bluuebunny/crossref_metadata_2025 huggingface dataset:
Localization of Varicella-Zoster Virus Gene 21 Protein in Virus-Infected Cells in Culture
Authors: Mahalingam Ravi , Lasher Robert , Wellish Mary , Cohrs Randall J. , Gilden Donald H.
Published: December 2019

Although four varicella-zoster virus (VZV) genes have been shown to be transcribed in latently infected human ganglia, their role in the development and maintenance of latency is unknown. To study these VZV transcripts, we decided first to localize their expression products in productively infected cells. We began with VZV gene 21, whose open reading frame (ORF) is 3,113 bp. We cloned the 5′ and 3′ ends and the predicted antigenic segments of the ORF as 1292-, 1280-, and 880-bp DNA fragments, respectively, into the prokaryotic expression vector pGEX-2T. The three VZV 21 ORFs were expressed as approximately 75-, 73-, and 59-kDa glutathione
S
-transferase fusion proteins in
Escherichia coli
. To prepare polyclonal antibodies that would recognize all potential epitopes on the VZV gene 21 protein, rabbits were inoculated with a mixture of the three fusion proteins, and antisera were obtained and affinity purified. Immunohistochemical and immunoelectron microscopic analyses using these antibodies revealed VZV ORF 21 protein in both the nucleus and cytoplasm of VZV-infected cells. When these antibodies were applied to purified VZV nucleocapsids, intense staining was seen in their central cores.
Circumvention of Immunity to the Adenovirus Major Coat Protein Hexon
Authors: Roy Soumitra , Shirley Pamela S. , McClelland Alan , Kaleko Michael
Published: December 2019

Immunity to adenoviruses is an important hurdle to be overcome for successful gene therapy. The presence of antibodies to the capsid proteins prevents efficacious adenovirus vector administration in vivo. We tested whether immunity to a particular serotype of adenovirus (Ad5) may be overcome with a vector that encodes the hexon sequences from a different adenovirus serotype (Ad12). We successfully constructed an adenovirus vector with a chimeric Ad5-Ad12 hexon which was not neutralized by plasma from C57BL/6 mice immunized with Ad5. The vector was also capable of transducing the livers of C57BL/6 mice previously immunized with Ad5.
A New Vaccinia Virus Intermediate Transcription Factor
Authors: Sanz Patrick , Moss Bernard
Published: December 2019

Transcription of the vaccinia virus genome is mediated by a virus-encoded multisubunit DNA-dependent RNA polymerase in conjunction with early-, intermediate-, and late-stage-specific factors. Previous studies indicated that two virus-encoded proteins (capping enzyme and VITF-1) and one unidentified cellular protein (VITF-2) are required for specific transcription of an intermediate promoter template in vitro. We have now extensively purified an additional virus-induced intermediate transcription factor with a native mass of approximately 100 kDa.
The Polyserine Tract of Herpes Simplex Virus ICP4 Is Required for Normal Viral Gene Expression and Growth in Murine Trigeminal Ganglia
Authors: Bates Patricia A. , DeLuca Neal A.
Published: December 2019

ICP4 of herpes simplex virus (HSV) is essential for productive infection due to its central role in the regulation of HSV transcription. This study identified a region of ICP4 that is not required for viral growth in culture or at the periphery of experimentally inoculated mice but is critical for productive growth in the trigeminal ganglia. This region of ICP4 encompasses amino acids 184 to 198 and contains 13 nearly contiguous serine residues that are highly conserved among the alphaherpesviruses. A mutant in which this region is deleted (ΔSER) was able to grow on the corneas of mice and be transported back to the trigeminal ganglia. ΔSER did not grow in the trigeminal ganglia but did express low levels of several immediate-early (ICP4 and ICP27) and early (thymidine kinase [
tk
] and UL42) genes. It expressed very low levels of the late gC gene and did not appear to replicate DNA. This pattern of gene expression was similar to that observed for a
tk
mutant,
dl
sptk. Both ΔSER and
dl
sptk expressed higher levels of the latency-associated transcript (LAT) per genome earlier in infected ganglia than did the wild-type virus, KOS. However, infected ganglia from all three viruses accumulated the same level of LAT per genome at 30 days postinfection (during latency). The data suggest that the polyserine tract of ICP4 provides an activity that is required for lytic infection in ganglia to progress to viral DNA synthesis and full lytic gene expression. In the absence of this activity, higher levels of LAT per genome accumulate earlier in infection than with wild-type virus.
The Early Region 4 orf4 Protein of Human Adenovirus Type 5 Induces p53-Independent Cell Death by Apoptosis
Authors: Marcellus Richard C. , Lavoie Josée N. , Boivin Dominique , Shore Gordon C. , Ketner Gary , Branton Philip E.
Published: December 2019

Previous studies by our group showed that infection of human and rodent cells by human adenovirus type 5 (Ad5) results in the induction of p53-independent apoptosis and cell death that are dependent upon transactivation of early region 4 (E4). To identify which E4 products are involved, studies were conducted with p53-deficient human SAOS-2 cells infected with various Ad5 E4 mutants. An E4orf6-deficient mutant was defective in cell killing, whereas another that expressed only E4orf6 and E4orf4 killed like wild-type virus, suggesting that E4orf6 may be responsible for cytotoxicity; however, a mutant expressing only E4orf4 induced high levels of cell death, indicating that this E4 product may also be able to induce cytotoxicity. To define the E4 cell death-inducing functions more precisely, cDNAs encoding individual E4 products were introduced into cells by DNA transfection in the absence of other Ad5 proteins. In cotransfections with a cDNA encoding firefly luciferase, enzymatic activity was high in all cases except with E4orf4, where luciferase levels were less than 20% of those in controls. In addition, drug selection of several cell types following transfection with retroviral vector DNA encoding individual E4 products as well as puromycin resistance yielded a large number of cell colonies except when E4orf4 was expressed. These data demonstrated that E4orf4 is the only E4 product capable of independent cell killing. Cell death induced by E4orf4 was due to apoptosis, as evidenced by 4′,6-diamidino-2-phenylindole (DAPI) staining of cell nuclei in E4orf4-expressing cells. Thus, although E4orf6 may play some role, these results suggested that E4orf4 may be the major E4 product responsible for induction of p53-independent apoptosis.
trans
-Complementation of Flavivirus RNA Polymerase Gene NS5 by Using Kunjin Virus Replicon-Expressing BHK Cells
Authors: Khromykh Alexander A. , Kenney Mark T. , Westaway Edwin G.
Published: December 2019

A BHK cell line persistently expressing a Kunjin (KUN) virus replicon RNA (repBHK, similar to our recently described ME/76Neo BHK cell line [A. A. Khromykh and E. G. Westaway, J. Virol. 71:1497–1505, 1997]) was used for rescue and propagation of KUN viruses defective in the RNA polymerase gene (NS5). A new infectious full-length KUN virus cDNA clone, FLSDX, prepared from our previously described cDNA clone pAKUN (A. A. Khromykh and E. G. Westaway, J. Virol. 68:4580–4588, 1994) and possessing ∼10
5
-fold higher specific infectivity than that of pAKUN, was used for preparation of defective mutants. Deletions of the predicted RNA polymerase motif GDD (producing FLd
GDD
) and of one of the predicted methyltransferase motifs (
S
-adenosylmethionine [SAM] binding site, producing FLdSAM) were introduced separately into FLSDX. Transcription and transfection of FLd
GDD
and FLdSAM RNAs into repBHK cells but not into normal BHK cells resulted in their replication and the recovery of defective viruses able to replicate only in repBHK cells. Reverse transcription-PCR and sequencing analyses showed retention of the introduced deletions in the genomes of the recovered viruses. Retention of these deletions, as well as our inability to recover viruses able to replicate in normal BHK cells after prolonged incubation (for 7 days) of FLd
GDD
- or FLdSAM-transfected repBHK cells, excluded the possibility that recombination had occurred between the deleted defective NS5 genes present in transfected RNAs and the functional NS5 gene present in the repBHK cells. An RNA with a point mutation in the GDD motif (FL
GVD
) was also complemented in transfected repBHK cells, and defective virus was recovered by day 3 after transfection. However, in contrast to the results with FLd
GDD
and FLdSAM RNAs, prolonged (4 days or more) incubation of FL
GVD
RNA in normal BHK cells allowed recovery of a virus in which the
GVD
mutation had reverted via a single base change to the wild-type
GDD
sequence. Overall, these results represent the first demonstration of
trans
-complementation of defective flavivirus RNAs with deleterious deletions in the flavivirus RNA polymerase gene NS5. The complementation system described here may prove to be useful for the in vivo complementation of deletions and mutations affecting functional domains or the essential secondary structure in any of the other flavivirus nonstructural proteins.
Epstein-Barr Virus Recombinant Lacking Expression of Glycoprotein gp150 Infects B Cells Normally but Is Enhanced for Infection of Epithelial Cells
Authors: Borza Corina M. , Hutt-Fletcher Lindsey M.
Published: December 2019

Glycoprotein gp150 is a highly glycosylated protein encoded by the BDLF3 open reading frame of Epstein-Barr virus (EBV). It does not have a homolog in the alpha- and betaherpesviruses, and its function is not known. To determine whether the protein is essential for replication of EBV in vitro, a recombinant virus which lacked its expression was made. The recombinant virus had no defects in assembly, egress, binding, or infectivity for B cells or epithelial cells. Infection of epithelial cells was, however, enhanced. The glycoprotein was sensitive to digestion with a glycoprotease that digests sialomucins, but no adhesion to cells that express selectins that bind to sialomucin ligands could be detected.
Preactivation of B Lymphocytes Does Not Enhance Mouse Mammary Tumor Virus Infection
Authors: Finke Daniela , Mortezavi Laure , Acha-Orbea Hans
Published: December 2019

We investigated whether mouse mammary tumor virus (MMTV) favors preactivated or naive B cells as targets for efficient infection. We have demonstrated previously that MMTV activates B cells upon infection. Here, we show that polyclonal activation of B cells leads instead to lower infection levels and attenuated superantigen-specific T-cell responses in vivo. This indicates that naive small resting B cells are the major targets of MMTV infection and that the activation induced by MMTV is sufficient to allow efficient infection.
Persistent Hz-1 Virus Infection in Insect Cells: Evidence for Insertion of Viral DNA into Host Chromosomes and Viral Infection in a Latent Status
Authors: Lin Chi-Long , Lee Jin-Ching , Chen Shih-Shun , Alan Wood H. , Li Ming-Liang , Li Chih-Fen , Chao Yu-Chan
Published: December 2019

Persistent/latent viral infections of insect cells are a prominent though poorly understood phenomenon. In this study, the long-term association between the Hz-1 virus and insect host cells, conventionally referred to as persistent viral infection, is described. With the aid of a newly developed fluorescent cell-labeling system, we found that productive viral replication occurs by spontaneous viral reactivation in fewer than 0.2% of persistently infected cell lines over a 5-day period. Once viral reactivation takes place, the host cell dies. The persistently infected cells contain various amounts of viral DNA, and, in an extreme case, up to 16% of the total DNA isolated from infected cells could be of viral origin. Both pulsed-field gel electrophoresis and in situ hybridization experiments showed that some of these viral DNA molecules are inserted into the host chromosomes but that the rest of viral DNA copies are free from host chromosomes. Thus, Hz-1 virus is the first nonretroviral insect virus known to insert its genome into the host chromosome during the infection process. These data also suggest that the previously described persistent infection of Hz-1 virus in insect cells should be more accurately referred to as latent viral infection.
Microglia Express CCR5, CXCR4, and CCR3, but of These, CCR5 Is the Principal Coreceptor for Human Immunodeficiency Virus Type 1 Dementia Isolates
Authors: Albright Andrew V. , Shieh Joseph T. C. , Itoh Takayuki , Lee Benhur , Pleasure David , O’Connor Michael J. , Doms Robert W. , González-Scarano Francisco
Published: December 2019

Microglia are the main human immunodeficiency virus (HIV) reservoir in the central nervous system and most likely play a major role in the development of HIV dementia (HIVD). To characterize human adult microglial chemokine receptors, we analyzed the expression and calcium signaling of CCR5, CCR3, and CXCR4 and their roles in HIV entry. Microglia expressed higher levels of CCR5 than of either CCR3 or CXCR4. Of these three chemokine receptors, only CCR5 and CXCR4 were able to transduce a signal in microglia in response to their respective ligands, MIP-1β and SDF-1α, as recorded by single-cell calcium flux experiments. We also found that CCR5 is the predominant coreceptor used for infection of human adult microglia by the HIV type 1 dementia isolates HIV-1DS-br, HIV-1RC-br, and HIV-1YU-2, since the anti-CCR5 antibody 2D7 was able to dramatically inhibit microglial infection by both wild-type and single-round luciferase pseudotype reporter viruses. Anti-CCR3 (7B11) and anti-CXCR4 (12G5) antibodies had little or no effect on infection. Last, we found that virus pseudotyped with the DS-br and RC-br envelopes can infect cells transfected with CD4 in conjunction with the G-protein-coupled receptors APJ, CCR8, and GPR15, which have been previously implicated in HIV entry.
Dynamics of Human Immunodeficiency Virus Type 1 Replication in Vertically Infected Infants
Authors: Luzuriaga Katherine , Wu Hulin , McManus Margaret , Britto Paula , Borkowsky William , Burchett Sandra , Smith Betsy , Mofenson Lynne , Sullivan John L. ,   the PACTG 356 Investigators
Published: December 2019

Plasma human immunodeficiency virus type 1 (HIV-1) turnover and kinetics were studied in children aged 15 days to 2 years following the initiation of a triple antiretroviral drug regimen consisting of zidovudine, lamivudine, and nevirapine. HIV-1 turnover was at least as rapid as that previously described in adults; turnover rates were more rapid in infants and children aged 3 months to 2 years than in infants less than 3 months of age. These data confirm the central role of HIV-1 replication in the pathogenesis of vertical HIV-1 infection and reinforce the importance of early, potent combination therapies for the long-term control of HIV-1 replication.
Targeting of the Visna Virus Tat Protein to AP-1 Sites: Interactions with the bZIP Domains of Fos and Jun In Vitro and In Vivo
Authors: Morse B. A. , Carruth L. M. , Clements J. E.
Published: December 2019

The visna virus Tat protein is required for efficient viral transcription from the visna virus long terminal repeat (LTR). AP-1 sites within the visna virus LTR, which can be bound by the cellular transcription factors Fos and Jun, are also necessary for Tat-mediated transcriptional activation. A potential mechanism by which the visna virus Tat protein could target the viral promoter is by protein-protein interactions with Fos and/or Jun bound to AP-1 sites in the visna virus LTR. Once targeted to the visna virus promoter, the Tat protein could then interact with basal transcription factors to activate transcription. To examine protein-protein interactions with cellular proteins at the visna virus promoter, we used an in vitro protein affinity chromatography assay and electrophoretic mobility shift assay, in addition to an in vivo two-hybrid assay, to show that the visna virus Tat protein specifically interacts with the cellular transcription factors Fos and Jun and the basal transcription factor TBP (TATA binding protein). The Tat domain responsible for interactions with Fos and Jun was localized to an alpha-helical domain within amino acids 34 to 69 of the protein. The TBP binding domain was localized to amino acids 1 to 38 of Tat, a region previously described by our laboratory as the visna virus Tat activation domain. The bZIP domains of Fos and Jun were found to be important for the interactions with Tat. Mutations within the basic domains of Fos and Jun abrogated binding to Tat in the in vitro assays. The visna virus Tat protein was also able to interact with covalently cross-linked Fos and Jun dimers. Thus, the visna virus Tat protein appears to target AP-1 sites in the viral promoter in a mechanism similar to the interaction of human T-cell leukemia virus type 1 Tax with the cellular transcription factor CREB, by binding the basic domains of an intact bZIP dimer. The association between Tat, Fos, and Jun would position Tat proximal to the viral TATA box, where the visna virus Tat activation domain could contact TBP to activate viral transcription.
Strong Human Immunodeficiency Virus (HIV)-Specific Cytotoxic T-Lymphocyte Activity in Sydney Blood Bank Cohort Patients Infected withnef-Defective HIV Type 1
Authors: Dyer Wayne B. , Ogg Graham S. , Demoitie Marie-Ange , Jin Xia , Geczy Andrew F. , Rowland-Jones Sarah L. , McMichael Andrew J. , Nixon Douglas F. , Sullivan John S.
Published: December 2019

Proposals for the use of live attenuated human immunodeficiency virus (HIV) type 1 (HIV-1) as a vaccine candidate in humans have been based on the protection afforded by attenuated simian immunodeficiency virus in the macaque model. Although it is not yet known if this strategy could succeed in humans, a study of the Sydney Blood Bank Cohort (SBBC), infected with an attenuated HIV-1 quasispecies with naturalnefandnef/long terminal repeat deletions for up to 17 years, could provide insights into the long-term immunological consequences of living with an attenuated HIV-1 infection. In this study, HIV-specific cytoxic T-lymphocyte (CTL) responses in an SBBC donor and six recipients were examined over a 3-year period with enzyme-linked immunospot, tetrameric complex binding, direct CTL lysis, and CTL precursor level techniques. Strong HIV-specific CTL responses were detected in four of seven patients, including one patient with an undetectable viral load. Two of seven patients had weak CTL responses, and in one recipient, no HIV-specific CTLs were detected. High levels of circulating effector and memory HIV-specific CTLs can be maintained for prolonged periods in these patients despite very low viral loads.
Enhancement of Human Immunodeficiency Virus Type 1 Infection by the CC-Chemokine RANTES Is Independent of the Mechanism of Virus-Cell Fusion
Authors: Gordon Cynthia J. , Muesing Mark A. , Proudfoot Amanda E. I. , Power Christine A. , Moore John P. , Trkola Alexandra
Published: December 2019

We have studied the effects of CC-chemokines on human immunodeficiency virus type 1 (HIV-1) infection, focusing on the infectivity enhancement caused by RANTES. High RANTES concentrations increase the infectivity of HIV-1 isolates that use CXC-chemokine receptor 4 for entry. However, RANTES can have a similar enhancing effect on macrophagetropic viruses that enter via CC-chemokine receptor 5 (CCR5), despite binding to the same receptor as the virus. Furthermore, RANTES enhances the infectivity of HIV-1 pseudotyped with the envelope glycoprotein of murine leukemia virus or vesicular stomatitis virus, showing that the mechanism of enhancement is independent of the route of virus-cell fusion. The enhancing effects of RANTES are not mediated via CCR5 or other known chemokine receptors and are not mimicked by MIP-1α or MIP-1β. The N-terminally modified derivative aminooxypentane RANTES (AOP-RANTES) efficiently inhibits HIV-1 infection via CCR5 but otherwise mimics RANTES by enhancing viral infectivity. There are two mechanisms of enhancement: one apparent when target cells are pretreated with RANTES (or AOP-RANTES) for several hours, and the other apparent when RANTES (or AOP-RANTES) is added during virus-cell absorption. We believe that the first mechanism is related to cellular activation by RANTES, whereas the second is an increase in virion attachment to target cells.
Adaptation of a CCR5-Using, Primary Human Immunodeficiency Virus Type 1 Isolate for CD4-Independent Replication
Authors: Kolchinsky Peter , Mirzabekov Tajib , Farzan Michael , Kiprilov Enko , Cayabyab Mark , Mooney Larissa J. , Choe Hyeryun , Sodroski Joseph
Published: December 2019

The gp120 envelope glycoprotein of the human immunodeficiency virus type 1 (HIV-1) promotes virus entry by sequentially binding CD4 and chemokine receptors on the target cell. Primary, clinical HIV-1 isolates require interaction with CD4 to allow gp120 to bind the CCR5 chemokine receptor efficiently. We adapted a primary HIV-1 isolate, ADA, to replicate in CD4-negative canine cells expressing human CCR5. The gp120 changes responsible for the adaptation were limited to alteration of glycosylation addition sites in the V2 loop–V1-V2 stem. The gp120 glycoproteins of the adapted viruses bound CCR5 directly, without prior interaction with CD4. Thus, a major function of CD4 binding in the entry of primary HIV-1 isolates can be bypassed by changes in the gp120 V1-V2 elements, which allow the envelope glycoproteins to assume a conformation competent for CCR5 binding.
trans-Dominant Interference with Human Immunodeficiency Virus Type 1 Replication and Transmission in CD4+Cells by an Envelope Double Mutant
Authors: Chen Steve S.-L. , Lee Sheau-Fen , Chuang Chin-Kai , Raj V. Samuel
Published: December 2019

We previously reported that a human immunodeficiency virus type 1 (HIV-1) envelope (Env) mutant with the whole cytoplasmic domain deleted, denoted mutant TC, is able to dominantly interfere with wild-type (wt) virus infectivity. In the present study, the feasibility of developing a dominant negative mutant-based genetic anti-HIV strategy targeting the gp41 cytoplasmic domain was investigated. Mutants TC and 427,TC, a TC derivative with a Trp-to-Ser substitution introduced into residue 427 in the CD4-binding site, and a series of mutants with deletions in the cytoplasmic domain, effectivelytrans-dominantly interfered with wt Env-mediated viral infectivity, as demonstrated by anenv trans-complementation assay. The syncytium formation-defective 427,TC double mutant not only inhibited heterologous LAV and ELI Env-mediated viral infectivity but also interfered with syncytium formation and infectivity mediated by the Env proteins of the two primary isolates 92BR and 92US. Stable HeLa-CD4-LTR-β-gal clones that harbored Tat-controlled expression cassettes encoding the control ΔKS, which had a deletion in theenvgene, wt, or mutantenvgene were generated. Viral transmission mediated by laboratory-adapted T-cell-tropic HXB2 and NL4-3 viruses was greatly reduced in the TC and 427,TC transfectants compared to that observed in the control ΔKS and wt transfectants. Viral replication caused by HXB2 and NL4-3 viruses and by macrophage-tropic ConB and ADA-GG viruses was delayed or reduced in human CD4+T cells transfected with the 427,TCenvconstruct compared to that observed in cells transfected with the control ΔKS or TCenvconstruct. The lack of significant interference by TC mutant was due neither to the lack of TCenvgene integration into host DNA nor to the lack of TC Env expression upon Tat induction. These results indicate that this 427,TC Env double mutant has a role in the development oftrans-dominant mutant-based genetic anti-HIV strategies.
Identification of an Internal Ribosome Entry Segment in the 5′ Region of the Mouse VL30 Retrotransposon and Its Use in the Development of Retroviral Vectors
Authors: López-Lastra Marcelo , Ulrici Sandrine , Gabus Caroline , Darlix Jean-Luc
Published: December 2019

Mouse virus-like 30S RNAs (VL30m) constitute a family of retrotransposons, present at 100 to 200 copies, dispersed in the mouse genome. They display little sequence homology to Moloney murine leukemia virus (MoMLV), do not encode virus-like proteins, and have not been implicated in retroviral carcinogenesis. However, VL30 RNAs are efficiently packaged into MLV particles that are propagated in cell culture. In this study, we addressed whether the 5′ region of VL30m could replace the 5′ leader of MoMLV functionally in a recombinant vector construct. Our data confirm that the putative packaging sequence of VL30 is located within the 5′ region (nucleotides 362 to 1149 with respect to the cap structure) and that it can replace the packaging sequence of MoMLV. We also show that VL30m contains an internal ribosome entry segment (IRES) in the 5′ region, as do MoMLV, Friend murine leukemia virus, Harvey murine sarcoma virus, and avian reticuloendotheliosis virus type A. Our data show that both the packaging and IRES functions of the 5′ region of VL30m RNA can be efficiently used to develop retrotransposon-based vectors.
Kinetics of the Development of Protective Immunity in Mice Vaccinated with a Live Attenuated Retrovirus
Authors: Dittmer Ulf , Race Brent , Hasenkrug Kim J.
Published: December 2019

Vaccination of mice with a live attenuated vaccine virus induces potent protection against subsequent challenge with pathogenic Friend retroviral complex. The kinetic studies presented here demonstrate protection from acute splenomegaly as early as 1 week postvaccination. At this time point virus-specific cytotoxic T lymphocytes (CTL) were demonstrable in direct chromium release assays. However, during the first 2 weeks after vaccination protection was incomplete since the mice were not protected against establishment of low-level persistent infections in the spleen. By 3 weeks postvaccination the animals were protected against the establishment of persistent virus as well as acute splenomegaly. The timing of this complete protection correlated with the presence of both virus-neutralizing antibodies and primed CTL in the immunized mice. Within 3 days of virus challenge, vaccinated mice showed high levels of activated B cells and CD4
+
and CD8
+
T cells, indicating an efficient priming of all lymphocyte subsets. Despite very limited replication of the vaccine virus, the protective effect was long lived and was still present 6 months after immunization.
Expression of Hepatitis C Virus Proteins Inhibits Signal Transduction through the Jak-STAT Pathway
Authors: Heim Markus H. , Moradpour Darius , Blum Hubert E.
Published: December 2019

Hepatitis C virus (HCV) infection is a leading cause of liver disease worldwide. Alpha interferon (IFN-α) therapy of chronic hepatitis C leads to a sustained response in 10 to 20% of patients only. The mechanisms of viral persistence and the pathogenesis of hepatitis C are poorly understood. We established continuous human cell lines, allowing the tightly regulated expression of the entire HCV open reading frame under the control of a tetracycline-responsive promoter. Using this in vitro system, we analyzed the effect of HCV proteins on IFN-induced intracellular signaling. Expression of HCV proteins in these cells strongly inhibited IFN-α-induced signal transduction through the Jak-STAT pathway. Inhibition occurred downstream of STAT tyrosine phosphorylation. Inhibition of the Jak-STAT pathway was not restricted to IFN-α-induced signaling but was observed in leukemia inhibitory factor-induced signaling through Stat3 as well. By contrast, tumor necrosis factor alpha-induced activation of the transcription factor NF-κB was not affected. Interference of HCV with IFN-α-induced signaling through the Jak-STAT pathway could contribute to the resistance to IFN-α therapy observed in the majority of patients and may represent a general escape strategy of HCV contributing to viral persistence and pathogenesis of chronic liver disease.
Transcription Originating in the Long Terminal Repeats of the Endogenous Mouse Mammary Tumor Virus MTV-3 Is Activated in Stat5a-Null Mice and Picks Up Hitchhiking Exons
Authors: Stegalkina Svetlana S. , Guerrero Annamaria , Walton Katherine D. , Liu Xiuwen , Robinson Gertraud W. , Hennighausen Lothar
Published: December 2019

The enhancer within the long terminal repeats (LTRs) of acquired somatic mouse mammary tumor viruses (MMTV) can activate juxtaposed genes and induce mammary tumors. In contrast, germ line proviral MMTV genomes are integrated in the host genome and considered to be genetically confined transcription units. Here we demonstrate that transcription initiated in an MMTV provirus proceeds into flanking host sequences. We discovered multiple polyadenylated transcripts which are induced in Stat5a null mice. These range from 1.5 kb to more than 8 kb and are specifically expressed in mammary tissue from pregnant and lactating mice from the 129 but not C57BL/6 strain. The RNAs emanate from both LTRs of the endogenous MTV-3 provirus on chromosome 11 and proceed at least 10 kb into the juxtaposed genomic territory. Transcripts originating in the 5′ LTR splice from the native splice site within the MMTV envelope gene into at least six exons, three of which contain functional internal splice sites. The combination of alternative splicing and the use of several polyadenylation sites ensure the generation of multiple transcripts. To date no significant open reading frame has been discovered. Furthermore, we demonstrate that transcription from the MMTV 5′ LTR is highly active in the absence of Stat5a, a transcription factor that had been shown previously to be required for transcription from the MMTV LTR.
Involvement of the Cytoplasmic Domain of the Hemagglutinin-Neuraminidase Protein in Assembly of the Paramyxovirus Simian Virus 5
Authors: Schmitt Anthony P. , He Biao , Lamb Robert A.
Published: December 2019

Efficient assembly of enveloped viruses at the plasma membranes of virus-infected cells requires coordination between cytosolic viral components and viral integral membrane glycoproteins. As viral glycoprotein cytoplasmic domains may play a role in this coordination, we have investigated the importance of the hemagglutinin-neuraminidase (HN) protein cytoplasmic domain in the assembly of the nonsegmented negative-strand RNA paramyxovirus simian virus 5 (SV5). By using reverse genetics, recombinant viruses which contain HN with truncated cytoplasmic tails were generated. These viruses were shown to be replication impaired, as judged by small plaque size, reduced replication rate, and low maximum titers when compared to those features of wild-type (wt) SV5. Release of progeny virus particles from cells infected with HN cytoplasmic-tail-truncated viruses was inefficient compared to that of wt virus, but syncytium formation was enhanced. Furthermore, accumulation of viral proteins at presumptive budding sites on the plasma membranes of infected cells was prevented by HN cytoplasmic tail truncations. We interpret these data to indicate that formation of budding complexes, from which efficient release of SV5 particles can occur, depends on the presence of an HN cytoplasmic tail.
Efficacy of Antiretroviral Agents against Murine Replication-Competent Retrovirus Infection in Human Cells
Authors: Powell Sharon K. , Artlip Moria , Kaloss Michele , Brazinski Scott , Lyons Russette , McGarrity Gerard J. , Otto Edward
Published: December 2019

Retroviral vectors for gene therapy are designed to minimize the occurrence of replication-competent retrovirus (RCR); nonetheless, it is possible that a vector-derived RCR could establish an infection in a patient. Since the efficacy of antiretroviral agents can be impacted by interactions between virus, host cell, and drug, five commonly used antiretroviral drugs were evaluated for their abilities to inhibit the replication of a murine leukemia virus (MLV)-derived RCR in human cells. The results obtained indicate that the combination of nucleoside analogs zidovudine and dideoxyinosine with the protease inhibitor indinavir effectively inhibits MLV-derived RCR replication in three human cell lines. In addition, MLV-derived RCR was found to be inherently resistant to the nucleoside analogs lamivudine and stavudine, suggesting that mutations conferring resistance to nucleoside analogs in human immunodeficiency virus type 1 have the same effect even in an alternative viral backbone.
Poliovirus Mutants at Histidine 195 of VP2 Do Not Cleave VP0 into VP2 and VP4
Authors: Hindiyeh Musa , Li Qi-Han , Basavappa Ravi , Hogle James M. , Chow Marie
Published: December 2019

The final stage of poliovirus assembly is characterized by a cleavage of the capsid precursor protein VP0 into VP2 and VP4. This cleavage is thought to be autocatalytic and dependent on RNA encapsidation. Analysis of the poliovirus empty capsid structure has led to a mechanistic model for VP0 cleavage involving a conserved histidine residue that is present in the surrounding environment of the VP0 cleavage site. Histidine 195 of VP2 (2195H) is hypothesized to activate local water molecules, thus initiating a nucleophilic attack at the scissile bond. To test this hypothesis, 2195H mutants were constructed and their phenotypes were characterized. Consistent with the requirement of VP0 cleavage for poliovirus infectivity, all 2195H mutants were nonviable upon introduction of the mutant genomes into HeLa cells. Replacement of 2195H with threonine or arginine resulted in the assembly of a highly unstable 150S virus particle. Further analyses showed that these particles contain genomic RNA and uncleaved VP0, criteria associated with the provirion assembly intermediate. These data support the involvement of 2195H in mediating VP0 cleavage during the final stages of virus assembly.
Establishment and Characterization of Cytopathogenic and Noncytopathogenic Pestivirus Replicons
Authors: Tautz N. , Harada T. , Kaiser A. , Rinck G. , Behrens S.-E. , Thiel H.-J.
Published: December 2019

Defective interfering particles (DIs) of bovine viral diarrhea virus (BVDV) have been identified and shown to be cytopathogenic (cp) in the presence of noncytopathogenic (noncp) helper virus. Moreover, a subgenomic (sg) RNA corresponding in its genome structure to one of those BVDV DIs (DI9) was replication competent in the absence of helper virus. We report here that an sg BVDV replicon which encodes from the viral proteins only the first three amino acids of the autoprotease Nproin addition to nonstructural (NS) proteins NS3 to NS5B replicates autonomously and also induces lysis of its host cells. This demonstrates that the presence of a helper virus is not required for the lysis of the host cell. On the basis of two infectious BVDV cDNA clones, namely, BVDV CP7 (cp) and CP7ins− (noncp), bicistronic replicons expressing proteins NS2-3 to NS5B were established. These replicons express, in addition to the viral proteins, the reporter gene encoding β-glucuronidase; the release of this enzyme from transfected culture cells was used to monitor cell lysis. Applying these tools, we were able to show that the replicon derived from CP7ins− does not induce cell lysis. Accordingly, neither Npronor any of the structural proteins are necessary to maintain the noncp phenotype. Furthermore, these sg RNAs represent the first pair of cp and noncp replicons which mimic complete BVDV CP7 and CP7ins− with respect to cytopathogenicity. These replicons will facilitate future studies aimed at the determination of the molecular basis for the cytopathogenicity of BVDV.
Effects of Targeting Herpes Simplex Virus Type 1 gD to the Endoplasmic Reticulum and
trans
-Golgi Network
Authors: Whiteley Alison , Bruun Birgitte , Minson Tony , Browne Helena
Published: December 2019

Glycoprotein D (gD) of herpes simplex virus type 1 (HSV-1) was modified to encode targeting signals known to localize proteins to either the endoplasmic reticulum (ER) or the
trans
-Golgi network. These motifs conferred the predicted targeting properties on gD in transfected cells as judged by immunofluorescence staining, and the exclusion of targeted gD from the cell surface was confirmed by the fact that these molecules exhibited substantially reduced activity in cell-cell fusion assays. Recombinant viruses expressing Golgi-targeted forms of gD grew to wild-type levels in noncomplementing cells, exhibited unaltered particle/infectivity ratios, and were found to contain wild-type levels of gD, whereas a recombinant expressing ER-retained gD was helper cell dependent and, when grown on noncomplementing cells, produced virions of low specific infectivity with greatly reduced levels of gD. These data imply that HSV-1 acquires its final membrane from a post-ER compartment and lend support to the view that the virus undergoes de-envelopment and reenvelopment steps during virus egress.
A Lentivirus Packaging System Based on Alternative RNA Transport Mechanisms To Express Helper and Gene Transfer Vector RNAs and Its Use To Study the Requirement of Accessory Proteins for Particle Formation and Gene Delivery
Authors: Srinivasakumar Narasimhachar , Schuening Friedrich G.
Published: December 2019

A lentivirus-based packaging system was designed to reduce the chance of recombination between helper and gene transfer vector sequences by using the constitutive transport element (CTE) derived from Mason-Pfizer monkey virus for expression of the viral proteins and the Rev-Rev response element (RRE) combination for expression of the gene transfer vector. Using this approach, we evaluated a series of human immunodeficiency virus type 1 packaging constructs that express one or more accessory proteins (Vif, Vpr, and Vpu), in addition to the Gag and Pol proteins, for particle formation and virus stock production for gene transfer. Constructs that also express Vpr or both Vpr and Vpu produced more particles, as measured by a p24 assay, than did plasmids that did not contain these sequences. Transactivation experiments showed that the packaging plasmids that encode Vpr or both Vpr and Vpu also expressed a functional single-exon Tat protein. For these constructs, high-titer virus stocks could be prepared in the absence of a cotransfected Tat-expressing plasmid. Amphotropic-envelope-pseudotyped virus stocks prepared with all of the packaging constructs, irrespective of whether any of the accessory proteins were coexpressed, were equally efficient in transducing growth-arrested HeLa cells. The combination/mixed packaging system was compared to systems that were based on either the CTE alone or Rev and RRE for expression of both the packaging plasmid as well as the gene transfer vector. The combination/mixed packaging system was comparable to the other systems for production of virus stocks, suggesting that this design may prove to be safer for the eventual deployment of lentivirus vectors for therapeutic purposes.
Overexpression of Cyclin A Inhibits Augmentation of Recombinant Adeno-Associated Virus Transduction by the Adenovirus E4orf6 Protein
Authors: Grifman Mirta , Chen Nancie N. , Gao Guang-ping , Cathomen Toni , Wilson James M. , Weitzman Matthew D.
Published: December 2019

The 34-kDa product of adenovirus E4 region open reading frame 6 (E4orf6) dramatically enhances transduction by recombinant adeno-associated virus vectors (rAAV). This is achieved by promoting the conversion of incoming single-stranded viral genomes into transcriptionally competent duplex molecules. The molecular mechanism for enhancing second-strand synthesis is not fully understood. In this study, we analyzed the cellular consequences of E4orf6 expression and the requirements for efficient rAAV transduction mediated by E4orf6. Expression of E4orf6 in 293 cells led to an inhibition of cell cycle progression and an accumulation of cells in S phase. This was preceded by specific degradation of cyclin A and p53, while the levels of other proteins involved in cell cycle control remained unchanged. In addition, the kinase activity of cdc2 was inhibited. We further showed that p53 expression is not necessary or inhibitory for augmentation of rAAV transduction by E4orf6. However, overexpression of cyclin A inhibited E4orf6-mediated enhancement of rAAV transduction. A cyclin A mutant incapable of recruiting protein substrates for cdk2 was unable to inhibit E4orf6-mediated augmentation. In addition, we created an E4orf6 mutant that is selectively defective in rAAV augmentation of transduction. Based on these findings, we suggest that cyclin A degradation represents a viral mechanism to disrupt cell cycle progression, resulting in enhanced viral transduction. Understanding the cellular pathways used during transduction will increase the utility of rAAV vectors in a wide range of gene therapy applications.
Adenoviruses Activate Human Dendritic Cells without Polarization toward a T-Helper Type 1-Inducing Subset
Authors: Rea Delphine , Schagen Frederik H. E. , Hoeben Rob C. , Mehtali Majid , Havenga Menzo J. E. , Toes Rene E. M. , Melief Cornelis J. M. , Offringa Rienk
Published: December 2019

Human monocyte-derived dendritic cells (DC) infected with recombinant adenoviruses (rAd) are promising candidate vaccines for inducing protective immunity against pathogens and tumors. However, since some viruses are known to negatively affect DC function, it is important to investigate the interactions between rAd and DC. We now show that infection by rAd enhances the immunostimulatory capacity of immature human monocyte-derived DC through the upregulation of the costimulatory molecules CD80, CD86, and CD40 and the major histocompatibility complex class I and II molecules. Although rAd infection fails to induce the secretion of interleukin-12 (IL-12) and only marginally induces the expression of the DC maturation marker CD83, it acts in synergy with CD40 triggering in rendering DC fully mature. rAd-infected DC triggered through CD40 produce more IL-12 and are more efficient in eliciting T-helper type 1 responses than DC activated by CD40 triggering only. rAd lacking one or more of the early regions, E1, E2A, E3, and E4, which play an important role in virus-host cell interactions are equally capable of DC activation. Efficient DC infection requires a high multiplicity of infection (>1,000), a fact which can be attributed to the absence of the coxsackievirus and adenovirus receptor on this cell type. Despite the poor ability of DC to be infected by rAd, which may be improved by targeting rAd to alternative DC surface molecules, DC infected with all currently tested rAd constitute potent immunostimulators. Our study provides new insights into the interactions between two highly promising vaccine components, rAd and DC, and indicates that their combination into one vaccine may be very advantageous for the stimulation of T-cell immunity.
Use of a gp120 Binding Assay To Dissect the Requirements and Kinetics of Human Immunodeficiency Virus Fusion Events
Authors: Doranz Benjamin J. , Baik Sarah S. W. , Doms Robert W.
Published: December 2019

Binding of the extracellular subunit of human immunodeficiency type 1 (HIV-1) envelope (Env) glycoprotein (gp120) to CD4 triggers the induction or exposure of a highly conserved coreceptor binding site in gp120 that helps mediate membrane fusion. Characterizing the structural features involved in gp120-coreceptor binding and the conditions under which binding occurs is important for understanding the fusion process, the evolution of pathogenic strains in vivo, the identification of novel anti-HIV compounds, and the development of HIV vaccines that utilize triggered structures of Env. Here we use the kinetics of interaction between CCR5 and gp120 to understand temporal and structural changes that occur during viral fusion. Using saturation binding and homologous competition analysis, we estimated the

K
d

of interaction between CCR5 and gp120 from the macrophage tropic HIV-1 strain JRFL to be 4 nM. Unlike Env-mediated fusion, gp120 binding to CCR5 did not require divalent cations or elevated temperatures. Binding was not significantly affected by the pH of binding, G-protein coupling of CCR5, or partial gp120 deglycosylation. Oligomeric, uncleaved JRFL gp140 failed to bind CCR5 despite its ability to bind CD4 and monoclonal antibody 17b, suggesting that the uncleaved ectodomain of gp41 interferes with full exposure of the chemokine receptor binding site. Exposure of the chemokine receptor binding site on gp120 could be induced rapidly by CD4, but exposure of this site was lost upon CD4 dissociation from gp120, indicating that the conformational changes in gp120 induced by CD4 binding are fully reversible. The functional gp120-soluble CD4 complex was remarkably stable over time and temperature ranges, offering the possibility that complexes in which the highly conserved coreceptor binding site in gp120 is exposed can be used for vaccine development.
Consistent Viral Evolutionary Changes Associated with the Progression of Human Immunodeficiency Virus Type 1 Infection
Authors: Shankarappa Raj , Margolick Joseph B. , Gange Stephen J. , Rodrigo Allen G. , Upchurch David , Farzadegan Homayoon , Gupta Phalguni , Rinaldo Charles R. , Learn Gerald H. , He Xi , Huang Xiao-Li , Mullins James I.
Published: December 2019

To understand the high variability of the asymptomatic interval between primary human immunodeficiency virus type 1 (HIV-1) infection and the development of AIDS, we studied the evolution of the C2-V5 region of the HIV-1envgene and of T-cell subsets in nine men with a moderate or slow rate of disease progression. They were monitored from the time of seroconversion for a period of 6 to 12 years until the development of advanced disease in seven men. Based on the analysis of viral divergence from the founder strain, viral population diversity within sequential time points, and the outgrowth of viruses capable of utilizing the CXCR4 receptor (X4 viruses), the existence of three distinct phases within the asymptomatic interval is suggested: an early phase of variable duration during which linear increases (∼1% per year) in both divergence and diversity were observed; an intermediate phase lasting an average of 1.8 years, characterized by a continued increase in divergence but with stabilization or decline in diversity; and a late phase characterized by a slowdown or stabilization of divergence and continued stability or decline in diversity. X4 variants emerged around the time of the early- to intermediate-phase transition and then achieved peak representation and began a decline around the transition between the intermediate and late phases. The late-phase transition was also associated with failure of T-cell homeostasis (defined by a downward inflection in CD3+T cells) and decline of CD4+T cells to ≤200 cells/μl. The strength of these temporal associations between viral divergence and diversity, viral coreceptor specificity, and T-cell homeostasis and subset composition supports the concept that the phases described represent a consistent pattern of viral evolution during the course of HIV-1 infection in moderate progressors. Recognition of this pattern may help explain previous conflicting data on the relationship between viral evolution and disease progression and may provide a useful framework for evaluating immune damage and recovery in untreated and treated HIV-1 infections.