bluuebunny/crossref_metadata_2025 huggingface dataset:
In Vivo Replication of an ICP34.5 Second-Site Suppressor Mutant following Corneal Infection Correlates with In Vitro Regulation of eIF2α Phosphorylation
Authors: Ward Stephen L. , Scheuner Donalyn , Poppers Jeremy , Kaufman Randal J. , Mohr Ian , Leib David A.
Published: March 2003

In animal models of herpes simplex virus type 1 (HSV-1) infection, ICP34.5-null viruses are avirulent and also fail to grow in a variety of cultured cells due to their inability to prevent RNA-dependent protein kinase (PKR)-mediated inhibition of protein synthesis. We show here that the inability of ICP34.5 mutants to grow in vitro is due specifically to the accumulation of phosphorylated eIF2α. Mutations suppressing the in vitro phenotype of ICP34.5-null mutants have been described which map to the unique short region of the HSV-1 genome, resulting in dysregulated expression of the US11 gene. Despite the inability of the suppressor mutation to suppress the avirulent phenotype of the ICP34.5-null parental virus following intracranial inoculation, the suppressor mutation enhanced virus growth in the cornea, trigeminal ganglia, and periocular skin following corneal infection compared to that with the ICP34.5-null virus. The phosphorylation state of eIF2α following in vitro infection with the suppressor virus was examined to determine if in vivo differences could be attributed to differential regulation of eIF2α phosphorylation. The suppressor virus prevented accumulation of phosphorylated eIF2α, while the wild-type virus substantially reduced eIF2α phosphorylation levels. These data suggest that US11 functions as a PKR antagonist in vivo, although its activity may be modulated by tissue-specific differences in translation regulation.
Human Papillomavirus Type 16 L1 Capsomeres Induce L1-Specific Cytotoxic T Lymphocytes and Tumor Regression in C57BL/6 Mice
Authors: Öhlschläger Peter , Osen Wolfram , Dell Kerstin , Faath Stefan , Garcea Robert L. , Jochmus Ingrid , Müller Martin , Pawlita Michael , Schäfer Klaus , Sehr Peter , Staib Caroline , Sutter Gerd , Gissmann Lutz
Published: March 2003

We analyzed capsomeres of human papillomavirus type 16 (HPV16) consisting of the L1 major structural protein for their ability to trigger a cytotoxic T-cell (CTL) response. To this end, we immunized C57BL/6 mice and used the L1
165-173
peptide for ex vivo restimulation of splenocytes prior to analysis (
51
Cr release assay and enzyme-linked immunospot assay [ELISPOT]). This peptide was identified in this study as a D
b
-restricted naturally processed CTL epitope by HPV16 L1 sequence analysis, major histocompatibility complex class I binding, and
51
Cr release assays following immunization of C57BL/6 mice with HPV16 L1 virus-like particles (VLPs). HPV16 L1 capsomeres were obtained by purification of HPV16 L1 lacking 10 N-terminal amino acids after expression in
Escherichia coli
as a glutathione
S
-transferase fusion protein (GST-HPV16 L1ΔN10). Sedimentation analysis revealed that the majority of the purified protein consisted of pentameric capsomeres, and assembled particles were not observed in minor contaminating higher-molecular-weight material. Subcutaneous (s.c.) as well as intranasal immunization of C57BL/6 mice with HPV16 L1 capsomeres triggered an L1-specific CTL response in a dose-dependent manner as measured by ELISPOT and
51
Cr release assay. Significant reduction of contaminating bacterial endotoxin (lipopolysaccharide) from the capsomere preparation did not diminish the immunogenicity. Antibody responses (serum and vaginal) were less robust under the experimental conditions employed. In addition, s.c. vaccination with HPV16 L1 capsomeres induced regression of established tumors expressing L1 determinants (C3 tumor cells). Our data demonstrate that capsomeres are potent inducers of CTL responses similar to completely assembled
T
=7 VLPs. This result is of potential relevance for the development of (combined prophylactic and therapeutic) HPV-specific vaccines, since capsomeres can be produced easily and also can be modified to incorporate heterologous sequences such as early HPV proteins.
Ability of the Matrix Protein of Vesicular Stomatitis Virus To Suppress Beta Interferon Gene Expression Is Genetically Correlated with the Inhibition of Host RNA and Protein Synthesis
Authors: Ahmed Maryam , McKenzie Margie O. , Puckett Shelby , Hojnacki Michael , Poliquin Laurent , Lyles Douglas S.
Published: March 2003

The vesicular stomatitis virus (VSV) matrix (M) protein plays a major role in the virus-induced inhibition of host gene expression. It has been proposed that the inhibition of host gene expression by M protein is responsible for suppressing activation of host interferon gene expression. Most wild-type (wt) strains of VSV induce little if any interferon gene expression. Interferon-inducing mutants of VSV have been isolated previously, many of which contain mutations in their M proteins. However, it was not known whether these M protein mutations were responsible for the interferon-inducing phenotype of these viruses. Alternatively, mutations in other genes besides the M gene may enhance the ability of VSV to induce interferons. These hypotheses were tested by transfecting cells with mRNA expressing wt and mutant M proteins in the absence of other viral components and determining their ability to inhibit interferon gene expression. The M protein mutations were the M51R mutation originally found in the
ts
O82 and T1026R1 mutant viruses, the double substitution V221F and S226R found in the TP3 mutant virus, and the triple substitution E213A, V221F, and S226R found in the TP2 mutant virus. wt M proteins suppressed expression of luciferase from the simian virus 40 promoter and from the beta interferon (IFN-β) promoter, while M proteins of interferon-inducing viruses were unable to inhibit luciferase expression from either promoter. The M genes of the interferon-inducing mutants of VSV were incorporated into the wt background of a recombinant VSV infectious cDNA clone. The resulting recombinant viruses were tested for their ability to activate interferon gene expression and for their ability to inhibit host RNA and protein synthesis. Each of the recombinant viruses containing M protein mutations induced expression of a luciferase reporter gene driven by the IFN-β promoter and induced production of interferon bioactivity more effectively than viruses containing wt M proteins. Furthermore, the M protein mutant viruses were defective in their ability to inhibit both host RNA synthesis and host protein synthesis. These data support the idea that wt M protein suppresses interferon gene expression through the general inhibition of host RNA and protein synthesis.
Contrasting Effects of Matrix Protein on Apoptosis in HeLa and BHK Cells Infected with Vesicular Stomatitis Virus Are due to Inhibition of Host Gene Expression
Authors: Kopecky Sarah A. , Lyles Douglas S.
Published: March 2003

Vesicular stomatitis virus (VSV) is a potent inducer of apoptosis in host cells. Recently, it has been shown that two VSV products are involved in the induction of apoptosis, the matrix (M) protein, and another viral product that has yet to be identified (S. A. Kopecky et. al., J. Virol. 75:12169-12181, 2001). Comparison of recombinant viruses containing wild-type (wt) or mutant M proteins showed that wt M protein accelerates VSV-induced apoptosis in HeLa cells, while wt M protein delays apoptosis in VSV-infected BHK cells. Our hypothesis to explain these results is that both effects of M protein are due to the ability of M protein to inhibit host gene expression. This hypothesis was tested by infecting cells with an M protein mutant virus defective in the inhibition of host gene expression (rM51R-M virus) in the presence or absence of actinomycin D, another inhibitor of host gene expression. Actinomycin D accelerated induction of apoptosis of HeLa cells infected with rM51R-M virus and delayed apoptosis in BHK cells infected with rM51R-M virus, similar to the effects of wt M protein. The idea that the induction of apoptosis by M protein in HeLa cells is due to its ability to inhibit host gene expression was further tested by comparing the activation of upstream caspase pathways by M protein versus that by actinomycin D or 5,6-dichlorobenzimidazole riboside (DRB). Expression of M protein activated both caspase-8 and caspase-9-like enzymes, as did treatment with actinomycin D or DRB. Induction of apoptosis by M protein, actinomycin D, and DRB was inhibited in stably transfected HeLa cell lines that overexpress Bcl-2, an antiapoptotic protein that inhibits the caspase-9 pathway. A synthetic inhibitor of caspase-8, Z-IETD-FMK, did not inhibit induction of apoptosis by M protein, actinomycin D, or DRB. Taken together, our data support the hypothesis that the induction of apoptosis by M protein is caused by the inhibition of host gene expression and that the caspase-9 pathway is more important than the caspase-8 pathway for the induction of apoptosis by M protein and other inhibitors of host gene expression.
Control of Central Nervous System Viral Persistence by Neutralizing Antibody
Authors: Ramakrishna Chandran , Bergmann Cornelia C. , Atkinson Roscoe , Stohlman Stephen A.
Published: March 2003

Replication of the neurotropic JHM strain of mouse hepatitis virus within the central nervous system is controlled by cellular immunity. However, following initial clearance, virus reactivates in the absence of humoral immunity. Viral recrudescence is prevented by the transfer of antiviral antibody (Ab). To characterize the specificity and biological functions of Ab critical for maintaining viral persistence, monoclonal Abs specific for the viral spike, matrix, and nucleocapsid proteins were transferred into infected B-cell-deficient mice following initial virus clearance. Neutralizing immunoglobulin G (IgG) but not IgA anti-spike Ab suppressed virus recrudescence, reduced viral antigen in most cell types except oligodendroglia, and was associated with reduced demyelination. Nonneutralizing monoclonal Abs specific for the spike, matrix, and nucleocapsid proteins did not prevent recrudescence, demonstrating that neutralization is critical for maintaining JHM mouse hepatitis virus persistence within the central nervous system. Ab-mediated protection was not associated with alterations in virus-specific T-cell function or inflammation. Furthermore, neutralizing Ab delayed but did not prevent virus recrudescence. These data indicate that following acute viral clearance cellular immunity is ineffective in controlling virus recrudescence and suggest that the continued presence of neutralizing Ab is the essential effector in maintaining viral persistence within the central nervous system.
Heat-Aggregated Noninfectious Influenza Virus Induces a More Balanced CD8+-T-Lymphocyte Immunodominance Hierarchy Than Infectious Virus
Authors: Cho Yunjung , Basta Sameh , Chen Weisan , Bennink Jack R. , Yewdell Jonathan W.
Published: March 2003

CD8+-T-cell (TCD8+) responses to infectious viruses are characterized by an immunodominance hierarchy in which the majority of TCD8+respond to one or a few immunodominant determinants, with a minority of TCD8+responding to a number of subdominant determinants. It is now well established that exogenous antigens are capable of inducing TCD8+to such immunodominant determinants, but the diversity of the response and the nature of the immunodominance hierarchy have not been examined. We addressed this issue by characterizing TCD8+responses to influenza virus preparations rendered inert by incubation for 10 min at 100°C, as first reported by Speidel et al. (Eur. J. Immunol. 27:2391-2399, 1997). Extending these findings, we show that the primary TCD8+response to boiled virus can be sufficiently robust to be detected ex vivo by intracellular cytokine staining and that the response encompasses many of the peptides recognized by TCD8+induced by infectious virus. Importantly, the immunodominance hierarchy elicited was leveled, and we were unable to detect TCD8+that were specific for boiled virus. We used peritoneal exudate cells as antigen-presenting cells in vitro, and a number of observations indicated that boiled virus is processed via a phagocytic route that is likely to be endosomal in nature. These findings suggest that the repertoires of immunogenic peptides generated by endosomes and cytosolic processes overlap to a surprising degree. Furthermore, they demonstrate that the form of antigen administered can influence immunodominance hierarchies and that exogenous-antigen vaccines can induce broad and balanced TCD8+responses.
Impact of the Central Polypurine Tract on the Kinetics of Human Immunodeficiency Virus Type 1 Vector Transduction
Authors: Van Maele Bénédicte , De Rijck Jan , De Clercq Erik , Debyser Zeger
Published: March 2003

Lentiviral vectors derived from human immunodeficiency virus type 1 (HIV-1) show great promise as gene carriers for future gene therapy. Insertion of a fragment containing the central polypurine tract (cPPT) in HIV-1 vector constructs is known to enhance transduction efficiency drastically, reportedly by facilitating the nuclear import of HIV-1 cDNA through a central DNA flap. We have studied the impact of the cPPT on the kinetics of HIV-1 vector transduction by real-time PCR. The kinetics of total HIV-1 DNA, two-long-terminal-repeat (2-LTR) circles, and, by an
Alu
-PCR, integrated proviral DNA were monitored. About 6 to 12 h after transduction, the total HIV-1 DNA reached a maximum level, followed by a steep decrease. The 2-LTR circles peaked after 24 to 48 h and were diluted upon cell division. Integration of HIV-1 DNA was first detected at 12 h postinfection. When HIV-1 vectors that contained the cPPT were used, DNA synthesis was similar but a threefold higher amount of 2-LTR circles was detected, confirming the impact on nuclear import. Moreover, a 10-fold increase in the amount of integrated DNA was observed in the presence of the cPPT. Only in the absence of the cPPT was a saturation in 2-LTR circle formation seen at a high multiplicity of infection, suggesting a role for the cPPT in overcoming a barrier to the nuclear import of HIV-1 DNA. A major effect of the central DNA flap on the juxtaposition of both LTRs is unlikely, since transduction with HIV-1 vectors containing ectopic cPPT fragments resulted in increased amounts of 2-LTR circles as well as integrated DNA. Inhibitors of transduction by cPPT-containing HIV vectors were also studied by real-time PCR. The reverse transcriptase inhibitor azidothymidine (AZT) and the nonnucleoside reverse transcriptase inhibitor α-APA clearly inhibited viral DNA synthesis, whereas integrase inhibitors such as the diketo acid L-708,906 and the pyranodipyrimidine V-165 specifically inhibited integration.
Global Dysfunction of CD4 T-Lymphocyte Cytokine Expression in Simian-Human Immunodeficiency Virus/SIV-Infected Monkeys Is Prevented by Vaccination
Authors: McKay Paul F. , Barouch Dan H. , Schmitz Jörn E. , Veazey Ronald S. , Gorgone Darci A. , Lifton Michelle A. , Williams Kenneth C. , Letvin Norman L.
Published: March 2003

Human immunodeficiency virus type 1 infection results in a dysfunction of CD4+T lymphocytes. The intracellular events contributing to that CD4+T-lymphocyte dysfunction remain incompletely elucidated, and it is unclear whether aspects of that dysfunction can be prevented. The present studies were pursued in a rhesus monkey model of AIDS to explore these issues. Loss of the capacity of peripheral blood CD4+T lymphocytes to express cytokines was first detected in simian immunodeficiency virus-infected monkeys during the peak of viral replication during primary infection and persisted thereafter. Moreover, infected monkeys with progressive disease had peripheral blood CD4+T lymphocytes that expressed significantly less cytokine than infected monkeys that had undetectable viral loads and intact CD4+T-lymphocyte counts. Importantly, CD4+T lymphocytes from vaccinated monkeys that effectively controlled the replication of a highly pathogenic immunodeficiency virus isolate following a challenge had a preserved functional capacity. These observations suggest that an intact cytokine expression capacity of CD4+T lymphocytes is associated with stable clinical status and that effective vaccines can mitigate against CD4+T-lymphocyte dysfunction following an AIDS virus infection.
Translational Compensation of a Frameshift Mutation Affecting Herpes Simplex Virus Thymidine Kinase Is Sufficient To Permit Reactivation from Latency
Authors: Griffiths Anthony , Chen Shun-Hua , Horsburgh Brian C. , Coen Donald M.
Published: March 2003

Herpes simplex virus thymidine kinase is important for reactivation of virus from its latent state and is a target for the antiviral drug acyclovir. Most acyclovir-resistant isolates have mutations in the thymidine kinase gene; however, how these mutations confer clinically relevant resistance is unclear. Reactivation from explanted mouse ganglia was previously observed with a patient-derived drug-resistant isolate carrying a single guanine insertion within a run of guanines in the thymidine kinase gene. Despite this mutation, low levels of active enzyme were synthesized following an unusual ribosomal frameshift. Here we report that a virus, generated from a pretherapy isolate from the same patient, engineered to lack thymidine kinase activity, was competent for reactivation. This suggested that the clinical isolate contains alleles of other genes that permit reactivation in the absence of thymidine kinase. Therefore, to establish whether thymidine kinase synthesized via a ribosomal frameshift was sufficient for reactivation under conditions where reactivation requires this enzyme, we introduced the mutation into the well-characterized strain KOS. This mutant virus reactivated from latency, albeit less efficiently than KOS. Plaque autoradiography revealed three phenotypes of reactivating viruses: uniformly low thymidine kinase activity, mixed high and low activity, and uniformly high activity. We generated a recombinant thymidine kinase-null virus from a reactivating virus expressing uniformly low activity. This virus did not reactivate, confirming that mutations in other genes that would influence reactivation had not arisen. Therefore, in strains that require thymidine kinase for reactivation from latency, low levels of enzyme synthesized via a ribosomal frameshift can suffice.
Template Dimerization Promotes an Acceptor Invasion-Induced Transfer Mechanism during Human Immunodeficiency Virus Type 1 Minus-Strand Synthesis
Authors: Balakrishnan Mini , Roques Bernard P. , Fay Philip J. , Bambara Robert A.
Published: March 2003

The biochemical mechanism of template switching by human immunodeficiency virus type 1 (HIV-1) reverse transcriptase and the role of template dimerization were examined. Homologous donor-acceptor template pairs derived from the HIV-1 untranslated leader region and containing the wild-type and mutant dimerization initiation sequences (DIS) were used to examine the efficiency and distribution of transfers. Inhibiting donor-acceptor interaction was sufficient to reduce transfers in DIS-containing template pairs, indicating that template dimerization, and not the mere presence of the DIS, promotes efficient transfers. Additionally, we show evidence that the overall transfer process spans an extended region of the template and proceeds through a two-step mechanism. Transfer is initiated through an RNase H-facilitated acceptor invasion step, while synthesis continues on the donor template. The invasion then propagates towards the primer terminus by branch migration. Transfer is completed with the translocation of the primer terminus at a site distant from the invasion point. In our system, most invasions initiated before synthesis reached the DIS. However, transfer of the primer terminus predominantly occurred after synthesis through the DIS. The two steps were separated by 60 to 80 nucleotides. Sequence markers revealed the position of primer terminus switch, whereas DNA oligomers designed to block acceptor-cDNA interactions defined sites of invasion. Within the region of homology, certain positions on the template were inherently more favorable for invasion than others. In templates with DIS, the proximity of the acceptor facilitates invasion, thereby enhancing transfer efficiency. Nucleocapsid protein enhanced the overall efficiency of transfers but did not alter the mechanism.
Foamy Virus Envelope Glycoprotein-Mediated Entry Involves a pH-Dependent Fusion Process
Authors: Picard-Maureau Marcus , Jarmy Gergely , Berg Angelika , Rethwilm Axel , Lindemann Dirk
Published: March 2003

In general, enveloped viruses use two different entry strategies and are classified accordingly into pH-dependent and pH-independent viruses. Different members of the retrovirus family use one or the other strategy. Little is known about the uptake of foamy viruses (FV), a special group of retroviruses, into the target cells. In this study, we examined the pH dependence of FV entry by analyzing FV envelope glycoprotein (Env)-mediated infection of target cells with murine leukemia virus or FV vector pseudotypes in the presence of various lysosomotropic agents. Similar to vesicular stomatitis virus glycoprotein G (VSV-G)-mediated uptake, FV Env-mediated entry was inhibited by various lysosomotropic agents, suggesting a pH-dependent endocytic pathway. However, in contrast to its effect on VSV-G pseudotypes, chloroquine failed to reduce the infectivity of FV Env pseudotypes, implying that the pathway is different from that of VSV-G. Glycoproteins of various other FV species showed inhibition profiles similar to that of the prototype FV (PFV) Env. Analysis of the pH dependence of the FV Env-mediated fusion process in a cell-to-cell fusion assay revealed an induction of syncytium formation by a short exposure to acidic pH, peaking around pH 5.5. Interestingly, of all FV Env species analyzed, only the PFV Env had a significant fusion activity at neutral pH. Taken together, these data suggest a pH-dependent endocytic pathway for infection of target cells by FV.
Human Cytomegalovirus US3 Chimeras Containing US2 Cytosolic Residues Acquire Major Histocompatibility Class I and II Protein Degradation Properties
Authors: Chevalier Mathieu S. , Johnson David C.
Published: March 2003

Human cytomegalovirus (HCMV) glycoprotein US2 increases the proteasome-mediated degradation of major histocompatibility complex (MHC) class I heavy chain (HC), class II DR-α and DM-α proteins, and HFE, a nonclassical MHC protein. US2-initiated degradation of MHC proteins apparently involves the recruitment of cellular proteins that participate in a process known as endoplasmic reticulum (ER)-associated degradation. ER-associated degradation is a normal process by which misfolded proteins are recognized and translocated into the cytoplasm for degradation by proteasomes. It has been demonstrated that truncated forms of US2, especially those lacking the cytoplasmic domain (CT), can bind MHC proteins but do not cause their degradation. To further assess how the US2 CT domain interacts with the cellular components of the ER-associated degradation pathway, we constructed chimeric proteins in which the US2 CT domain or the CT and transmembrane (TM) domains replaced those of the HCMV glycoprotein US3. US3 also binds both class I and II proteins but does not cause their degradation. Remarkably, chimeras containing the US2 CT domain caused the degradation of both MHC class I and II proteins although this degradation was less than that by wild-type US2. Therefore, the US2 CT and TM domains can confer on US3 the capacity to degrade MHC proteins. We also analyzed complexes containing MHC proteins and US2, US3, US11, or US3/US2 chimeras for the presence of cdc48/p97 ATPase, a protein that binds polyubiquitinated proteins and likely functions in the extraction of substrates from the ER membrane before the substrates meet proteasomes. p97 ATPase was present in immunoprecipitates containing US2, US11, and two chimeras that included the US2 CT domain, but not in US3 complexes. Therefore, it appears that the CT domain of US2 participates in recruiting p97 ATPase into ER-associated degradation complexes.
Poliovirus CRE-Dependent VPg Uridylylation Is Required for Positive-Strand RNA Synthesis but Not for Negative-Strand RNA Synthesis
Authors: Murray Kenneth E. , Barton David J.
Published: March 2003

The
cis
-acting replication element (CRE) is a 61-nucleotide stem-loop RNA structure found within the coding sequence of poliovirus protein 2C. Although the CRE is required for viral RNA replication, its precise role(s) in negative- and positive-strand RNA synthesis has not been defined. Adenosine in the loop of the CRE RNA structure functions as the template for the uridylylation of the viral protein VPg. VPgpUpU
OH
, the predominant product of CRE-dependent VPg uridylylation, is a putative primer for the poliovirus RNA-dependent RNA polymerase. By examining the sequential synthesis of negative- and positive-strand RNAs within preinitiation RNA replication complexes, we found that mutations that disrupt the structure of the CRE prevent VPg uridylylation and positive-strand RNA synthesis. The CRE mutations that inhibited the synthesis of VPgpUpU
OH
, however, did not inhibit negative-strand RNA synthesis. A Y3F mutation in VPg inhibited both VPgpUpU
OH
synthesis and negative-strand RNA synthesis, confirming the critical role of the tyrosine hydroxyl of VPg in VPg uridylylation and negative-strand RNA synthesis.
trans
-replication experiments demonstrated that the CRE and VPgpUpU
OH
were not required in
cis
or in
trans
for poliovirus negative-strand RNA synthesis. Because these results are inconsistent with existing models of poliovirus RNA replication, we propose a new four-step model that explains the roles of VPg, the CRE, and VPgpUpU
OH
in the asymmetric replication of poliovirus RNA.
Consequences of DNA-Dependent Protein Kinase Catalytic Subunit Deficiency on Recombinant Adeno-Associated Virus Genome Circularization and Heterodimerization in Muscle Tissue
Authors: Duan Dongsheng , Yue Yongping , Engelhardt John F.
Published: March 2003

Circular concatemerization of the recombinant adeno-associated virus (rAAV) genome has been suggested as the predominant process facilitating long-term rAAV transduction in muscle. A recent study (S. Song, P. J. Laipis, K. I. Berns, and T. R. Flotte, Proc. Natl. Acad. Sci. USA 98:4084-4088, 2001) with SCID mice, which are defective in the DNA-dependent protein kinase catalytic subunit (DNA-PKcs), has suggested that DNA-PKcs regulates the removal of free rAAV vector ends in muscle tissue. In the present study, we have sought to evaluate whether a lack of DNA-PKcs activity reduces circularization of rAAV genomes in SCID muscle and whether such a reduction alters the directivity of heterodimerization. Consistent with the previous report, linear rAAV genomes and free vector ends were detected only in DNA-PKcs-deficient muscle by Southern blotting. Appreciable amounts of circular rAAV genomes were detected in both DNA-PKcs-deficient and wild-type muscle samples by Southern blotting and bacterial trapping experiments. The existence of double-D inverted terminal repeat circular intermediates in SCID and wild-type muscles was also supported by their sensitivity to T7 endonuclease I digestion. However, DNA-PKcs-deficient muscle did demonstrate a ∼50% reduction in the abundance of rescued circular genomes, despite equivalent levels of single rAAV transduction seen in wild-type animals. Dual
trans
-splicing
lacZ
vectors were used to functionally evaluate directional head-to-tail intermolecular viral genome concatamerization in vivo. Although AAV genomes are processed differently in SCID and wild-type muscles, a comparable level of
trans
-splicing-mediated β-galactosidase expression was observed in both strains, suggesting that both circular and linear AAV concatemers may have contributed to the
trans
-splicing-mediated transgene expression. In summary, we have shown that SCID skeletal muscle retains a fairly high capacity to form circular genomes, despite a significant increase in linear vector genomes. Furthermore, the alteration in equilibrium between circular and linear concatemer genomes caused by the lack of DNA-PKcs activity does not appear to significantly affect the efficiency of dual-vector gene expression from head-to-tail linear and/or circular heterodimers.
Strain Variations in Single Amino Acids of the 86-Kilodalton Human Cytomegalovirus Major Immediate-Early Protein (IE2) Affect Its Functional and Biochemical Properties: Implications of Dynamic Protein Conformation
Authors: Barrasa M. Inmaculada , Harel Noam , Yu Yongjun , Alwine James C.
Published: March 2003

The 86-kDa major immediate-early protein, IEP86 (IE2, IE2
579aa
, or ppUL122a), from the Towne and AD169 strains of human cytomegalovirus show four amino acid variations, namely, R68Q, K455E, T541A, and seven consecutive serines beginning at position 258 in Towne and eight serines in AD169. A commonly utilized IEP86 cDNA expression clone (herein called the original cDNA) (E. Baracchini, E. Glezer, K. Fish, R. M. Stenberg, J. A. Nelson, and P. Ghazal, Virology 188:518-529, 1992) shows the Towne R68 and seven serines but contains the AD169 E455 and A541 plus two amino acid mutations, M242I and A463T. In transcriptional activation analyses using several promoters, the IEP86 produced by the original cDNA was 40 to 60% less active than wild-type (WT) Towne IEP86, whereas AD169 IEP86 was two to three times more active than WT Towne IEP86. To determine which amino acid variations or mutations accounted for the differences in transcriptional activation, they were individually tested in the WT Towne IEP86 background. K455E, M242I, and the eighth serine had little effect on transcriptional activation or sumoylation when inserted into the Towne background. T541A significantly increased transcriptional activation on all promoters tested and showed increased sumoylation; T541A is the primary reason that WT AD169 IEP86 has increased activity over WT Towne IEP86. The increased sumoylation seen with T541A was quantitatively reduced to WT Towne levels when the K455E alteration was present, suggesting that K455 may be a sumoylation site or that E455 may cause alterations in the IEP86 structure which affect overall sumoylation. A463T was very deleterious to transcriptional activation and caused reduced sumoylation. The A436T mutation in the original cDNA is partially compensated by the presence of the T541A variation. Phosphopeptide mapping suggests that a threonine at 463 or 541 does not introduce a phosphorylation site. However, the A463T mutation does affect phosphorylation at a distant site, suggesting that it alters the conformation of the protein. Promoter-specific effects were noted with some of the amino acid variations, particularly T541A. Structural modeling is presented which suggests how A463T and T541A alter the functional structure of WT Towne IEP86. A hydrophobic core containing A463 is predicted to be responsible for the functional integrity of the carboxy-terminal region of IEP86 between amino acids 344 and 579.
Changes in Rhinovirus Protein 2C Allow Efficient Replication in Mouse Cells
Authors: Harris Julie R. , Racaniello Vincent R.
Published: March 2003

Rhinovirus type 16 was found to replicate in mouse L cells that express the viral receptor, human intercellular adhesion molecule 1 (ICAM-1). However, infection of these cells at a low multiplicity of infection leads to no discernible cytopathic effect, and low virus titers are produced. A variant virus, 16/L, was isolated after alternate passage of rhinovirus 16 between HeLa and ICAM-1 L cells. Infection of mouse cells with 16/L leads to higher virus titers, increased production of RNA, and total cytopathic effect. Three amino acid changes were identified in the P2 region of virus 16/L, and the adaptation phenotype mapped to two changes in protein 2C. The characterization of a rhinovirus host range mutant will facilitate the investigation of cellular proteins required for efficient viral growth and the development of a murine model for rhinovirus infection.
Role of ESCRT-I in Retroviral Budding
Authors: Martin-Serrano Juan , Zang Trinity , Bieniasz Paul D.
Published: March 2003

Retroviral late-budding (L) domains are required for the efficient release of nascent virions. The three known types of L domain, designated according to essential tetrapeptide motifs (PTAP, PPXY, or YPDL), each bind distinct cellular cofactors. We and others have demonstrated that recruitment of an ESCRT-I subunit, Tsg101, a component of the class E vacuolar protein sorting (VPS) machinery, is required for the budding of viruses, such as human immunodeficiency virus type 1 (HIV-1) and Ebola virus, that encode a PTAP-type L domain, but subsequent events remain undefined. In this study, we demonstrate that VPS28, a second component of ESCRT-I, binds to a sequence close to the Tsg101 C terminus and is therefore recruited to the plasma membrane by HIV-1 Gag. In addition, we show that Tsg101 exhibits a multimerization activity. Using a complementation assay in which Tsg101 is artificially recruited to sites of HIV-1 assembly, we demonstrate that the integrity of the VPS28 binding site within Tsg101 is required for particle budding. In addition, mutation of a putative leucine zipper or residues important for Tsg101 multimerization also impairs the ability of Tsg101 to support HIV-1 budding. A minimal multimerizing Tsg101 domain is a dominant negative inhibitor of PTAP-mediated HIV-1 budding but does not inhibit YPDL-type or PPXY-type L-domain function. Nevertheless, YDPL-type L-domain activity is inhibited by expression of a catalytically inactive mutant of the class E VPS ATPase VPS4. These results indicate that all three classes of retroviral L domains require a functioning class E VPS pathway in order to effect budding. However, the PTAP-type L domain appears to be unique in its requirement for an intact, or nearly intact, ESCRT-I complex.
Interactions between Papillomavirus L1 and L2 Capsid Proteins
Authors: Finnen Renée L. , Erickson Kimberly D. , Chen Xiaojiang S. , Garcea Robert L.
Published: March 2003

The human papillomavirus (HPV) capsid consists of 360 copies of the major capsid protein, L1, arranged as 72 pentamers on a T=7 icosahedral lattice, with substoichiometric amounts of the minor capsid protein, L2. In order to understand the arrangement of L2 within the HPV virion, we have defined and biochemically characterized a domain of L2 that interacts with L1 pentamers. We utilized an in vivo binding assay involving the coexpression of recombinant HPV type 11 (HPV11) L1 and HPV11 glutathione
S
-transferase (GST) L2 fusion proteins in
Escherichia coli
. In this system, L1 forms pentamers, GST=L2 associates with these pentamers, and L1+L2 complexes are subsequently isolated by using the GST tag on L2. The stoichiometry of L1:L2 in purified L1+L2 complexes was 5:1, indicating that a single molecule of L2 interacts with an L1 pentamer. Coexpression of HPV11 L1 with deletion mutants of HPV11 L2 defined an L1-binding domain contained within amino acids 396 to 439 near the carboxy terminus of L2. L2 proteins from eight different human and animal papillomavirus serotypes were tested for their ability to interact with HPV11 L1. This analysis targeted a hydrophobic region within the L1-binding domain of L2 as critical for L1 binding. Introduction of negative charges into this hydrophobic region by site-directed mutagenesis disrupted L1 binding. L1-L2 interactions were not significantly disrupted by treatment with high salt concentrations (2 M NaCl), weak detergents, and urea concentrations of up to 2 M, further indicating that L1 binding by this domain is mediated by strong hydrophobic interactions. L1+L2 protein complexes were able to form virus-like particles in vitro at pH 5.2 and also at pH 6.8, a pH that is nonpermissive for assembly of L1 protein alone. Thus, L1/L2 interactions are primarily hydrophobic, encompass a relatively short stretch of amino acids, and have significant effects upon in vitro assembly.
Complexes of Poliovirus Serotypes with Their Common Cellular Receptor, CD155
Authors: He Yongning , Mueller Steffen , Chipman Paul R. , Bator Carol M. , Peng Xiaozhong , Bowman Valorie D. , Mukhopadhyay Suchetana , Wimmer Eckard , Kuhn Richard J. , Rossmann Michael G.
Published: March 2003

Structures of all three poliovirus (PV) serotypes (PV1, PV2, and PV3) complexed with their cellular receptor, PV receptor (PVR or CD155), were determined by cryoelectron microscopy. Both glycosylated and fully deglycosylated CD155 exhibited similar binding sites and orientations in the viral canyon for all three PV serotypes, showing that all three serotypes use a common mechanism for cell entry. Difference maps between the glycosylated and deglycosylated CD155 complexes determined the sites of the carbohydrate moieties that, in turn, helped to verify the position of the receptor relative to the viral surface. The proximity of the CD155 carbohydrate site at Asn105 to the viral surface in the receptor-virus complex suggests that it might interfere with receptor docking, an observation consistent with the properties of mutant CD155. The footprints of CD155 on PV surfaces indicate that the south rim of the canyon dominates the virus-receptor interactions and may correspond to the initial CD155 binding state of the receptor-mediated viral uncoating. In contrast, the interaction of CD155 with the north rim of the canyon, especially the region immediately outside the viral hydrophobic pocket that normally binds a cellular “pocket factor,” may be critical for the release of the pocket factor, decreasing the virus stability and hence initiating uncoating. The large area of the CD155 footprint on the PV surface, in comparison with other picornavirus-receptor interactions, could be a potential limitation on the viability of PV escape mutants from antibody neutralization. Many of these are likely to have lost their ability to bind CD155, resulting in there being only three PV serotypes.
Infection of Cattle with a Bovine Herpesvirus 1 Strain That Contains a Mutation in the Latency-Related Gene Leads to Increased Apoptosis in Trigeminal Ganglia during the Transition from Acute Infection to Latency
Authors: Lovato Luciane , Inman Melissa , Henderson Gail , Doster Alan , Jones Clinton
Published: March 2003

Bovine herpesvirus 1 (BHV-1) is an important pathogen of cattle and infection is usually initiated via the ocular or nasal cavity. After acute infection, the primary site for BHV-1 latency is sensory neurons in the trigeminal ganglia (TG). Reactivation from latency occurs sporadically, resulting in virus shedding and transmission to uninfected cattle. The only abundant viral transcript expressed during latency is the latency-related (LR) RNA. An LR mutant was constructed by inserting three stop codons near the beginning of the LR RNA. This mutant grows to wild-type (wt) efficiency in bovine kidney cells and in the nasal cavity of acutely infected calves. However, shedding of infectious virus from the eye and TG was dramatically reduced in calves infected with the LR mutant. Calves latently infected with the LR mutant do not reactivate after dexamethasone treatment. In contrast, all calves latently infected with wt BHV-1 or the LR rescued mutant reactivate from latency after dexamethasone treatment. In the present study, we compared the frequency of apoptosis in calves infected with the LR mutant to calves infected with wt BHV-1 because LR gene products inhibit apoptosis in transiently transfected cells. A sensitive TUNEL (terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end labeling) assay and an antibody that detects cleaved caspase-3 were used to identify apoptotic cells in TG. Both assays demonstrated that calves infected with the LR mutant for 14 days had higher levels of apoptosis in TG compared to calves infected with wt BHV-1 or to mock-infected calves. Viral gene expression, except for the LR gene, is extinguished by 14 days after infection, and thus this time frame is operationally defined as the establishment of latency. Real-time PCR analysis indicated that lower levels of viral DNA were present in the TG of calves infected with the LR mutant throughout acute infection. Taken together, these results suggest that the antiapoptotic properties of the LR gene play an important role during the establishment of latency.
Binding of Adenovirus Capsid to Dipalmitoyl Phosphatidylcholine Provides a Novel Pathway for Virus Entry
Authors: Balakireva Larissa , Schoehn Guy , Thouvenin Eric , Chroboczek Jadwiga
Published: March 2003

Adenovirus (Ad) is an airborne, nonenveloped virus infecting respiratory epithelium. To study the mechanism of Ad entry, we used alveolar adenocarcinoma A549 cells, which have retained the ability of alveolar epithelial type II cells to synthesize the major component of pulmonary surfactant, disaturated phosphatidylcholine. Stimulation of phosphatidylcholine secretion by calcium ionophore or phorbol ester augmented the susceptibility of these cells to Ad. Both Ad infection and recombinant-Ad-mediated transfection increased in the presence of dipalmitoyl phosphatidylcholine (DPPC) liposomes in culture medium. Importantly, in the presence of DPPC liposomes, virus penetrates the cells independently of virus-specific protein receptors. DPPC vesicles bind Ad and are efficiently incorporated by A549 lung cells, serving as a virus vehicle during Ad penetration. To identify the viral protein(s) mediating Ad binding, a flotation of liposomes preincubated with structural viral proteins was employed, showing that the only Ad protein bound to DPPC vesicles was a hexon. The hexon preserved its phospholipid-binding properties upon purification, confirming its involvement in virus binding to the phospholipid. Given that disaturated phosphatidylcholine not only covers the inner surface of alveoli in the lungs but also reenters alveolar epithelium during lung surfactant turnover, Ad binding to this phospholipid may provide a pathway for virus entry into alveolar epithelium in vivo.
Characterization and Comparison of Recombinant Simian Immunodeficiency Virus from Drill (
Mandrillus leucophaeus
) and Mandrill (
Mandrillus sphinx
) Isolates
Authors: Hu Jinjie , Switzer William M. , Foley Brian T. , Robertson David L. , Goeken Robert M. , Korber Bette T. , Hirsch Vanessa M. , Beer Brigitte E.
Published: March 2003

Since simian immunodeficiency virus (SIV) was found to be the source of the human AIDS pandemic, a major goal has been to characterize the diversity of SIV strains in the wild and to assess their potential for crossover into humans. In the present study, SIV was isolated from a seropositive drill (
Mandrillus leucophaeus
) and three seropositive mandrills (
Mandrillus sphinx
) by using macaque peripheral blood mononuclear cells (PBMC). Full-length sequences were obtained from a drill and mandrill and designated SIVdrl1FAO and SIVmnd5440, respectively. A 182-bp fragment of the
pol
genes of the two remaining mandrill SIV isolates was also analyzed. Phylogenetic analyses demonstrated that SIVdrl1FAO formed a monophyletic clade with SIVmnd5440 and SIVmndM14, recently designated SIVmnd type 2. Both the SIVdrl and SIVmnd type 2 genomes carried a
vpx
gene and appeared to share a common ancestor with SIVrcm in the 5′ region of the genome and with SIVmndGB1 (type 1) in the 3′ region of the genome. A statistically significant recombination breakpoint was detected at the beginning of envelope, suggesting that the viruses were descendents of the same recombinant. Phylogenetic analysis of
vpx
and
vpr
genes demonstrated that the
vpx
genes formed a monophyletic cluster that grouped with
vpr
from SIVagm. In addition, both SIVdrl1FAO and SIVmnd5440 replicated in human PBMC and therefore could pose a risk of transmission to the human population.
Packaging of Human Chromosome 19-Specific Adeno-Associated Virus (AAV) Integration Sites in AAV Virions during AAV Wild-Type and Recombinant AAV Vector Production
Authors: Hüser Daniela , Weger Stefan , Heilbronn Regine
Published: March 2003

Adeno-associated virus type 2 (AAV-2) establishes latency by site-specific integration into a unique locus on human chromosome 19, called AAVS1. During the development of a sensitive real-time PCR assay for site-specific integration, AAV-AAVS1 junctions were reproducibly detected in highly purified AAV wild-type and recombinant AAV vector stocks. A series of controls documented that the junctions were packaged in AAV capsids and were newly generated during a single round of AAV production. Cloned junctions displayed variable AAV sequences fused to AAVS1. These data suggest that packaged junctions represent footprints of AAV integration during productive infection. Apparently, AAV latency established by site-specific integration and the helper virus-dependent, productive AAV cycle are more closely related than previously thought.
Membrane Association of VP22, a Herpes Simplex Virus Type 1 Tegument Protein
Authors: Brignati Michael J. , Loomis Joshua S. , Wills John W. , Courtney Richard J.
Published: March 2003

Tegument proteins of herpes simplex virus type 1 (HSV-1) are hypothesized to contain the functional information required for the budding or envelopment process proposed to occur at cytoplasmic compartments of the host cell. One of the most abundant tegument proteins of HSV-1 is the U
L
49 gene product, VP22, a 38-kDa protein of unknown function. To study its subcellular localization, a VP22-green fluorescent protein chimera was expressed in transfected human melanoma (A7) cells. In the absence of other HSV-1 proteins, VP22 localizes to acidic compartments of the cell that may include the
trans
-Golgi network (TGN), suggesting that this protein is membrane associated. Membrane pelleting and membrane flotation assays confirmed that VP22 partitions with the cellular membrane fraction. Through truncation mutagenesis, we determined that the membrane association of VP22 is a property attributed to amino acids 120 to 225 of this 301-amino-acid protein. The above results demonstrate that VP22 contains specific information required for targeting to membranes of acidic compartments of the cell which may be derived from the TGN, suggesting a potential role for VP22 during tegumentation and/or final envelopment.
Regulation of Host Cell Transcriptional Physiology by the Avian Pneumovirus Provides Key Insights into Host-Pathogen Interactions
Authors: Munir Shirin , Kapur Vivek
Published: March 2003

Infection with a viral pathogen triggers several pathways in the host cell that are crucial to eliminating infection, as well as those that are used by the virus to enhance its replication and virulence. We have here used suppression subtractive hybridization and cDNA microarray analyses to characterize the host transcriptional response in an avian pneumovirus model of infection. The results of our investigations reveal a dynamic host response that includes the regulation of genes with roles in a vast array of cellular functions as well as those that have not been described previously. The results show a considerable upregulation in transcripts representing the interferon-activated family of genes, predicted to play a role in virus replication arrest. The analysis also identified transcripts for proinflammatory leukocyte chemoattractants, adhesion molecules, and complement that were upregulated and may account for the inflammatory pathology that is the hallmark of viral respiratory infection. Interestingly, alterations in the transcription of several genes in the ubiquitin and endosomal protein trafficking pathways were observed, suggesting a role for these pathways in virus maturation and budding. Taken together, the results of our investigations provide key insights into individual genes and pathways that constitute the host cell's response to avian pneumovirus infection, and they have enabled the development of resources and a model of host-pathogen interaction for an important avian respiratory tract pathogen.