an alternative protocol, we substituted trypsin dispersion of an infected monolayer, followed by washing to remove the trypsin. When we subsequently sonicated the pellet of trypsin-dis- persed cells, we obtained little or no infectious cell-free virus ( ⬍ 100 PFU/25 sq cm). Our results confirmed an earlier, similar result reported by Brunell (2). From these results, we surmised that cell-free virus consisted of virus that had entered the outer cell membrane, only to be separated from the membrane by sonication. Trypsin treatment presumably removed or de- stroyed this virus. We had previously demonstrated that infec- tious virus is easily disassociated from an infectedcell mono- layer. Based on these cumulative observations, we decided to enumerate the viral particles detectable in the outer cell mem- brane of infected cells (Fig. 1). We postulated that by counting * Corresponding author. Mailing address: University of Iowa Hos-
Characterization of a new BDV variant from persistently infected cells. Based on the hypothesis that a certain percent- age of cells within a population of BDV-infected cells may harbor virus variants in addition to wild-type virus, we specu- lated that cocultivation of uninfected cells with a single BDV- infectedcell might allow the characterization of such variants. To test this hypothesis, we seeded uninfected C6 cells onto a single He/80-infected C6 cell to allow the spread of virus to neighboring cells. Monitoring of infection by indirect IFA in- dicated that after 3 to 4 weeks of passaging, 50 to 90% of the cells were infected with BDV (data not shown). Subsequent RPA screening using total RNA of 30 independent infected C6 cell cultures revealed that one culture contained a BDV vari- ant (designated 2/1), as indicated by a dramatic change in the signal pattern (Fig. 2A, lane 5). To confirm this observation we determined the nucleotide sequence of the variant by sequenc- ing nested RT-PCR amplification products corresponding to the M-G coding sequence (nt 1975 to 2510). Two nucleotide changes were identified in 2/1 at positions 2433 and 2435 com- pared to He/80 (Fig. 2B), one of which resulted in a noncon- servative change of isoleucine to threonine at position 67 of the G protein.
TNFR superfamily (34). We showed their ability to impair both specific signaling pathways and phenotypes induced by TNF and LMP1 in epithelial and endothelial cells. Here, to investigate the potential role of our DNs in EBV-infectedcell lines, we analyzed lymphocytic (NC5) (11) and monocytic (TE1) (27) cell lines in- fected and transformed by EBV. We used doxycycline (Dox)- inducible vectors (pRT1) (2) to conditionally express the short versions of the dominant negative LMP1-TES1S and LMP1- TES2S. The resulting constructs, pRT1-LMP1-TES1S and pRT1- LMP1-TES2S, and the control vector pRT1-GFP (Fig. 1A) express proteins that are, in contrast to wt LMP1, monomeric and cyto- plasmic. These constructs were stably transfected into the TE1 and NC5 cell lines. To check the inducibility of LMP1-DN constructs with Dox, stable NC5 cells were incubated with or without 2 g/ml Dox for 24 h, and then cell lysates were analyzed by Western blotting as shown in Fig. 1B. To evaluate the dose responsiveness of LMP1-DN expression, we incubated NC5 cells containing LMP1-DNs or GFP with increasing doses of Dox (0, 0.2, 0.4, 1, 2, and 4 g/ml). Twenty-four hours after Dox administration, cells were analyzed by flow cytometry. The results (expressed as the fold increase in the mean fluorescence intensity [MFI] for each condition over the MFI for control nontransfected cells) are re- ported in histograms (Fig. 1C). Except at 0.2 g/ml Dox, the in- ducibilities of LMP1-TES1 and LMP1-TES2 were similar. To check the ability of LMP1-DNs to bind to LMP1 adapters in our models (NC5 and TE1), we performed DN immunoprecipitation (using an anti-GFP antibody) followed by immunoblot analysis of adapters. The data indicate that LMP1-DNs bind adapters in both TE1 and NC5 cells, in which LMP1 is constitutively expressed and activated (11, 27). LMP1-TES1S binds TRAF2, whereas LMP1- TES2 binds TRADD and, to a lesser extent, TRAF2 in NC5 and TE1 cells (Fig. 1D; also data not shown). In addition, immunopre- cipitation of wt LMP1 in the presence of LMP1-DNs showed that wt LMP1 binds TRAF2 and TRADD (Fig. 1E, left, lane GFP) and that LMP1-DNs are able to impair these interactions. LMP1- TES1S reduces the binding of TRAF2 to wt LMP1, whereas LMP1- TES2S reduces TRADD and TRAF2 binding (Fig. 1E for NC5 cells; data not shown for TE1 cells). Similar results were obtained in LCLs. In these cells, immunoprecipitation of wt LMP1 in the pres-
The rapid decay of the viral load after drug treatment in patients infected with human immunodeficiency virus type 1 (HIV-1) has been shown to result from the rapid loss of infected cells due to their high turnover, with a generation time of around 1 to 2 days. Traditionally, viral decay dynamics after drug treatment is investigated using models of differential equations in which both the death rate of infected cells and the viral production rate are assumed to be constant. Here, we describe age-structured models of the viral decay dynamics in which viral production rates and death rates depend on the age of the infected cells. In order to investigate the effects of age-dependent rates, we compared these models with earlier descriptions of the viral load decay and fitted them to previously published data. We have found no supporting evidence that infected- cell death rates increase, but cannot reject the possibility that viral production rates increase, with the age of the cells. In particular, we demonstrate that an exponential increase in viral production with infected-cell age is perfectly consistent with the data. Since an exponential increase in virus production can compensate for the exponential loss of infected cells, the death rates of HIV-1-infected cells may be higher than previously anticipated. We discuss the implications of these findings for the life span of infected cells, the viral generation time, and the basic reproductive number, R 0 .
Herpes simplex virus 1 (HSV-1) has evolved numerous mechanisms to evade or counteract innate and specific host immune responses (reviewed in references 23, 28, and 51). These strategies promote escape from phagocytes, comple- ment, NK cells, and cytotoxic T cells (CTL). In particular, CTL have been shown to be crucial for control of both productive and latent virus infections, and this is mirrored in the plethora of mechanisms which members of the Herpesviridae family have adopted to evade major histocompatibility complex (MHC) class I-mediated immune responses (39). HSV-1 and HSV-2 encode at least one protein, the infectedcell protein (ICP) 47, which is dedicated to the evasion of CTL-mediated responses to infection (53). ICP47 disrupts normal peptide loading of MHC class I molecules by disabling peptide trans- port mediated by TAP1/TAP2 (13, 19). However, the extent to which HSV-1 can interfere directly with the MHC class II antigen presentation pathway is not known, despite the role of CD4 ⫹ T cells in coordinating immune responses and in limit-
lates. A common feature of the Deltaretrovirus genus is the use of proline tRNA as a primer for the complemen- tary minus-strand DNA synthesis. tRNA genes are highly conserved between different species . Alignment of the collected sequences showed a very high degree of con- servation of this functionally important region. Addition- ally, a second highly conserved region was identified approximately 1.8 kb 3' of the tRNA binding site (Fig. 1). The pol ORF of HTLV-I/-II/BLV is expressed by using two ribosomal -1 frame shifts, and the second frame shift with the transcription start site of the pol ORF lies within this region . The high degree of conservation of this region is thus understandable. A phylogenetic tree constructed from the aligned region illustrates the genetic relation- ships (Fig. 2). No other genomic regions with a similarly high degree of conservation were identified (see Addi- tional File 1). Degenerate primers complementary to these regions were constructed (Fig. 1). The degeneracy of the primers was moderate (4-fold for (+) and 12-fold for (-)). The PCR was tested using serial dilutions of Deltaret- rovirus-infectedcell line DNA in human leukocyte DNA under various conditions (Fig. 3). Retrovirus-infectedcell
Human papillomaviruses (HPVs) are small-DNA viruses that infect cutaneous and mucosal squamous epithelium and produce benign or malignant tumors. The HPV infectious cy- cle is initiated in keratinocytes of the basal cell layer (13). In these cells the viral genome is maintained at a low copy num- ber by synchronous replication with chromosomal DNA. When the infectedcell moves up from the basal layer and enters the terminal differentiation program, the virus switches to vegeta- tive replication and the HPV genome is amplified to very high levels. Further differentiation is accompanied by expression of the capsid proteins and eventual virion formation in the most differentiated cells. The early protein, E1 E4, expressed from a spliced mRNA transcript, is abundant in cells that have switched to productive infection (29, 30, 33). Loss of E1 E4 expression in experimental systems that recapitulate the infec- tious cycle correlates with reduced viral genome amplification and late gene expression in differentiating keratinocytes (28, 32, 49), indicating that E1 E4 has an important role in the vegetative cycle of HPVs. Knowledge of E1 E4 functions is limited, however, precluding a complete understanding of the role of this viral protein in the HPV life cycle.
We obtained 3,575,737 and 617,535 high-quality reads from the normal and infectedcell samples, respectively, remained for miRNA analysis. The length distribution of the high-quality reads ranged 16–30 nt. Most sequence reads ranged 21–24 nt, which belonged to the typical size range (Fig. 1). We identified 533 and 286 porcine miRNAs in normal PK-15 cells and infected PK-15 cells, respect- ively. This indicates that the normal cells contained more miRNAs than the infected cells. The change of expression of miRNAs between normal and infected PK-15 cells reflects that miRNAs can play key roles during the viral infection process, where virus can affect cellular miRNAs expression profile on their own benefit. ssc-miR-21 was the most abundantly expressed miRNA, followed by ssc- miR-30a-5p. miRNAs were considered differentially expressed when the fold change (FC) difference between groups was >2 or ≤0.5 and P ≤ 0.01, or when a miRNA was not expressed in either the infected or control group. There were 193 differentially expressed miRNAs; 128 were
Transfection of cells with a plasmid expressing a caspase-3–GFP fu- sion protein. Cell lines latently infected with herpesviruses were trans- fected with a plasmid, pcasp3-WT-GFP, that expresses functional wild- type (WT) caspase-3 fused to green fluorescent protein (GFP), a kind gift from Shinji Kamada, Biosignal Research Center, Kobe University (35). pUC19 was used as a negative control. APC-annexin V (BD Pharmingen) was used to detect apoptosis. Monoclonal antibodies against EBV p52, HHV-6A gp116, HHV-6B gp116, and HHV-7 KR4 (NIH AIDS Research and Reference Reagent Program) and KSHV ORFK8.1 (Advanced Bio- technologies, Inc.), each at 1:400 dilution, and goat anti-mouse secondary antibody labeled with PerCP (Advanced Biotechnologies) were used to detect viral protein expression. Cells were seeded in 24-well plates, and 2 h prior to transfection, the medium was replaced by RPMI 1640 medium with no FBS and antibiotics. Lipofectamine 2000 (Invitrogen) was mixed with Opti-MEM I medium (Invitrogen) at a 1:25 dilution and kept for 10 min at RT. This mixture was then complexed with 500 ng of pcasp3-WT- GFP at a ratio of 1:1 and kept at RT for 30 min. Two hundred microliters of this complex was added for transfection in each well. The plates were gently rocked at 37°C for 5 h. TPA (20 ng/ml) was added to selected wells and incubated for 1 h. DCPE (final concentration, 50 nM) was added to selected samples.
The diagnostic performance of the InR flag for the diagnosis of Plasmodium was evaluated. Samples with malaria parasites show a distinct cluster and unique loca- tion on three-dimensional cell plots. The characteristics of the cluster are dependent on the species of Plasmo- dium as well as the size and number of the parasite pre- sent. The sensitivity and specificity for detecting P. vivax was not significant for P. falciparum. This may be a result of early trophozoites of P. falciparum that developed over 10 h in the peripheral blood concealed in the microvas- culature, sinusoids or other slow blood flow before being Fig. 1 White blood cell scattergrams generated by the Mindray BC-6800 haematology analyzer. a An example of the gates lymphocyte, monocyte, neutrophil, infected RBC (yellow spots) and eosinophil in a representative patient with malaria; b the distinct cluster of malaria parasites (yellow spots) in three-dimensional analysis scattergrams
Adoptive transfer of T-cell clones produces CNS degenera- tion. We investigated whether TMEV-induced T-cell clones could initiate CNS pathology. T-cell clones 8a-1A and 8b-1C were transferred into naı¨ve mice. During the 1-week observa- tion period, 25% of the mice showed a mildly impaired righting reflex (mean righting reflex score of all mice injected with T cell clones ⫾ standard error of the mean [SEM], 0.9 ⫾ 0.1). Histologically, T-cell clones induced CNS pathology, including meningitis, perivascular cuffing, and gliosis in the corpus cal- losum, caudoputamen, internal capsule, basal forebrain, cere- bellar peduncle, cerebellum, midbrain, and brain stem (Fig. 4a). Five of eight mice developed large CNS degenerative lesions with loss of myelin. Lesions were located not only close to the injection site, such as the thalamus, fimbria hippocampi, and corpus callosum, but also some distance from the injection site, including the posterior funiculus of the spinal cord (Fig. 4d). No difference was seen in CNS pathology between mice injected with the two separate CD8 ⫹ T-cell clones. Control mice that received normal spleen cells or PBS had no lesions but only mild gliosis around the injection site (Fig. 4c).
Plasmids. The EBER1 and EBER2 genes are located within the BamHI C fragment of Akata EBV DNA. pUC-A/C⌬PstI/EcoK contained the part of the BamHI C fragment of Akata EBV genome (corresponding to the EcoRI-PstI fragment spanning nucleotides 4163 through 9699 of EBV B95-8 strain). A hygromycin resistance marker gene driven by simian virus 40 early enhancer and promoter was cloned into the BamHI site of pBS246 (Life Technologies) to make pBS246/hyg so that the hygromycin resistance marker gene was flanked by two loxP sites. The AccI fragment of pUC-A/C⌬PstI/EcoK spanning the EBER genes (corresponding to nucleotides 6612 through 7263 of EBV B95-8 strain) was replaced with the NotI fragment of pBS246/hyg to make pEBER-KO. pEBER-KO was used as a targeting construct to generate EBER knockout EBV. The NotI fragment of pBS246/hyg was inserted into the SacI site (nucleotide 6285 of EBV B95-8 strain) of pUC-A/C ⌬ PstI/EcoK to make pEBER-KI, which was used as a targeting construct to generate EBER knock-in EBV. Other known EBV open reading frames were not affected by this insertion. pSG-Cre was constructed by cloning the blunted XhoI-MluI fragment of pBS185 (Life Technologies), con- taining a cre recombinase gene, into the blunted BglII site of pSG5 (Stratagene). Generation of homologously recombined EBVs. Twenty micrograms of the targeting construct was digested with BstPI and EcoRV and introduced into EBV-infected Akata cells by an electroporation method as described previously (33). Transfected cells were cultured for 2 days and plated in 96-well plates at 10 4
Cancer cells have been increasingly grown in pharmaceutical research to understand tumorigenesis and develop new therapeutic drugs. Currently, cells are typically grown using two-dimensional (2-D) cell culture approaches, where the native tumor microenvironment is difficult to recapitulate. Thus, one of the main obstacles in oncology is the lack of proper infection models that recount main features present in tumors. In recent years, microtechnology-based platforms have been employed to generate three-dimensional (3-D) models that better mimic the native microenvironment in cell culture. Here, we present an innovative approach to culture Kaposi’s sarcoma-associated herpesvirus (KSHV) infected human B cells in 3-D using a microwell array system. The results demonstrate that the KSHV-infected B cells can be grown up to 15 days in a 3-D culture. Compared with 2-D, cells grown in 3-D had increased numbers of KSHV latency-associated nuclear antigen (LANA) dots, as detected by immunofluorescence microscopy, indicating a higher viral genome copy number. Cells in 3-D also demonstrated a higher rate of lytic reactivation. The 3-D microwell array system has the potential to improve 3-D cell oncology models and allow for better-controlled studies for drug discovery.
Several types of mutations might confer the trypsin-indepen- dent phenotype selected during persistent rotavirus infection of MA104 cells. Trypsin cleaves outer capsid protein VP4, resulting in the generation of particle-associated cleavage frag- ments VP5* and VP8* (15, 18, 21, 34). It is possible that mutations selected during persistent infection alter the VP4 cleavage site, rendering the protein susceptible to cleavage by other host proteases, such as those present on the cell surface (reviewed in reference 12). Alternatively, mutations selected during persistent infection might alter the fusion domain of VP5* to allow it to mediate membrane penetration in the absence of trypsin-mediated VP4 cleavage. It seems unlikely that progeny virions exiting infected cells contain cleaved VP4 proteins, as experiments using SDS-PAGE to assess viral struc- tural proteins indicate that progeny virions contain intact VP4. However, the electrophoretic mobility of PI virus VP4 protein is altered in comparison to that of wt virus, which suggests that mutations were selected in PI virus VP4 during persistant infection. It is conceivable that mutations in other rotavirus proteins that interact with VP4, such as outer capsid protein VP7 or inner capsid protein VP6 (45, 52), might confer trypsin-
Despite efficient translocation of the viral DNA to the nu- cleus, B19V infection in the presence of antibodies is abortive. We demonstrated that ␣ -B19V antibodies dramatically increased B19V uptake into endothelial cells in the presence of heat-labile serum factors, such as C1q. An important issue that remained to be clarified, however, was whether the virus internalized by this route was efficiently translocated to the nucleus and could subse- quently initiate a productive infection. To monitor nuclear trans- location, cytoplasmic and nuclear fractions from U937 and EA.hy926 cells and HCAEC infected with B19V were assayed for the number of B19V genomes. Infections were performed with or without prior incubation with IgG fractions from anti-B19V-neg- ative or -positive sera, as described previously. For cell fraction- ation, special emphasis was put on avoiding possible contamina- tion of the nuclei with residual cytoplasmic fraction by repeated washing of the nuclei with hypotonic buffer. In line with these considerations, no tubulin, which was used as a cytoplasmic marker protein, was detectable in the nuclear fractions by Western blot analysis (Fig. 8A to C, bottom right). Whereas contamination of the nuclear fraction by cytoplasmic proteins could largely be ruled out in the Western analysis, the MCM7 replication protein used as a nuclear marker protein was also found in the cytoplasmic fraction, especially in U937 cells (Fig. 8A to C, bottom left). How- ever, despite the resulting overestimation of the amount of B19V DNA present in the cytoplasm, more than 99% of the B19V DNA internalized in the presence of anti-B19V antibodies was actually found in the nuclear fraction in the monocytic U937 cell line, and also in the EA.hy926 and HCAEC endothelial cells (Fig. 8A to C, top, compare the cytoplasm with the nucleus). Thus, the B19V virions internalized by the antibody-mediated route, or at least the associated genomes, seem to be very efficiently transported to the nucleus.
Our results also indicate that IL-6 can let the LCLs escape from apoptotic cell death. The results suggest that IL-6 per- haps contributes to LCL growth via two different mechanisms: one by working as an autocrine growth factor and the other by letting the LCLs escape from apoptotic cell death. We also tried to complement the lack of EBER2 gene by trans-supply- ing IL-6 in the B-cell transformation assay. The result revealed that trans-supplied IL-6 failed to completely complement the lack of EBER2 gene. One possible explanation is that exog- enously added IL-6 may be less effective than endogenous autocrine IL-6. Alternatively, the result can be interpreted that IL-6 is not the only cellular factor that is induced by EBER2 and contributes to B-cell transformation. Presumably, IL-6 must cooperate with other EBER2-induced cellular factors to accelerate the initial phase of B-cell growth transformation.