scholarly journals Serum extracellular vesicle depletion processes affect release and infectivity of HIV-1 in culture

2016 ◽  
Author(s):  
Zhaohao Liao ◽  
Dillon C. Muth ◽  
Erez Eitan ◽  
Meghan Travers ◽  
Elin Lehrmann ◽  
...  
Keyword(s):  
Culture Media ◽  
Virus Production ◽  
Cell Types ◽  
Extracellular Vesicle ◽  
Substantial Effect ◽  
Primary Cells ◽  
Antiviral Responses ◽  
Health And Disease ◽  
Infected Cells ◽  
Hiv 1

ABSTRACTExtracellular vesicles (EVs, including exosomes and microvesicles) are involved in intercellular communication in health and disease and affect processes including immune and antiviral responses. Ultracentrifuged serum is depleted of EVs and, when used in culture media, reduces growth and viability of some cell types. In this study, we examined the effects of serum EV depletion processes on HIV-1 replication in primary cells and cell lines, including two HIV-1 latency models. Increased HIV-1 production was observed in certain EV-depleted conditions, along with cell morphology changes and decreased cell viability. Add-back of ultracentrifuge pellets rescued baseline HIV-1 production. Primary cells appeared to be less sensitive to EV depletion. ACH-2 and U1 latency models produced more HIV-1 under EV-depleted conditions, while virus produced under processed serum conditions was more infectious. Finally, changes in cellular metabolism and gene expression were associated with EV-depleted culture. In conclusion, the EV environment of HIV-1 infected cells has a substantial effect on virus production and infectivity. EV-dependence of cell cultures should be examined carefully along with other experimental variables. However, EVs may not be the only particles depleted by ultracentrifugation or other processes. Effects of EVs may be accompanied by or confused with those of closely associated or physically similar particles.

scholarly journals Effects of inducing or inhibiting apoptosis on Sindbis virus replication in mosquito cells

2008 ◽  
Vol 89 (11) ◽  
pp. 2651-2661 ◽  
Author(s):  
Hua Wang ◽  
Carol D. Blair ◽  
Ken E. Olson ◽  
Rollie J. Clem
Keyword(s):  
Virus Replication ◽  
Persistent Infection ◽  
Sindbis Virus ◽  
Actinomycin D ◽  
Virus Production ◽  
Cell Types ◽  
Lytic Infection ◽  
Mosquito Cells ◽  
Infected Cells ◽  
Apoptotic Genes

Sindbis virus (SINV) is a mosquito-borne virus in the genus Alphavirus, family Togaviridae. Like most alphaviruses, SINVs exhibit lytic infection (apoptosis) in many mammalian cell types, but are generally thought to cause persistent infection with only moderate cytopathic effects in mosquito cells. However, there have been several reports of apoptotic-like cell death in mosquitoes infected with alphaviruses or flaviviruses. Given that apoptosis has been shown to be an antiviral response in other systems, we have constructed recombinant SINVs that express either pro-apoptotic or anti-apoptotic genes in order to test the effects of inducing or inhibiting apoptosis on SINV replication in mosquito cells. Recombinant SINVs expressing the pro-apoptotic genes reaper (rpr) from Drosophila or michelob_x (mx) from Aedes aegypti caused extensive apoptosis in cells from the mosquito cell line C6/36, thus changing the normal persistent infection observed with SINV to a lytic infection. Although the infected cells underwent apoptosis, high levels of virus replication were still observed during the initial infection. However, virus production subsequently decreased compared with persistently infected cells, which continued to produce high levels of virus over the next several days. Infection of C6/36 cells with SINV expressing the baculovirus caspase inhibitor P35 inhibited actinomycin D-induced caspase activity and protected infected cells from actinomycin D-induced apoptosis, but had no observable effect on virus replication. This study is the first to test directly whether inducing or inhibiting apoptosis affects arbovirus replication in mosquito cells.

Regulation and mechanisms of extracellular vesicle biogenesis and secretion

2018 ◽  
Vol 62 (2) ◽  
pp. 125-133 ◽  
Author(s):  
Crislyn D’Souza-Schorey ◽  
Jeffrey S. Schorey
Keyword(s):  
Tumor Microenvironment ◽  
Intercellular Communication ◽  
Membrane Vesicles ◽  
Cell Types ◽  
Extracellular Vesicle ◽  
Systemic Delivery ◽  
Heterogeneous Membrane ◽  
Vesicle Biogenesis ◽  
Infected Cells ◽  
Pathogenic Agents

EV (extracellular vesicle) biology is a rapidly expanding field. These heterogeneous membrane vesicles, which are shed from virtually all cell types, collectively represent a new dimension of intercellular communication in normal physiology and disease. They have been shown to deliver infectious and pathogenic agents to non-infected cells whereas in cancers they are thought to condition the tumor microenvironment. Their presence in body fluids and inherent capacity for systemic delivery point to their clinical promise. All of the above only intensifies the need to better understand the classification, mode of biogenesis, and contents of the different subtypes of EVs. This article focusses on vesicle subtypes labeled as exosomes and MVs (microvesicles) and discusses the biogenesis and release of these vesicles from cells.

scholarly journals Reinfection results in accumulation of unintegrated viral DNA in cytopathic and persistent human immunodeficiency virus type 1 infection of CEM cells.

1990 ◽  
Vol 172 (4) ◽  
pp. 1035-1042 ◽  
Author(s):  
C D Pauza ◽  
J E Galindo ◽  
D D Richman
Keyword(s):  
Human Immunodeficiency Virus ◽  
Human Immunodeficiency Virus Type ◽  
Virus Type ◽  
Virus Production ◽  
Neutralizing Antibody ◽  
Viral Dna ◽  
Immunodeficiency Virus ◽  
Infected Cells ◽  
Hiv 1

High levels of unintegrated viral DNA accumulate during human immunodeficiency virus type 1 (HIV-1) infection of CEM T cells. Reinfection of already infected cells is required to attain these levels and reinfection also promotes the development of HIV-induced cytopathology. Rates of virus production, however, are independent of the accumulation of unintegrated viral DNA. Neutralizing antibody added soon after infection reduced viral DNA levels without appreciably affecting the production of cell-free viral p24 antigen or reverse transcriptase activity. Only 50 pM AZT were required to reduce the accumulation of unintegrated viral DNA by 50% in contrast to the 25 nM required to inhibit virus production by 50%. Cytopathology, as measured by number of syncytia in infected cell cultures, was correlated with highly elevated levels of unintegrated viral DNA. The minimal levels of unintegrated viral DNA present constitutively in the persistently infected HCEM cell line were consonant with the absence of cytopathic effects in these cells. These data demonstrate that inhibiting the reinfection of already infected cells modulates cytopathic HIV-1 infection to a form that is persistent and noncytopathic.

scholarly journals Human cytomegalovirus modulation of CCR5 expression on myeloid cells affects susceptibility to human immunodeficiency virus type 1 infection

2006 ◽  
Vol 87 (8) ◽  
pp. 2171-2180 ◽  
Author(s):  
Christine A. King ◽  
Joan Baillie ◽  
John H. Sinclair
Keyword(s):  
Human Immunodeficiency Virus ◽  
Human Cytomegalovirus ◽  
Cell Types ◽  
Ccr5 Expression ◽  
Immunodeficiency Virus ◽  
Infected Cells ◽  
Bystander Cells ◽  
Hiv 1

For some time there has been evidence suggesting an interaction between human cytomegalovirus (HCMV) and Human immunodeficiency virus (HIV) in the pathogenesis of AIDS. Here, the interaction of HCMV and HIV-1 was examined in monocyte/macrophage cells, two cell types known to be targets for both viruses in vivo. Infection experiments demonstrated that prior infection with HCMV impeded subsequent superinfection with HIV-1. In contrast, uninfected bystander cells within the population were still permissive for HIV-1 infection and were also found to express increased levels of Gag after HIV-1 superinfection. Analysis of CCR5, a co-receptor for HIV-1, on HCMV-infected and bystander cells showed a substantial loss of surface CCR5 expression on infected cells due to HCMV-induced reduction of total cellular CCR5. In contrast, uninfected bystander cells displayed increased surface CCR5 expression. Furthermore, the data suggested that soluble factor(s) secreted from HCMV-infected cells were responsible for the observed upregulation of CCR5 on uninfected bystander cells. Taken together, these results suggest that, whilst HCMV-infected monocytes/macrophages are refractory to infection with HIV-1, HCMV-uninfected bystander cells within a population are more susceptible to HIV-1 infection. On this basis, HCMV infection may contribute to the pathogenesis of HIV-1.

scholarly journals Infection of monocyte-derived macrophages with human immunodeficiency virus type 1 (HIV-1). Monocyte-tropic and lymphocyte-tropic strains of HIV-1 show distinctive patterns of replication in a panel of cell types.

1989 ◽  
Vol 170 (4) ◽  
pp. 1149-1163 ◽  
Author(s):  
R Collman ◽  
N F Hassan ◽  
R Walker ◽  
B Godfrey ◽  
J Cutilli ◽  
...  
Keyword(s):  
T Cell ◽  
Host Range ◽  
Cell Line ◽  
Cell Types ◽  
Primary Cells ◽  
Primary Monocyte ◽  
Single Cell Type ◽  
Different Strains ◽  
Different Cell Types ◽  
Hiv 1

To characterize the host range of different strains of HIV-1, we have used four types of cells, primary monocyte-derived macrophages (MDM), primary PBL, a promonocyte cell line (U937), and a CD4+ T cell line (SUP-T1). These cells were infected with three prototype strains of HIV-1, a putative lymphocyte-tropic strain (IIIB), and two putative monocyte-tropic strains (SF162 and DV). Infections were monitored by assays for infectious virus, for cell-free and cell-associated viral antigen (p24), and for the proportion of cells infected by immunohistochemical staining. It was concluded that: (a) the use of four different cell types provides a useful biological matrix for distinguishing the tropism of different strains of HIV-1; this matrix yields more information than the infection of any single cell type. (b) A monocyte-tropic strain of HIV-1, such as strain SF162, shows a reciprocal host range when compared with a lymphocyte-tropic strain such as IIIB; strain SF162 replicates well in primary MDM but not in U937 or SUP-T1 cells, while strain IIIB replicates well in both U937 and SUP-T1 cells but not in MDM. (c) Both lymphocyte-tropic and monocyte-tropic strains of HIV-1 replicate well in PBL. (d) The promonocyte cell line, U937, and the T cell line, SUP-T1, differ markedly from primary cells, such as MDM and PBL, in their ability to support the replication of different strains of HIV-1; these cell lines cannot be used as surrogates for primary cells in host range studies of HIV-1 strains.

scholarly journals 2549 Characterizing the expression kinetics of HIV-1 envelope protein

2018 ◽  
Vol 2 (S1) ◽  
pp. 7-7
Author(s):  
Djin-Ye Oh ◽  
Lihong Liu ◽  
Benjamin Trinité ◽  
En-Wei Hu-Van Wright ◽  
Vincent Sahi ◽  
...  
Keyword(s):  
Cell Surface ◽  
Virus Production ◽  
Cellular Level ◽  
Hiv Treatment ◽  
Cell Surface Expression ◽  
Substantial Impact ◽  
Infected Cells ◽  
Kinetics Of ◽  
Hiv 1 ◽  
Expression Kinetics

OBJECTIVES/SPECIFIC AIMS: Characterize the expression kinetics of HIV-1 Envelope and their relationship to virus production at the cellular level. METHODS/STUDY POPULATION: In vitro and ex vivo laboratory analyses. RESULTS/ANTICIPATED RESULTS: Initial studies addressing the kinetics of cell surface. Envelope (Env) expression reveal that Env expression to peaks on day 2 post infection. Next steps include a series of experiments to compare the kinetics of Env cell surface expression with broadly neutralizing antibody (bNAb)-mediated ADCC and the characterization of virus production kinetics in this same context. To be maximally effective, ADCC elimination of infected cells should occur before peak Env expression. DISCUSSION/SIGNIFICANCE OF IMPACT: Potent bNAbs to HIV-1 recognize vulnerable sites on the HIV-1 Envelope (Env) protein and are of great clinical interest due to their potential use in the prevention and treatment of HIV-1 infection. Their effectiveness depends not only on the neutralization of viral infectivity, but also on the elimination of productively infected cells via antibody-dependent cellular cytotoxicity (ADCC). On a cellular level, ADCC dynamics are determined by the timing and level of Env expression on the surface of HIV-infected cells. This study aims to delineate the expression kinetics of HIV-1 Envelope and their relationship to virus production. We expect that it will provide new insights into the utility of bNAb-mediated ADCC in treating and possibly curing HIV-1 infection; therefore results might have substantial impact on future HIV treatment strategies.

scholarly journals HTLV-1 contains a high CG dinucleotide content and is susceptible to the host antiviral protein ZAP

Retrovirology ◽  
2019 ◽  
Vol 16 (1) ◽  
Author(s):  
Paola Miyazato ◽  
Misaki Matsuo ◽  
Benjy J. Y. Tan ◽  
Michiyo Tokunaga ◽  
Hiroo Katsuya ◽  
...  
Keyword(s):  
T Cell ◽  
Virus Production ◽  
Host Cells ◽  
Cell Leukaemia ◽  
Leukaemia Virus ◽  
Delta Type ◽  
Infected Cells ◽  
Cg Dinucleotide ◽  
Hiv 1

Abstract Background Human T cell leukaemia virus type 1 (HTLV-1) is a retrovirus associated with human diseases such as adult T-cell leukaemia/lymphoma and HTLV-1 associated myelopathy/tropical spastic paraparesis. In contrast to another human retrovirus, human immunodeficiency virus type 1 (HIV-1), HTLV-1 persists in the host not via vigorous virus production but mainly via proliferation and/or long-term survival in the form of silent proviruses in infected host cells. As a result, HTLV-1-infected cells rarely produce virus particles in vivo even without anti-retroviral treatment. That should be an advantage for the virus to escape from the host immune surveillance by minimizing the expression of viral antigens in host cells. However, why HIV-1 and HTLV-1 behave so differently during natural infection is not fully understood. Results We performed cap analysis of gene expression (CAGE) using total RNAs and nascent, chromatin-associated, RNAs in the nucleus and found that HTLV-1 RNAs were processed post-transcriptionally in infected cells. RNA processing was evident for the sense viral transcripts but not the anti-sense ones. We also found a higher proportion of CG di-nucleotides in proviral sequences of HTLV-1-infected cells, when compared to the HIV-1 genomic sequence. It has been reported recently that CG dinucleotide content of viral sequence is associated with susceptibility to the antiviral ZC3HAV1 (ZAP), suggesting the involvement of this protein in the regulation of HTLV-1 transcripts. To analyse the effect of ZAP on HTLV-1 transcripts, we over-expressed it in HTLV-1-infected cells. We found there was a dose-dependent reduction in virus production with ZAP expression. We further knocked down endogenous ZAP with two independent targeting siRNAs and observed a significant increase in virus production in the culture supernatant. Other delta-type retroviruses such as simian T-cell leukaemia virus and bovine leukaemia virus, also contain high CG-dinucleotide contents in their viral genomes, suggesting that ZAP-mediated suppression of viral transcripts might be a common feature of delta-type retroviruses, which cause minimal viremia in their natural hosts. Conclusions The post-transcriptional regulatory mechanism involving ZAP might allow HTLV-1 to maintain a delicate balance required for prolonged survival in infected individuals.

Coxsackievirus B transmission and possible new roles for extracellular vesicles

2013 ◽  
Vol 41 (1) ◽  
pp. 299-302 ◽  
Author(s):  
Jameel M. Inal ◽  
Samireh Jorfi
Keyword(s):  
Calcium Concentration ◽  
Rna Virus ◽  
Extracellular Vesicle ◽  
Host Cells ◽  
Physical Interaction ◽  
Enveloped Viruses ◽  
Coxsackievirus B ◽  
Infected Cells ◽  
Adenovirus Receptor ◽  
Hiv 1

Coxsackievirus B1, a member of the Picornaviridae family is a non-enveloped single-stranded RNA virus associated with human diseases including myocarditis and pancreatitis. Infection of the intestinal mucosa, lined by polarized epithelial cells, requires interaction of coxsackievirus with apically located DAF (decay-accelerating factor) before transport to the basolaterally located CAR (coxsackie and adenovirus receptor), where entry is mediated by endocytosis. As with many other non-enveloped viruses, coxsackievirus has to induce lysis of host cells in order to perpetuate infection. However, recent evidence indicates that virus spread to secondary sites is not only achieved by a lytic mechanism and a non-lytic cell–cell strategy has been suggested for coxsackievirus B3. A physical interaction between infected and non-infected cells has been shown to be an efficient mechanism for retroviral transmission and one type of extracellular vesicle, the exosome, has been implicated in HIV-1 transmission. HIV-1 also takes advantage of depolymerization of actin for spread between T-cells. Calpain-mediated depolymerization of the actin cytoskeleton, as a result of increases in intracellular calcium concentration during coxsackievirus infection, would result in a release of host cell-derived microvesicles. If so, we speculate that maybe such microvesicles, increasingly recognized as major vehicles mediating intercellular communication, could play a role in the intercellular transmission of non-enveloped viruses.

scholarly journals Antibody-Dependent Cellular Cytotoxicity against Reactivated HIV-1-Infected Cells

2015 ◽  
Vol 90 (4) ◽  
pp. 2021-2030 ◽  
Author(s):  
Wen Shi Lee ◽  
Jonathan Richard ◽  
Marit Lichtfuss ◽  
Amos B. Smith ◽  
Jongwoo Park ◽  
...  
Keyword(s):  
Immune Responses ◽  
Autologous Serum ◽  
Cellular Cytotoxicity ◽  
Primary Cells ◽  
Antibody Dependent Cellular Cytotoxicity ◽  
Serum Antibodies ◽  
Latently Infected ◽  
Infected Cells ◽  
Antibody Epitopes ◽  
Hiv 1

ABSTRACTLifelong antiretroviral therapy (ART) for HIV-1 does not diminish the established latent reservoir. A possible cure approach is to reactivate the quiescent genome from latency and utilize immune responses to eliminate cells harboring reactivated HIV-1. It is not known whether antibodies within HIV-1-infected individuals can recognize and eliminate cells reactivated from latency through antibody-dependent cellular cytotoxicity (ADCC). We found that reactivation of HIV-1 expression in the latently infected ACH-2 cell line elicited antibody-mediated NK cell activation but did not result in antibody-mediated killing. The lack of CD4 expression on these HIV-1 envelope (Env)-expressing cells likely resulted in poor recognition of CD4-induced antibody epitopes on Env. To examine this further, cultured primary CD4+T cells from HIV-1+subjects were used as targets for ADCC. Theseex vivo-expanded primary cells were modestly susceptible to ADCC mediated by autologous or heterologous HIV-1+serum antibodies. Importantly, ADCC mediated against these primary cells could be enhanced following incubation with a CD4-mimetic compound (JP-III-48) that exposes CD4-induced antibody epitopes on Env. Our studies suggest that with sufficient reactivation and expression of appropriate Env epitopes, primary HIV-1-infected cells can be targets for ADCC mediated by autologous serum antibodies and innate effector cells. The results of this study suggest that further investigation into the potential of ADCC to eliminate reactivated latently infected cells is warranted.IMPORTANCEAn HIV-1 cure remains elusive due to the persistence of long-lived latently infected cells. An HIV-1 cure strategy, termed “shock and kill,” aims to reactivate HIV-1 expression in latently infected cells and subsequently eliminate the reactivated cells through immune-mediated killing. While recent research efforts have focused on reversing HIV-1 latency, it remains unclear whether preexisting immune responses within HIV-1+individuals can efficiently eliminate the reactivated cells. HIV-1-specific antibodies can potentially eliminate cells reactivated from latency via Fc effector functions by recruiting innate immune cells. Our study highlights the potential role that antibody-dependent cellular cytotoxicity might play in antilatency cure approaches.

scholarly journals Extracellular Vesicles from Infected Cells Are Released Prior to Virion Release

Cells ◽  
2021 ◽  
Vol 10 (4) ◽  
pp. 781
Author(s):  
Yuriy Kim ◽  
Gifty A. Mensah ◽  
Sarah Al Sharif ◽  
Daniel O. Pinto ◽  
Heather Branscome ◽  
...  
Keyword(s):  
Leukemia Virus ◽  
Viral Proteins ◽  
Extracellular Vesicle ◽  
Virion Release ◽  
Human T Cell ◽  
Rich Media ◽  
T Cell Leukemia Virus ◽  
Infected Cells ◽  
Related Proteins ◽  
Hiv 1

Here, we have attempted to address the timing of EV and virion release from virally infected cells. Uninfected (CEM), HIV-1-infected (J1.1), and human T cell leukemia virus-1 (HTLV-1)-infected (HUT102) cells were synchronized in G0. Viral latency was reversed by increasing gene expression with the addition of serum-rich media and inducers. Supernatants and cell pellets were collected post-induction at different timepoints and assayed for extracellular vesicle (EV) and autophagy markers; and for viral proteins and RNAs. Tetraspanins and autophagy-related proteins were found to be differentially secreted in HIV-1- and HTLV-1-infected cells when compared with uninfected controls. HIV-1 proteins were present at 6 h and their production increased up to 24 h. HTLV-1 proteins peaked at 6 h and plateaued. HIV-1 and HTLV-1 RNA production correlated with viral protein expression. Nanoparticle tracking analysis (NTA) showed increase of EV concentration over time in both uninfected and infected samples. Finally, the HIV-1 supernatant from the 6-h samples was found not to be infectious; however, the virus from the 24-h samples was successfully rescued and infectious. Overall, our data indicate that EV release may occur prior to viral release from infected cells, thereby implicating a potentially significant effect of EVs on uninfected recipient cells prior to subsequent viral infection and spread.

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