This revealed 33 CD81 interactions, ten of which were previously described. Both proteins are expressed intracellularly and seem to affect a post-binding entry step during HCV infection. Finally, we report a whole cell proteome expression dataset for human hepatoma cells and show that CAPN5 is strongly associated with CD Pathogens engage host cell protein networks to gain access into replication competent intracellular compartments [ 21 — 23 ]. Mass spectrometry based-proteomics has matured into a powerful technology to comprehensively analyze protein-protein interactions PPIs from cell culture and primary cell material [ 22 — 24 ].
This allowed us to use antibodies against endogenous or tag epitopes to pull out different versions of the bait. We confirmed expression of the respective CD81 protein by immunoblot, immunofluorescence microscopy and flow cytometry.
Wildtype and HA-tagged CD81 were expressed at the cell surface and in intracellular compartments comparable to endogenous CD81 in Huh Lunet N cells lacking detectable CD81 expression served as negative control in all infection and proteomics experiments [ 25 ]. A Schematic overview of the experimental setup used to define the CDinteractome in human hepatoma cell lines.
GAPDH served as loading control. Representative of 4 biological replicates. Median of 4 biological replicates.
D Number of proteins significantly enriched in the indicated co-IPs and membrane associated fraction. For each protein the t-test difference log 10 of CD81 versus control co-IP of 4 biological replicates is plotted against the p value -log Proteins significantly enriched are highlighted in dark grey.
Next we determined which co-IP methods preserved known CD81 interactions. Moreover, we evaluated the use of the homobifunctional amine-reactive cell membrane impermeable crosslinker bis-sulfosuccinimidyl suberate, BS3. This reduced capture may result from crosslinking of large protein aggregates, which are lost during preclearing of lysates or from masking of antibody epitopes. We therefore decided to primarily analyze CD81 networks from Brij lysed, non-crosslinked cells.
Moreover, we observed even weaker protein enrichment when using an anti-HA antibody, indicating that pulling on the ectodomain of CD81 may preserve more interactions than pulling on the C-terminus. CD81 is a tetraspanin consisting of four transmembrane domains, one small and one large extracellular loop LEL and short cytosolic domains. This underlines the important function of the CD81 LEL not only in virion binding, but also in molecular interactions with host proteins.
Taken together we discovered 29 CD81 interactors in human hepatoma cells, 17 of which are novel CD81 interactors without a previously reported function in CD81 binding or HCV cell invasion see S1 Table for full dataset. Here due to lack of control cells negative for CD81 expression, we performed isotype control co-IPs to determine nonspecific background binding of proteins to the IP resin Fig 2B.
We analyzed primary human hepatocytes from five donors. More than proteins were enriched more than fold over controls in both donors. Twenty-three CD81 interactors found in primary human hepatocytes overlapped with the set of 29 hepatoma cell interactors Fig 2F and Table 1.
Of note, these ten hits were also enriched in anti-CD81 co-IPs from CD81HA expressing cells, but not to an extent to fulfill our very stringent hit inclusion criteria of roughly fold enrichment Table 1. The 10 novel hits together with the 23 hits from the first overlap analysis resulted in 33 high confidence CD81 interactors in primary hepatocytes and hepatoma cells Fig 2G and Table 1.
Fig 2H summarizes the protein abundance of the CD81 bait protein and the 33 CD81 interactors in co-IPs from hepatoma cells and primary human hepatocytes. From this network, we included the closest nine nodes in our hit list for follow up, resulting in a final hit list of 42 proteins. In addition, the LFQ approach and extensive comparison of hepatoma and primary cell datasets elucidated 27 CD81 interactors without a previously reported role in HCV entry or CD81 binding. Actin served as loading control.
Among the 26 proteins, 16 overlapped with the analysis in F , resulting in a total of 33 stringent CD81 interactors in PHH and hepatoma cells. H Heat map showing protein abundance as median intensity log 10 for the 33 hits and the CD81 bait in indicated co-IP samples.
Red and blue colors indicate high or low intensity difference, respectively. See also S3 Table. Nine highest scoring additional nodes indicated by asterisk were included for follow up analysis. The full set of identified proteins is depicted in S3A Fig.
Infectivity was measured 48 hpi as luciferase activity and normalized for cell viability and plate effects. See also S3 Fig. As CD81 mediates its molecular functions through PPIs, we hypothesized that a subset of CD81 interactions is required by both pathogens to productively infect liver cells.
To unravel yet unknown CD81 interacting proteins acting as pathogen entry facilitators we investigated human hepatoma cell susceptibility to HCV after silencing each factor Fig 3B. Moreover, we found two novel putative entry facilitators.
Silencing efficiency was not assessed in this RNAi screen. The discovery of CBL and CBLB as HCV host factors are in line with findings showing that ubiquitination events are required in endocytosis and entry of viruses of diverse families including influenza virus and adenoviruses [ 30 , 31 ].
Next we set out to understand whether a similar or distinct set of CD81 PPIs was necessary for hepatoma cell entry of the malaria parasite Plasmodium yoelii.
Here, we used P. First, we evaluated the P. In contrast, P. Using CDdependent P. Silencing of CD81 more pronouncedly affected P. Thus, despite the usage of CD81 as entry factor by HCV and Plasmodium sporozoites, a distinct subset of CD81 interactors seems to aid entry into liver cells.
Development of exoerythrocytic forms indicated by co-localization of GFP with the parasitophorous vacuole marker UIS4. Mean and SEM of 4 biological replicates shown. C Schematic overview of the experimental setup used to analyze the role of CD81 interacting proteins in P.
D Human hepatoma cells were transfected with a pool of three siRNAs as described in Fig 3C , followed by infection with sporozoites of a P. Infectivity was measured 24 hpi as formation of exoerythrocytic forms by microscopy. Knock down of CD81 significantly decreased P. See also S4 and S7 Figs. However, we could clearly show protein expression in hepatoma cells by flow cytometry, immunofluorescence microscopy and immunoblot Figs 5D , S5D and S5H. Albumin black dot shown as additional positive control.
Dotted lines indicate median values of all detected proteins. Nuclei were stained with DAPI. Both proteins have multiple cellular component annotations including plasma membrane, cytosol and nucleus. In human hepatoma cells we detected both proteins in intracellular compartments but not exposed to the cell surface Fig 5C and 5D.
Immunofluorescence analysis further confirmed expression in cytoplasmic and nuclear compartments as reported for other cell types S5C and S5D Fig. CD81 knockout cells served as positive control. Parental cells black served as positive control. Isotype control stainings or stainings with secondary antibody only white as negative controls. Infectivity normalized to particles without envelope protein negative control , to particles with VSV-G envelope positive control and to infection of cells transduced with non-targeting scrambled sgRNA.
E Quantification of HCV fusion activity at the plasma membrane. A pH 7 buffer wash served to determine the background infection rate. Green: NS5A. Blue: DAPI. Replication quantified as luciferase activity at the indicated time point post electroporation. Results normalized to the 4 h time point to account for electroporation efficiency. See also S6 Fig.
Therefore, we took advantage of lentiviral pseudoparticles displaying the HCV surface glycoproteins E1 and E2 and thus mimicking the receptor dependent entry steps [ 16 , 34 ]. The final steps in the HCV entry process are pH-dependent fusion of viral envelope and endosomal membrane and subsequent uncoating of the viral nucleocapsid.
In this assay, entry through the natural route is prevented by concanamycin A blockage of endosomal acidification. To address HCV postentry steps, i. Transfection efficiencies were comparable in all tested cell lines Fig 6F. We observed a slightly reduced replication at 72 h post transfection in the CBLB knockout cells.
Subgenomic genotype 1b and full length genotype 2a replicons showed comparable results as genotype 2a subgenomes S6B and S6D Fig. A slight reduction in infectious titers was observed for CAPN5 knockout cells. Together, these data suggest that CAPN5 and CBLB affect primarily a post-binding but pre-fusion life cycle step, which is not mimicked by lentiviral pseudoparticles.
Similarly, P. We confirmed these findings using non-reporter genotype 2 HCV strain Jc1. B Infection with hCoV expressing a luciferase reporter. Infectivity quantified 24 hpi as luciferase activity RLU, relative light units.
Infectivity analyzed 16 hpi by flow cytometry as mean fluorescence intensity MFI. Infectivity measured 72 hpi as luciferase activity and normalized to infection of Lunet N hCD81 cells transduced with non-targeting scrambled sgRNA. Scr: scrambled sgRNA. Infection of knockout and complemented cell lines with HCV genotype 2 reporter virus upper panel.
The protein expression level in knockout and complemented cell lines was analyzed by immunoblot lower panel. Representative of 3 independent experiments. LOQ: limit of quantification. Shown are three two for CAPN5 dead independent experiments with technical duplicates each. Infection was quantified 72 hpi by luciferase assay. Ub: ubiquitin, P: phosphate group. AP2: adaptor protein complex 2. We demonstrate that HCV binding to CD81 on the liver cell surface is not an isolated event, but steady state CD81 protein interactions are required for virion uptake.
Specifically, we mapped protein interactions of CD81 in resting human hepatocytes and demonstrate that a subset of preexisting CD81 interactions is necessary for HCV infection. Tetraspanins organize membrane microdomains and signaling platforms in a cell type specific manner.
In B lymphocytes CD81 serves as a clamp for the B cell receptor complex [ 37 , 38 ]. In liver cells only little information on the CD81 guided membrane protein complexes is available and this is reflected by a limited knowledge on endogenous CD81 functions in the liver [ 15 , 39 ].
Here, we identified 33 CD81 associated proteins and their relative strength of interaction with CD81 as determined by abundance in CD81 co-IPs depicted in a centered network in S3A Fig plus nine closest neighbor network proteins. Notably, ephrin receptor serves as entry facilitator for HCV and other viruses such as Nipah virus and Kaposi sarcoma herpesvirus [ 14 , 40 , 41 ].
The top four associated diseases and disorders reflect a reported role of CD81 in cell migration and include organismal injury 33 molecules , cancer 28 molecules , inflammatory response 17 molecules and infectious disease 15 molecules S3C Fig. The notion of an involvement of CD81 receptor complexes in cell migration is further strengthened by the association of the CD81 interactome with the following molecular and cellular functions: cell-to-cell signaling 33 molecules , cell movement 25 molecules and cell morphology 23 molecules S3D Fig.
The two top ranked cellular networks associated with the CD81 interactome in liver cells are cell-to-cell signaling and movement score 36, 18 focus molecules and infectious disease score 16, 10 focus molecules S3E Fig. The latter two receptors are HCV entry facilitators, highlighting once more that the here-defined CD81 interactome can indeed reveal host factors with a role in pathogen invasion in the liver.
However, we did not control silencing efficiency and used serum containing conditions, therefore especially growth factor and transferrin dependent entry facilitators presumably escaped detection. Presumably, transient binding of E3 ligases and subsequent targeting of the protein complex to proteasomal degradation hampered the detection.
Ubiquitin ligase substrate trapping methods may overcome such experimental limitations [ 47 ]. In this study, we could overcome this caveat by in silico network analysis and inclusion of closest network nodes in the functional validation.
The model of CD81 linked signaling platforms serving as entry microdomains is in line with recent reports on influenza virus entry. Influenza virus interacts with sialylated molecules on the cell surface, which causes clustering of lipid rafts and activation of raft-associated signaling molecules including EGFR.
The latter then activates its endocytosis together with influenza virus [ 48 ]. Tetraspanins moreover play a role in entry and spread of HIV-1, human papillomaviruses and human cytomegalovirus [ 49 — 52 ].
In fact we think that the unbiased interaction proteomics provides a different perspective on virus entry than functional knockout screens, as it opens avenues for diverse functional follow up studies and hypothesis generation. In the absence of infection CD81 is thought to regulate migration of liver cells [ 39 ].
In confirmation of this notion, in silico analysis maps here identified CD81 network proteins to cellular movement pathways. Progression of HCV infected tissue to liver cancer is a slow, indirect and multifactorial process. Our network analysis suggests that cellular PPIs engaged by the virus during entry might be linked to tumor development.
One other problem is that although livers are primarily composed of hepatocytes, they also contain large numbers of Kupffer cells macrophages , sinusoid cells endothelial , and other cell types in this portal system.
The liver studies using PCR did not distinguish the contribution of different infected cell types to the result. Although liver cells always tested positive for the negative strand of HCV, none of the studies determined which liver cell type, hepatocytes, Kupffer, sinusoidal cells, or other cell types, harbored replicating HCV. One last factor that may have affected this analysis is publication bias. If researchers did not find extrahepatic replication, they may not have published their results.
This would cause an overestimate of the studies that found extrahepatic replication. Although it is likely there was some publication bias, we are unable to determine how much of this occurred. This bias would affect the proportion of studies finding extrahepatic replication but not how many studies found it.
As 57 different studies using PCR found negative strands in extrahepatic tissues, even if there was a lot of publication bias, the published studies would still provide very strong evidence for extrahepatic replication. In situ hybridization ISH can be used to detect viral nucleic acids, and can often distinguish infected cell types, and it can also determine where in the cell the nucleic acid is located.
Although the technique can provide nice data when it works, there are a number of problems with the technique. In addition, the detection of negative strands is more difficult due to its lower concentration than positive strands. A second problem with detecting negative strands is that the presence of both positive and negative HCV RNA in cells can cause them to form double strands.
Both of these problems will cause an underestimation of the number of infected cells and interfere with determining which cells are infected. In addition, there is also natural variability between patients, stage and manifestations of disease, and tissue types. These and other potential problems may make it difficult to compare results and sort out what is actually happening in particular tissues and cells [ 35 ]. We are primarily interested in learning if there are conclusive evidence for the determination of replication in particular cell types or tissues.
Fifteen studies listed in Table 5 have investigated this question. Of these, ten looked at which cell types were positive for negative strands. Nine stated that hepatocytes were infected, while nine also mentioned infection of other liver cells.
Five that mentioned hepatocytes were infected appeared to assume only hepatocytes were infected, as they didn't mention any other cell types in the liver, infected or not.
One study stated that all the positive cells were not hepatocytes, while most suggested that other cell types were infected along with hepatocytes. These studies vary considerably in their methodologies, patient populations, and results. However, it appears likely that both hepatocytes and mononuclear cells harbor replicating HCV in the liver.
In most of these studies, hepatocytes were more likely to be positive than other cell types. Studies using ISH also have investigated whether replication occurs in extrahepatic sites.
Five studies were analyzed to determine whether negative strands could be found in PBMC Table 5 [ 36 — 40 ]. In all cases, samples contained negative strands of HCV. Studies also found replicating HCV in perihepatic lymph nodes PLN [ 41 ], salivary gland ductal and acinar epithelial cells [ 42 , 43 ], and oral epithelial cells [ 44 ].
The results, therefore, suggest that PBMC is likely a site of extrahepatic replication. In addition, other tissues are likely sites of HCV replication. As the NS proteins are not found in the virus, evidence that NS proteins are in cells suggests that replication occurred in those cells.
In addition, NS proteins may be released from cells and diffuse in the interstitial space to nearby cells. Lastly, due to the variability of different isolates of HCV, antibodies may react against a particular NS protein in cells from one patient but not with proteins from another patient due to epitope specificity. This usually causes researchers to test a variety of antibodies to find the one they believe gives the best results.
Frozen tissues generally give better results than formalin-fixed samples. As for ISH, researchers used a variety of methods and antibodies, making comparisons difficult.
We were again interested in consistent results from various tissues or cell types. Studies on liver cells using immunohistochemistry IHC or immunofluorescence IF for the detection of NS proteins were evaluated, and eight are listed in Table 6.
Studies that investigated which cells contain replicating HCV in livers have yielded inconsistent results. Two studies found only hepatocytes stained positive for HCV NS proteins, although one didn't indicate whether they investigated other cell types. Four studies found both hepatocytes and lymphocytes or monocytes containing HCV NS proteins, one found only macrophages, and another found lymphocytes contained them. A study of brain cells using IF found NS5A stained astrocytes and microglial cells macrophages [ 47 ].
Two of the other studies listed above also found macrophages staining positive for NS proteins. Studies have also investigated replication in other cell types. These found NS proteins in B cells and other cell types found in lymph nodes [ 41 ], and others found intestinal epithelial cells [ 48 ] and kidney cells [ 49 ] stained positive for NS proteins. Studies of liver infections are inconclusive regarding which cell types primarily contain replicating HCV.
A variety of other methods have been used to investigate the infection of various tissues and cell types by HCV. Although they don't make definitive statements about which cells are infected by HCV, they broaden the range of evidence of replication.
In this technique, cells were isolated, fixed with formaldehyde, and permeabilized. RT is performed, and then tagged PCR is performed using fluorescent primers. Two studies have looked at the kinetics of viral infection after liver transplantation. The first investigated levels of HCV after liver transplantation, noting that serum levels of HCV declined for two days, and then increased [ 51 ].
They suggested that viral half life was short, and extrahepatic replication contributed little to the level of total virus in the serum. This observation was based primarily on the decline of HCV concentration for two days. However, they did not consider that the repopulation of the liver by HCV infected cells could reduce HCV in the serum, thereby affecting their conclusions. The second group of researchers noticed that the kinetics of HCV concentration in the serum usually exhibited a biphasic curve [ 52 ].
This suggested that there were two viral compartments, each with different replication kinetics. In other words, the kinetics suggested extrahepatic replication of HCV occurs. In our case, we were interested in the percentage of cells that contained replicating HCV. However, only a few papers have determined these percentages.
A study that investigated infection of different cell types in occult infections measured the percentage of cells infected in PBMC by confocal microscopy and found 0. These studies show that only a few percent of cells are infected by HCV. This number varies widely with study design and cell type, so a more precise number is currently not available.
Occult infections show a much lower rate of infected cells. Quasispecies analysis of HCV has been performed by single-strand conformational polymorphism analysis SSCP and by sequencing variants that have been cloned. Comparisons between various tissues or cell types were then performed. For SSCP analysis, researchers usually sequence bands that vary between tissues. As one band can harbor more than one variant, these analyses therefore usually underestimate the variation in samples [ 59 ].
It is not clear, however, whether this problem would alter the overall conclusions of the studies described below as to whether different tissues harbor different major variants. Several studies have investigated whether liver variants are the same as other tissues Table 7a and 7b. Variants were found in each of the tissues that weren't in the other two tissues. All but one of the studies found differences between the liver, serum, and PBMC, which found small differences in the 5'UTR for some of the patients [ 61 ].
These studies show that each of these tissues contain different variants. These studies did not include liver variants. In all cases compartmentalization between serum or plasma and PBMC was seen in many or all of the patients analyzed. Studies noted lower variability in PBMC than in sera [ 64 , 65 ], that the compartmentalization was stable for over two years [ 63 ], and that for liver transplant recipients, the more variable of the donor or recipient strains later became the dominant strain in the transplanted liver [ 66 ].
Others noted patients with different genotypes in PBMC and plasma [ 67 , 68 ]. Various combinations of tissues have been examined by a number of researchers Table 7d. Studies showed that various extrahepatic tissues each had variants that were more alike than variants in the other cell types, suggesting that each cell type has a pool of HCV that was a little different than the pool in the other cell types [ 58 , 69 — 74 ].
Three of the studies noted that different compartments at times contained different genotypes, as measured by the INNO-LiPA line probe assay. If tissues contain different variants, do these differences matter?
Both studies found mDC variants differed from liver variants. One of the studies investigated in vitro translation efficiency of the variants, and mDC variants showed reduced translation compared to the liver variants. To determine if different variants affected replication of HCV, quasispecies from brain, serum, and liver were analyzed for in vitro translation efficiency. They found that brain derived variants were translated less efficiently than serum or liver.
These studies therefore suggest that the quasispecies differences can affect translation efficiency, and presumably other aspects of HCV replication. Since HCV infection is associated with dementia, investigators have also examined infection of nervous system tissues. Two studies investigated sequences in liver, plasma, and brain and found minor differences in the 5'UTR and major differences in the E1 region [ 80 , 81 ]. Brain specific sequences were identified.
Two other studies looked at cerebral spinal fluid CSF and found different variants than serum in some patients [ 75 , 82 ], while a third, using SSCP, found CSF and plasma very similar [ 83 ].
Since SSCP isn't as sensitive as sequencing for detecting differences and only three patients were studied, it is unclear whether this study could have detected significant differences between these two tissues. In two related studies, brain tissue was compared to serum sequences by SSCP and sequencing from tissue obtained from cadavers [ 84 , 85 ]. Brain and serum variants were significantly different. The kinetics of HCV infection of tissues has also been investigated. Studies investigating the quasispecies in liver and serum found that some patients had significantly different quasispecies in the two samples, including the consensus sequences [ 86 , 87 ], but that the quasispecies complexity varied considerably over time [ 88 ].
Variants seemed to appear first in liver, then spread to serum, suggesting most originated in the liver and not in the serum.
However, the study could not answer which cells in the liver produce most of the HCV. One study which compared positive and negative strand liver variants and serum variants by SSCP and sequencing showed that four of the patients' liver negative strand variants and serum variants were very similar, while the other two patients had low titers of virus and unreliable results [ 89 ]. As the liver positive and negative strand variants differed significantly, it may be that most of the HCV RNA found in the liver may be inside cells but not replicating.
As there is 10 to times as much positive as negative strand in the liver, most of the virus could be in cells that don't productively replicate HCV, while it does replicate in another cell type. Nevertheless, this needs further study. A second study of HIV-HCV co-infected women compared cervical cytobrush and plasma and found unique variants in each tissue [ 91 ]. Overall, we analyzed 44 studies, with 43 showing distinct differences between variants found in two or more tissues in many of the patients examined.
Some also showed different genotypes in different tissues. One possibility is that these tissues bind and take in different HCV variants, but the virus doesn't replicate in these tissues or cell types. Since different tissues contain different quasispecies, studies have investigated how these populations change over time. A recent study followed quasispecies found in four patients over a span of up to 18 years [ 93 ].
They suggested there were four stages of HCV evolution: 1 HCV establishes an infection; 2 incremental evolution of variants within subpopulations; 3 diversification into new subpopulations; and 4 strong negative selection in these subpopulations reduces variation and HCV achieves a stable adaptation to the host.
Although the small number of patients in the study was a drawback, the model needs further investigation. A different method of studying temporal variation is to study the repopulation of livers after transplantation. Liver specimens had detectable amounts of negative strands by RT-PCR within 7 days after transplantation [ 94 ].
Levels of negative strand in the liver after transplants do not correlate with serum levels of HCV [ 94 , 95 ], and negative strands were more likely to be found in transplanted PBMC than in PBMC from individuals with chronic HCV infection [ 40 , 96 ].
We also examined seven studies that compared quasispecies before and after liver transplantation. These studies compared donor and recipient HCV, and determined which type was present in the livers of transplant recipients at various times after transplantation. Studies of liver re-infection by HCV have examined the donor and recipient strains to analyze this process. One early study showed that initially after one transplantation, both the donor and recipient HCV strains could be found in PBMC, but only the recipient strain was found in serum [ 97 ], and, after a week, only the recipient strain was still found in both serum and PBMC.
This suggested that one strain can take over the infection in an individual. Another comparison of variants with those found in the new livers showed that, for 3 of the 4 patients, liver variants were most similar to those found in serum [ 98 ], while another study compared variants in plasma and PBMC after transplantation and found frequent compartmentalization and a decrease in quasispecies complexity, which suggests a genetic bottleneck [ 99 ].
Two studies of liver re-infection compared donor and recipient HCV strains over time to show that one strain can overtake or exclude another [ 66 , ], with the more variable strain at the time of transplant overtaking the other strain.
A study by another group reached the same conclusions: the most variable strain s at the time of transplant was the one that was present much later [ ]. Overall, the data suggests that newly transplanted livers are infected by free virus or cells from the blood.
The strain of HCV with the most variants is more likely to be able to adapt to the new liver and outcompetes the other strain. How one strain out competes another is unknown. The vector can then be used in an in vitro system or inserted into cells using transfection. Luciferase is produced by translation of the reporter vector, so increased luminescence results from increased translation of the reporter gene. One study analyzed in vitro translation of HCV variants found in serum [ ].
The variants had different efficiencies for in vitro translation and also after transfection into Vero monkey kidney , HepG2 liver carcinoma , and Jurkat T cells. Some of the variants translated better in HepG2 and Vero cells, others in Jurkat cells, suggesting that some variants were better adapted to particular cell types. As B cells have frequently been found to be infected by HCV, one group investigated the in vitro translation efficiency of variants found in B cells and compared them to variants found in plasma but not B cells [ ].
These studies show that particular variants translate better in different cell types, supporting the hypothesis that HCV replicates extrahepatically. Although these studies have been detailed above relating to detection of HCV and quasispecies analysis, the effects of the co-infection on HCV replication have not been detailed. Here we examined two issues: 1 Does HCV replication increase or decrease in co-infected individuals? Only studies that compared HCV-mono-infected and co-infected individuals were examined.
Some studies have looked at extrahepatic replication in co-infected individuals. Three studies investigated the HCV positive strand load in extrahepatic tissues and found no significant differences in the average viral load in either tissue in HCV-infected and co-infected individuals [ 83 , ] or in the percentage of infected cells [ 45 ]. They did find a correlation of HCV negative-strands in the liver tissue and disease levels such as liver inflammation and fibrosis.
This could be due to reduced immune pressure on these individuals, or a greater host range of the virus in co-infected individuals. They suggested that the increased heterogeneity may be due to an enlarged viral replicative space, i.
Therefore, there do not appear to be dramatic differences in RNA levels, but co-infection appears to affect quasispecies and which cell types are infected by HCV. The effect of co-infection on the CNS has also been studied. One study of CSF in co-infected individuals found that of five individuals that had genotype 3a in serum and PBMC, two had only genotype 1b in CSF, while two others had a mix of 3a and 1b [ 75 ]. These studies suggest that co-infection may affect infection of the CNS.
Although the amount of HCV replication in co-infected individuals may not be significantly different, most quasispecies analyses showed variants in CSF, genital tracts, and PBMC that are not present in serum or liver.
In addition, co-infected individuals are more likely to have detectable HCV in extrahepatic tissues. Studies of how these viruses interact in these cell types will help us understand the interactions between these two viruses. We have analyzed studies that have used RT-PCR to detect negative strands in over a dozen different extrahepatic sites.
These studies provide overwhelming evidence that HCV negative strands are found outside the liver. Studies that used hybridization or antibody binding and detection were used to observe replication in individual cells. These studies, in theory, can determine exactly which cells are infected and contain replicating HCV.
Studies using liver biopsies haven't consistently found hepatocytes infected, and often find other positive cell types.
The sensitivity of these methods is low, and the possibility of both false positives and false negatives are high. The one consistent result from these studies is that only a small percentage of cells are infected by HCV in any particular tissue. Another method of investigating HCV replication is to sequence and compare HCV found in various tissues or cell types.
The analysis presented above shows rather conclusively that the sequences found in different tissues often differ from each other. Although one explanation is that different tissues bind different variants of HCV, the combination of the presence of negative strands of HCV and different sequences in tissues is very strong evidence that HCV replicates in a variety of tissues and cell types.
Although hepatocytes are definitely infected by HCV, we were unable to find significant evidence for replication in them.
However, the liver contains Kupffer cells macrophages , B cells, T cells, endothelial cells and others in addition to hepatocytes. Most studies used cell morphology to identify cell types, while only three studies that we examined used cell markers to identify infected cells. The second used antibodies to cell receptors to determine which cells were infected in livers [ ]. They found CD68 positive cells contained NS4.
Researchers should perform studies using double-labeling to determine which cell types in livers contain replicating HCV.
Liver transplants also provide a window into HCV replication. After liver transplantation, the transplanted liver is almost immediately repopulated by HCV. Studies we examined suggested that the variants repopulating the liver are derived originally from the liver that was in the individual. However, studies investigating the order of infection are scant.
Which cells are first infected in the liver? Does the reinfection come from cells in the blood or from free virus? Answers to these questions would either confirm or deny the prevailing idea that hepatocytes are the most important cell type for HCV replication. The significance of extrahepatic replication needs a lot more study. Some studies have suggested that extrahepatic replication occurs, but is of minor significance. Other studies suggest it has roles in a variety of diseases associated with HCV infection, including B cell lymphomas, hepatocellular carcinoma, and other diseases.
A number of studies have investigated the presence of negative strands and treatment outcome. One study, for example, suggested that negative strands in PBMC of mothers are correlated with transmission to their offspring, presumably through the presence of lymphocytes in breast milk [ 30 ].
This needs confirmation and further study. To fully understand the significance of extrahepatic replication, models must be proposed and in vitro studies performed. Studies analyzing in vitro replication can be broken down into two major groups: 1 short term cell culture demonstrating the presence of HCV in particular cell types and for studies of HCV replication; and 2 long term cell culture that has the goal of studying all aspects of HCV.
We will look at examples of each type. We can also break down studies by whether the investigators cultured infected cells from individuals, or infected fresh cultured cells with virus from patient serum or plasma. Last, studies of infection of various cell types have been conducted.
There have been numerous reports of in vitro systems, particularly using cultured hepatocytes. Most of these haven't proven useful for further study. Therefore, we have chosen to only discuss systems that have been used enough to produce more than one paper, or ones that illustrate methods or infections of extrahepatic cell types.
One method of studying HCV replication in vitro is to start with a mixture of cell types found in blood. One early study showed that PBMC could sustain an infection by HCV for about 25 days, with a peak of infection around the second or third week [ ]. Viral titers were low. This suggested that the culture mimicked conditions in the blood.
In vitro translation of these variants showed greater translation in B and T cell lines compared to HCV strain H77, but not in monocytes or granulocytes. Some HCV-infected individuals that have been treated have undetectable amounts of HCV in the serum and plasma using standard methods.
The viral RNA prepares to reproduce. It may also prevent your liver cell from functioning properly. Sometimes, the viral RNA causes your liver cell to reproduce, too. Things amp up as the viral RNA constructs a template for replicating itself. The viral RNA is cloned over and over to create new viruses.
These are developed by ribosomes, or cell protein builders, during this stage and released. Protein units called capsomeres come together and form new particles around the viral RNA. These make a covering shaped like a sphere, known as a capsid. In the final stage, the new virus creates a bud with itself inside. A protective coating surrounds the bud.
This process continues until the infected liver cell dies. RNA viruses evolve faster than other organisms. For some, the virus will clear up on its own. About 75 to 85 percent of people infected with HCV go on to develop chronic infection. Recent advances in HCV treatment have made it possible for people with chronic hepatitis C to clear the virus. These advances have made treatment more effective, as well as reducing side effects and shortening the duration of therapy.
If you have hepatitis C, talk with your doctor about the best treatment plan for you. After determining the correct course of medication, they can help you navigate the different programs and policies available to help cover the cost of treatment. Keep reading: What you should know about hepatitis C treatment costs ».
Viremia is a medical term for viruses present in the bloodstream. Learn about the effects of various viruses along with symptoms and treatment. The terms in vivo and in vitro refer to how certain studies, laboratory experiments, and medical procedures are performed. One example is in vitro….
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