integrin receptors and induce and transmit the disease to susceptible animals.
Essential Elements of the Capsid Protein for Self-Assembly into Empty Virus-Like Particles of Hepatitis E Virus
Authors: Li Tian-Cheng , Takeda Naokazu , Miyamura Tatsuo , Matsuura Yoshiharu , Wang Joseph C. Y. , Engvall Helena , Hammar Lena , Xing Li , Cheng R. Holland
Published: September 2005

Hepatitis E virus (HEV) is a noncultivable virus that causes acute liver failure in humans. The virus's major capsid protein is encoded by an open reading frame 2 (ORF2) gene. When the recombinant protein consisting of amino acid (aa) residues 112 to 660 of ORF2 is expressed with a recombinant baculovirus, the protein self-assembles into virus-like particles (VLPs) (T.-C. Li, Y. Yamakawa, K. Suzuki, M. Tatsumi, M. A. Razak, T. Uchida, N. Takeda, and T. Miyamura, J. Virol. 71:7207-7213, 1997). VLPs can be found in the culture medium of infected Tn5 cells but not in that of Sf9 cells, and the major VLPs have lost the C-terminal 52 aa. To investigate the protein requirement for HEV VLP formation, we prepared 14 baculovirus recombinants to express the capsid proteins truncated at the N terminus, the C terminus, or both. The capsid protein consisting of aa residues 112 to 608 formed VLPs in Sf9 cells, suggesting that particle formation is dependent on the modification process of the ORF2 protein. In the present study, electron cryomicroscopy and image processing of VLPs produced in Sf9 and Tn5 cells indicated that they possess the same configurations and structures. Empty VLPs were found in both Tn5 and Sf9 cells infected with the recombinant containing an N-terminal truncation up to aa residue 125 and C-terminal to aa residue 601, demonstrating that the aa residues 126 to 601 are the essential elements required for the initiation of VLP assembly. The recombinant HEV VLPs are potential mucosal vaccine carrier vehicles for the presentation of foreign antigenic epitopes and may also serve as vectors for the delivery of genes to mucosal tissue for DNA vaccination and gene therapy. The results of the present study provide useful information for constructing recombinant HEV VLPs having novel functions.
Identification of an RNA Silencing Suppressor from a Plant Double-Stranded RNA Virus
Authors: Cao Xuesong , Zhou Peng , Zhang Xiaoming , Zhu Shifeng , Zhong Xuehua , Xiao Qi , Ding Biao , Li Yi
Published: September 2005

RNA silencing is a mechanism which higher plants and animals have evolved to defend against viral infection in addition to regulation of gene expression for growth and development. As a counterdefense, many plant and some animal viruses studied to date encode RNA silencing suppressors (RSS) that interfere with various steps of the silencing pathway. In this study, we report the first identification of an RSS from a plant double-stranded RNA (dsRNA) virus. Pns10, encoded by S10 of
Rice dwarf phytoreovirus
(RDV), exhibited RSS activity in coinfiltration assays with the reporter green fluorescent protein (GFP) in transgenic
Nicotiana benthamiana
line 16c carrying GFP. The other gene segments of the RDV genome did not have such a function. Pns10 suppressed local and systemic silencing induced by sense RNA but did not interfere with local and systemic silencing induced by dsRNA. Expression of Pns10 also increased the expression of β-glucuronidase in transient assays and enhanced
Potato virus X
pathogenicity in
N. benthamiana
. Collectively, our results establish Pns10 as an RSS encoded by a plant dsRNA virus and further suggest that Pns10 targets an upstream step of dsRNA formation in the RNA silencing pathway.
Herpes Simplex Virus 1 Envelopment Follows Two Diverse Pathways
Authors: Leuzinger Helene , Ziegler Urs , Schraner Elisabeth M. , Fraefel Cornel , Glauser Daniel L. , Heid Irma , Ackermann Mathias , Mueller Martin , Wild Peter
Published: September 2005

Herpesvirus envelopment is assumed to follow an uneconomical pathway including primary envelopment at the inner nuclear membrane, de-envelopment at the outer nuclear membrane, and reenvelopment at the
trans
-Golgi network. In contrast to the hypothesis of de-envelopment by fusion of the primary envelope with the outer nuclear membrane, virions were demonstrated to be transported from the perinuclear space to rough endoplasmic reticulum (RER) cisternae. Here we show by high-resolution microscopy that herpes simplex virus 1 envelopment follows two diverse pathways. First, nuclear envelopment includes budding of capsids at the inner nuclear membrane into the perinuclear space whereby tegument and a thick electron dense envelope are acquired. The substance responsible for the dense envelope is speculated to enable intraluminal transportation of virions via RER into Golgi cisternae. Within Golgi cisternae, virions are packaged into transport vacuoles containing one or several virions. Second, for cytoplasmic envelopment, capsids gain direct access from the nucleus to the cytoplasm via impaired nuclear pores. Cytoplasmic capsids could bud at the outer nuclear membrane, at membranes of RER, Golgi cisternae, and large vacuoles, and at banana-shaped membranous entities that were found to continue into Golgi membranes. Envelopes originating by budding at the outer nuclear membrane and RER membrane also acquire a dense substance. Budding at Golgi stacks, designated wrapping, results in single virions within small vacuoles that contain electron-dense substances between envelope and vacuolar membranes.