The cell cycle status is a key determinant for completion of these early stages of infection [ 2 ], and retroviruses have been classified based on their ability to productively infect non-cycling cells. Gammaretroviruses, like murine leukemia virus, require mitosis for proviral integration [ 3 ], while lentiviruses such as human immunodeficiency virus type 1 HIV-1 show almost no difference between dividing and non-dividing cells [ 4 ].
Indeed, HIV-1 and other lentiviruses can replicate in terminally differentiated and post-mitotic cells such as neurons or macrophages [ 5 , 6 ]. However, like murine leukemia virus, HIV-1 cannot infect naive quiescent CD4-positive T cells or monocytes isolated from peripheral blood that are in the G0 stage of the cell cycle [ 2 , 7 ]. Since reverse transcription does occur in these conditions [ 8 ], it is conceivable that other host cell proteins or processes are necessary for the completion of the viral cycle [ 9 ].
Foamy viruses FVs are complex retroviruses isolated from different animal species, mainly in non-human primates. FVs share with all retroviruses the same organization of the genome, which encodes Gag, Pol, and Env proteins. In addition, the FV genome encodes at least two other proteins, Tas and Bet, which are not incorporated into the viral particle.
Remarkably, FVs exhibit some features related to the hepatitis B virus [ 10 ]. In particular, they reverse transcribe their RNA genome during the late stages of infection, leading to the presence of infectious viral DNA in extracellular virions [ 11 ].
Moreover, the structural FV Gag presents specific characteristics that set it clearly apart from other retroviral Gag proteins. In particular, FV Gag maturation by the viral protease does not lead to the formation of the canonical matrix, capsid, and nucleocapsid products.
Rather, the Gag precursor is partially cleaved by the viral protease near its C terminus into a mature product, before or during budding. This results in the presence of two Gag proteins of 71 kDa and 68 kDa in extracellular virions [ 12 ]. We have previously reported that upon entry into target cells and prior to nuclear translocation, incoming FV capsids traffic along the microtubule network to reach the microtubule-organizing centre MTOC [ 13 , 14 ], which includes the centrosome in animal cells [ 15 ].
A similar route has been described for HIV-1 [ 16 ]. Drugs disrupting the microtubule network, such as nocodazole or colchicine, largely prevent early intracellular FV trafficking [ 13 ], as well as that of HIV-1 [ 16 ].
Gag also targets the pericentrosomal region for capsid assembly during the late stages of infection [ 17 ]. Similar to murine leukemia virus, productive FV infection requires passage through mitosis [ 18 , 19 ]. Like all other animal retroviruses, FVs do not productively infect cells arrested in the G0 stage of the cell cycle, such as peripheral T lymphocytes or growth-arrested fibroblasts in vitro [ 19 ]. To gain insight into this restriction, we have focused our attention on early stages of FV replication in G0 cells.
Here, we demonstrate that incoming FVs stably localize at the vicinity of the MTOC as structured and assembled capsids for several weeks in resting cultures in vitro. Upon cell stimulation, Gag proteolysis and capsid disassembly take place, allowing infection to proceed. Altogether, these data demonstrate that the centrosome represents a cellular site around which incoming viruses persist as a stable pre-integration intermediate in resting cells, and that virus uncoating is the rate-limiting step for FV infection in growth-arrested cells.
Cycling and resting human primary MRC5 fibroblastic cells were infected with the prototypic primate foamy virus PFV at a multiplicity of infection m. Consistent with previous reports [ 18 , 19 ], we found that PFV does not productively infect G0 resting cells unpublished data. To investigate the molecular mechanisms involved in the restriction of FV replication cycle in G0 resting cells, we first analyzed the distribution of incoming viral components in resting MRC5 cells.
As early as 4 h post-infection p. These findings are consistent with previous observations showing the pericentrosomal concentration of incoming FVs after trafficking along the microtubule network in cycling cells [ 13 ]. Remarkably, while in cycling cells, incoming Gag antigens are no longer observed near this organelle 10 h p. Figure 1 A. We then investigated the localization of the viral genome in infected resting cells.
Figure 1 B. A Sub-cellular localization of incoming Gag proteins studied by confocal microscopy following indirect immunofluorescence at 3, 15, and 30 d p. Nuclei are stained with DAPI blue. B Sub-cellular localization of the viral DNA genome from incoming PFV analyzed by confocal microscopy following in situ hybridization 15 d p. To visualize the status assembled capsids or not of incoming viruses in resting cells, MRC5 cells were analyzed by electron microscopy EM at different time points after infection.
In cycling cells Figure 2 A , incoming FVs were observed at the centrosome 4 h p. At later time points, these viral capsids completely disassembled in cycling cells as already reported [ 20 ], and viral capsids were never detected in uninfected cells Figure 2 A. Importantly, assembled and structured viral capsids were observed around the centrosome in resting cells 5 or 15 d p. Figure 2 A , strongly suggesting that virus uncoating is impaired under these settings.
A Electron micrographs of uninfected and infected cycling and resting cells. Normally shaped intracellular capsids pointed out by a black arrow are observed in PFV-infected cycling cells at 4 h p.
In resting cells at 5 or 15 d p. High magnifications are presented in the right corner. No viral capsids are detected in uninfected cells. B Western blot analysis of protein extracts from PFV-infected cycling and resting cells using anti-Gag abs.
Analysis of protein lysates performed 7 h p. In resting cells, 5 or 15 d p. FV uncoating requires the enzymatic activity of both viral and cellular proteases, which cleave the major structural components of viral capsids the 71—68 kDa Gag doublet , into shorter fragments [ 20 ].
Among these cleavage products, a kDa Gag-derived product specifically results from the action of the viral protease [ 20 ]. To confirm at the biochemical level that FV uncoating is inhibited in resting cells, the status of the Gag polyprotein was determined.
Figure 2 B shows that Gag cleavage products, in particular the kDa fragment, were easily detected in cycling cells. These results demonstrate the absence of Gag cleavage in resting cells, reflecting the absence of viral uncoating observed by EM. To assess whether incoming FV capsids at the centrosome of resting cells constitute a stable pre-integration intermediate, which can later be reactivated for productive infection, resting PFV-infected MRC5 cell cultures were stimulated to divide by splitting and serum addition.
At different time points following this activation, Gag expression and distribution were analyzed by indirect immunofluorescence using mouse anti-Gag abs. Gag was detected in both compartments in the entire culture 48 h after cell activation, and the formation of numerous syncytia was detected 96 h post-activation Figure 3 A. These results demonstrate that viral replication, which was inhibited in resting cells, resumes upon cell activation.
To confirm that cell activation actually triggered virus uncoating, the status of the Gag polyprotein was studied by Western blot. To exclusively analyze incoming viral antigens, avoiding contamination from Gag synthesis and degradation, which might occur following reactivation, these experiments were performed under cycloheximide CHX treatment, a translation inhibitor.
At 24 h post-reactivation, several Gag cleavage products, notably the kDa fragment [ 20 ], were clearly detected in CHX-treated cells Figure 3 B. Moreover, under these settings, accumulation of Gag was observed only in untreated reactivated cells.
Altogether, these observations demonstrated that entry into the cell cycle triggered virus uncoating, as assessed by Gag cleavage, and productive infection. A Sub-cellular localization of Gag proteins green studied by confocal microscopy following indirect immunofluorescence using anti-Gag antiserum.
Thirty days p. In contrast, Gag proteins are detected both in the cytoplasm and the nucleus of these cells at 24, 48, and 96 h post-activation by splitting and serum addition. Syncitia, characteristic of a productive FV infection, appear 96 h post-reactivation. Therefore, the intracellular distribution of incoming FVs was assessed in one of its natural targets. In these cells, localization of incoming capsids was analyzed by confocal microscopy. Consistent with our previous observation of infected resting MRC5 cells, incoming Gag strictly localized at the centrosome from day 2 to day 5 p.
Figure 4 B. In April , we removed the 25 MB limit for scanned files. When a malicious file is uploaded to OneDrive, it will be synced to the local machine before it's marked as malware.
After it's marked as malware, the user can't open the synced file anymore from their local machine. Microsoft organizations that have Microsoft Defender for Office included in their subscription or purchased as an add-on can enable Safe Attachments for SharePoint, OneDrive, and Microsoft Teams for enhanced reporting and protection. Malware and ransomware protection in Microsoft Skip to main content. This browser is no longer supported. Download Microsoft Edge More info. Contents Exit focus mode.
Is this page helpful? Vaccine requirements were a major issue in statewide elections in Virginia earlier this month. Democratic gubernatorial candidate Terry McAuliffe repeatedly criticized Republican Glenn Youngkin for not embracing a strict vaccine mandate. Youngkin, who went on to win in November's election, has said he does not support a vaccine mandate. Sears repeatedly referenced commonly held falsehoods about the Covid vaccine and the virus itself.
She suggested people who've been infected with Covid did not need to get the vaccine, and questioned the benefits that masks provide for protecting against transmission of the virus. One doesn't follow the other," Sears said, ignoring scientific data showing the Covid vaccine prevents hospitalizations and deaths. Health experts have said people who have tested positive for Covid will have some antibody protection, but those antibodies will fade away over time. People who've tested positive for Covid should still get the vaccine for that reason, according to experts, and evidence shows that people may receive better protection by being fully vaccinated compared with having recovered from Covid Scientists also have said universal masking for people in close proximity can reduce Covid transmission significantly, more so than when some people or nobody is wearing masks.
Studies show that masks can help prevent people from spreading the coronavirus to others and may help protect the wearer from becoming infected themselves. New Ohio congressional map struck down. Load Error. VA Lt.
0コメント