Two strategies underlying the trade-off of hepatitis C virus proliferation: stay-at-home or leaving-home?
Shoya IwanamiKosaku KitagawaYusuke AsaiHirofumi OhashiKazane NishiokaHisashi InabaShinji NakaokaTakaji WakitaOdo DiekmannShingo IwamiKoichi Watashi
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Abstract Viruses proliferate through both genome replication inside infected cells and transmission to new target cells or to new hosts. Each viral genome molecule in infected cells is used either for amplifying the intracellular genome as a template (“stay-at-home strategy”) or for packaging into progeny virions to be released extracellularly (“leaving-home strategy”). The balance between these strategies is important for both initial growth and transmission of viruses. In this study, we used hepatitis C virus (HCV) as a model system to study the functions of viral genomic RNA in both RNA replication in cells and in progeny virus assembly and release. Using viral infection assays combined with mathematical modelling, we characterized the dynamics of two different HCV strains (JFH-1, a clinical isolate, and Jc1-n, a laboratory strain), which have different viral assembly and release characteristics. We found that 1.27% and 3.28% of JFH-1 and Jc1-n intracellular viral RNAs, respectively, are used for producing and releasing progeny virions. Analysis of the Malthusian parameter of the HCV genome (i.e., initial growth rate) and the number of de novo infections (i.e., initial transmissibility) suggests that the leaving-home strategy provides a higher level of initial transmission for Jc1-n, while, in contrast, the stay-at-home strategy provides a higher initial growth rate for JFH-1. Thus, theoretical-experimental analysis of viral dynamics enables us to better understand the proliferation strategies of viruses. Ours is the first study to analyze stay-leave trade-offs during the viral life cycle and their significance for viral proliferation.ABSTRACT Although antiviral agents which block human immunodeficiency virus (HIV) replication can result in long-term suppression of viral loads to undetectable levels in plasma, long-term therapy fails to eradicate virus, which generally rebounds after a single treatment interruption. Multiple structured treatment interruptions (STIs) have been suggested as a possible strategy that may boost HIV-specific immune responses and control viral replication. We analyze viral dynamics during four consecutive STI cycles in 12 chronically infected patients with a history (>2 years) of viral suppression under highly active antiretroviral therapy. We fitted a simple model of viral rebound to the viral load data from each patient by using a novel statistical approach that allows us to overcome problems of estimating viral dynamics parameters when there are many viral load measurements below the limit of detection. There is an approximate halving of the average viral growth rate between the first and fourth STI cycles, yet the average time between treatment interruption and detection of viral loads in the plasma is approximately the same in the first and fourth interruptions. We hypothesize that reseeding of viral reservoirs during treatment interruptions can account for this discrepancy, although factors such as stochastic effects and the strength of HIV-specific immune responses may also affect the time to viral rebound. We also demonstrate spontaneous drops in viral load in later STIs, which reflect fluctuations in the rates of viral production and/or clearance that may be caused by a complex interaction between virus and target cells and/or immune responses.
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Efforts to treat HCV patients are focused on developing antiviral combinations that lead to the eradication of infection. Thus, it is important to identify optimal combinations from the various viral inhibitor classes. Based on viral dynamic models, HCV entry inhibitors are predicted to reduce viral load in a monophasic manner reflecting the slow death rate of infected hepatocytes (t1/2 = 2-70 days) and the protection of naïve, un-infected cells from HCV infection. In contrast, replication inhibitors are predicted to reduce viral load in a biphasic manner. The initial rapid reduction phase is due to the inhibition of virus production and elimination of plasma virus (t1/2∼3 hours). The second, slower reduction phase results from the elimination of infected hepatocytes. Here we sought to compare the ability of HCV entry and replication inhibitors as well as combinations thereof to reduce HCV infection in persistently-infected Huh7 cells. Treatment with 5 × EC50 of entry inhibitors anti-CD81 Ab or EI-1 resulted in modest (≤ 1 log10 RNA copies/ml), monophasic declines in viral levels during 3 weeks of treatment. In contrast, treatment with 5 × EC50 of the replication inhibitors BILN-2016 or BMS-790052 reduced extracellular virus levels more potently (~2 log10 RNA copies/ml) over time in a biphasic manner. However, this was followed by a slow rise to steady-state virus levels due to the emergence of resistance mutations. Combining an entry inhibitor with a replication inhibitor did not substantially enhance the rate of virus reduction. However, entry/replication inhibitor and replication/replication inhibitor combinations reduced viral levels further than monotherapies (up to 3 log10 RNA copies/ml) and prolonged this reduction relative to monotherapies. Our results demonstrated that HCV entry inhibitors combined with replication inhibitors can prolong antiviral suppression, likely due to the delay of viral resistance emergence.
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HIV entry inhibitors such as maraviroc (MVC) prevent cell-free viruses from 24 entering the cells. In clinical trials, patients who were treated with MVC often 25 displayed viral loads that were above the limit of conventional viral load detection 26 in comparison with efavirenz-based regminens. We hypothesize that viruses 27 blocked by entry inhibitors may be redistri buted to plasma where they artificially 28 increase viral load measurements in contrast to use of antiretroviral drugs 29 (ARVs) that act intracellularly. 30 We infected PM-1 cells with CCR5-tropi c HIV-1 BaL or CXCR 4-tropic HIV-1 NL4-31 3 in the presence of inhibitory concentrati ons of efavirenz, ralt egravir, enfuvirtide, 32 maraviroc, and AMD3100, the latter three being entry inhibitors. Supernatant 33 viral load, reverse transcriptase enzyme activity, and intracellular nucleic acid 34 levels were measured at times up to 24 hr post-infection. Infectivity of 35 redistributed dual-tropic HIV-1 wa s assessed using TZM-bl cells. 36 Extracellular viral load analysis revealed that entry inhibitor-treated cells had 37 higher levels of virus in supernatant versus the other ARVs at 8 hours post-38 infection. By 24 hours, supernatant viral load was still higher for entry inhibitors
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The cytoplasmic tail (gp41CT) of the HIV-1 envelope (Env) mediates Env incorporation into virions and regulates Env intracellular trafficking. Little is known about the functional impact of variability in this domain. To address this issue, we compared the replication of recombinant virus pairs carrying the full Env (Env viruses) or the Env ectodomain fused to the gp41CT of NL4.3 (EnvEC viruses) (12 subtype C and 10 subtype B pairs) in primary CD4+ T-cells and monocyte-derived-macrophages (MDMs). In CD4+ T-cells, replication was as follows: B-EnvEC = B-Env>C-EnvEC>C-Env, indicating that the gp41CT of subtype C contributes to the low replicative capacity of this subtype. In MDMs, in contrast, replication capacity was comparable for all viruses regardless of subtype and of gp41CT. In CD4+ T-cells, viral entry, viral release and viral gene expression were similar. However, infectivity of free virions and cell-to-cell transmission of C-Env viruses released by CD4+ T-cells was lower, suggestive of lower Env incorporation into virions. Subtype C matrix only minimally rescued viral replication and failed to restore infectivity of free viruses and cell-to-cell transmission. Taken together, these results show that polymorphisms in the gp41CT contribute to viral replication capacity and suggest that the number of Env spikes per virion may vary across subtypes. These findings should be taken into consideration in the design of vaccines.
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Reliance on hepatitis C virus (HCV) replicon systems and protein-based screening assays has led to treatments that target HCV viral replication proteins. The model does not encompass other viral replication cycle steps such as entry, processing, assembly and secretion, or viral host factors. We previously applied a phenotypic high-throughput screening platform based on an infectious HCV system and discovered an aryloxazole-based anti-HCV hit. Structure-activity relationship studies revealed several compounds exhibiting EC50 values below 100 nM. Lead compounds showed inhibition of the HCV pseudoparticle entry, suggesting a different mode of action from existing HCV drugs. Hit 7a and lead 7ii both showed synergistic effects in combination with existing HCV drugs. In vivo pharmacokinetics studies of 7ii showed high liver distribution and long half-life without obvious hepatotoxicity. The lead compounds are promising as preclinical candidates for the treatment of HCV infection and as molecular probes to study HCV pathogenesis.
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In a typical HIV-1-infected patient, plasma viral load (pVL) increases steeply in the first week after acute infection, then decreases as the immune system becomes activated, resulting in antibody seroconversion 3–13 days after infection and a full western blot pattern approximately 3 months later [1–3]. The so-called viral set point or steady-state viral load is reached after approximately 40–276 days from the acute infection moment [1]. Especially in the first few weeks of infection, differences are obvious in patients, especially with regard to time to peak load and time to viral load drop from peak to nadir [1], but also in the absolute viral RNA count. The viral set point is thought to represent a trade-off between viral replication capacity and repression of the virus by the host immune system. HIV-1 RNA levels vary considerably between individuals and also throughout the infection course in a particular individual. The viral load at set point is an important parameter, as it is strongly predictive of clinical progression [4,5]. Both the innate replicating capacity (fitness) of the virus strain and the strength of the host immune system would intuitively be the most obvious contributors, but it has been suggested that age, sex, shared human leukocyte antigen (HLA) alleles and duration of infection also contribute to the phenomenon [6]. The involvement of virus characteristics could easily be measured by analyzing the HIV replication capacity in donor–recipient pairs, wherein the viral load should be similar if viral replication fitness is the main determinant of pVL. A cohort of transmission pairs, necessary to study comparative HIV-1 viral load dynamics, is not easy to establish. Viral relationships indicative of transmission should first be determined by phylogenetic analysis. Then, an acute phase plasma sample (to minimize the effect of immune pressure) of the recipient and a matching sample from the donor should be available. Hecht et al. [7] have analyzed early plasma samples from 24 such transmission pairs, all comprising men having sex with men (MSM), and reported a significant correlation between the HIV-1 RNA levels within the transmission pairs. However, they cautioned that these results should be reproduced in other cohorts to validate the finding. We here report a similar analysis in early samples from 56 sequence-verified HIV-1 transmission pairs, 60% MSM and 40% heterosexual, from The Netherlands. Recipients were sampled during primary infection, 20 recipients were in Fiebig et al. [8] stages III–IV (viral RNA+/− or indeterminate western blot) and 36 recipients were in Fiebig et al. [8] stages V (viral RNA+/western blot p31−) and VI (viral RNA+/western blot fully developed). HIV-1 blood pVL measurements were done using the Versant HIV-1 RNA 3.0 assay (Bayer Diagnostics Division, Tarrytown, New York, USA), NucliSens HIV-1 RNA (bioMérieux, Boxtel, The Netherlands) or m2000rt (Abbott Molecular Inc., Des Plaines, Illinois, USA). Viral loads of all couples were measured using the same assay. Samples from donors matched the time point when recipient samples were taken. Linear regression analysis was done with GraphPad Prism, version 5.01 (GraphPad Software, San Diego, California, USA) and correlation coefficients were calculated. In contrast to Hecht et al. [7], we do not find a strong correlation between plasma viral RNA levels within the pairs (Fig. 1). The Pearson coefficient of correlation (r) in our cohort was 0.25 for all 56 transmission pairs, 0.29 (range −0.17 to 0.65) for pairs when the recipients were in Fiebig et al. [8] stages III–IV and 0.06 (range −0.27 to 0.39) for pairs when the recipients were in Fiebig et al. [8] stages V–VI, suggesting that the correlation is completely lost when the infection progresses. The correlation coefficient (r) between viral RNA levels in donors and recipients was 0.55 in the 24 pairs studied by Hecht et al. [7], which were in similar early stages of HIV infection. A correlation coefficient (r) above 0.8 is usually denoted as strong and below 0.5 as weak, whereas r is equal to 1 represents a perfect correlation. So, in our transmission pairs, we detect only a weak correlation between viral RNA levels in acutely infected recipients and donors. Similar results were obtained for a transmission cohort [6] in Zambia where the viral RNA levels between 115 donor and seroconverting recipient pairs had a correlation coefficient (r) of 0.21 (P = 0.03). In this study, factors such as sex, age, HLA markers and duration of infection were also shown to contribute.Fig. 1: Relationship of HIV-1 RNA levels in 56 transmission pairs. Viral RNA levels in blood plasma from source individuals were correlated with viral RNA levels in recipients in the acute or early stages of infection. Correlations are shown for all 56 transmission pairs or for sources and recipients when the latter are separated according to the primary infection stage criteria of Fiebig et al. [8].The low correlation between pVL in donors and recipients suggests that viral traits do contribute to pVL early in infection, but that other factors are equally or more important.
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Two to three percent of the world's population is chronically infected with hepatitis C virus (HCV) and thus at risk of developing liver cancer. Although precise mechanisms regulating HCV entry into hepatic cells are still unknown, several cell surface proteins have been identified as entry factors for this virus. Among these molecules, the tetraspanin CD81 is essential for HCV entry. Here, we have identified a partner of CD81, EWI-2wint, which is expressed in several cell lines but not in hepatocytes. Ectopic expression of EWI-2wint in a hepatoma cell line susceptible to HCV infection blocked viral entry by inhibiting the interaction between the HCV envelope glycoproteins and CD81. This finding suggests that, in addition to the presence of specific entry factors in the hepatocytes, the lack of a specific inhibitor can contribute to the hepatotropism of HCV. This is the first example of a pathogen gaining entry into host cells that lack a specific inhibitory factor.
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