HIV pseudotypes bearing native hepatitis C virus (HCV) glycoproteins (strain H and Con1) are infectious for the human hepatoma cell lines Huh-7 and PLC͞PR5. Infectivity depends on coexpression of both E1 and E2 glycoproteins, is pH-dependent, and can be neutralized by mAbs mapping to amino acids 412-447 within E2. Cell-surface expression of one or all of the candidate receptor molecules (CD81, low-density lipoprotein receptor, scavenger receptor class B type 1, and dendritic cell-specific intercellular adhesion molecule 3 grabbing nonintegrin) failed to confer permissivity to HIV-HCV pseudotype infection. However, HIV-HCV pseudotype infectivity was inhibited by a recombinant soluble form of CD81 and a mAb specific for CD81, suggesting that CD81 may be a component of a receptor complex. Hepatitis C virus (HCV) is an enveloped, positive-stranded RNA virus classified in the family Flaviviridae. Infection is often associated with chronic disease, sometimes resulting in cirrhosis and hepatocellular carcinoma. The principal site of replication is thought to be the liver, although several laboratories have suggested that HCV may infect a wider range of cell types including monocytes͞macrophages and B cells (1, 2).HCV encodes two putative envelope glycoproteins (gps), E1 and E2, which are believed to be type I integral transmembrane proteins. In vitro expression studies have shown that both gps associate to form heterodimers, which accumulate in the endoplasmic reticulum (ER), the proposed site for HCV assembly and budding (reviewed in ref.3). The lack of in vitro systems for HCV propagation has hampered biological and physiochemical studies on the virion and its mechanism(s) of cell entry, and the cellular receptors remain unknown. HCV purified from plasma has been reported to exist in association with plasma lipoproteins, suggesting that the virus may use the low-density lipoprotein receptor (LDLR) to gain entry into cells (4-6).The selective association of a virus with a target cell is usually determined by an interaction between the viral gps and specific cell-surface receptor(s) and is an essential step in the initiation of infection. Such interaction(s) often define the host range and cellular or tissue tropism of a virus and have a role in determining virus pathogenicity. In the absence of native HCV particles, truncated version(s) of the E2 gp (7, 8), E1E2 gp-liposomes (9), and virus-like particles expressed in insect cell systems (10, 11) have been used as mimics to study virus-cell interactions. Truncated soluble versions of E2 have been reported to bind specifically to human cells and were used to identify interactions with CD81 (7, 8), scavenger receptor class B type 1 (SR-B1) (12), and dendritic cell-specific intercellular adhesion molecule 3 grabbing nonintegrin (DC-SIGN) (13,14). One limitation with these studies is that they measure only HCV gp-cell attachment and not virus-mediated cell fusion. To overcome the lack of a conventional cell culture system for the propagation of infectious HCV particles, ps...

Monotherapy with (-)2',3'-dideoxy-3'-thiacytidine (3TC) leads to the appearance of a drug-resistant variant of human immunodeficiency virus-type 1 (HIV-1) with the methionine-184 --> valine (M184V) substitution in the reverse transcriptase (RT). Despite resulting drug resistance, treatment for more than 48 weeks is associated with a lower plasma viral burden than that at baseline. Studies to investigate this apparent contradiction revealed the following. (i) Titers of HIV-neutralizing antibodies remained stable in 3TC-treated individuals in contrast to rapid declines in those treated with azidothymidine (AZT). (ii) Unlike wild-type HIV, growth of M184V HIV in cell culture in the presence of d4T, AZT, Nevirapine, Delavirdine, or Saquinavir did not select for variants displaying drug resistance. (iii) There was an increase in fidelity of nucleotide insertion by the M184V mutant compared with wild-type enzyme.

Comprehensive monitoring of genotypic and phenotypic antiviral resistance was performed on 673 entecavir (ETV)-treated nucleoside naïve hepatitis B virus (HBV) patients. ETV reduced HBV DNA levels to undetectable by PCR (<300 copies/mL, <57 IU/mL) in 91% of hepatitis B e antigen (HBeAg)-positive and -negative patients by Week 96. Thirteen percent (n ‫؍‬ 88) of the comparator lamivudine (LVD)-treated patients experienced a virologic rebound (>1 log increase from nadir by PCR) in the first year, with 74% of these having LVD resistance (LVDr) substitutions evident. In contrast, only 3% (n ‫؍‬ 22) of ETV-treated patients exhibited virologic rebound by Week 96. Three ETV rebounds were attributable to LVDr virus present at baseline, with one having a S202G ETV resistance (ETVr) substitution emerge at Week 48. None of the other rebounding patients had emerging genotypic resistance or loss of ETV susceptibility. Genotyping all additional ETV patients with PCR-detectable HBV DNA at Weeks 48, 96, or end of dosing identified seven additional patients with LVDr substitutions, including one with simultaneous emergence of LVDr/ETVr. Generally, ETV patients with LVDr were detectable at baseline (8/10) and most subsequently achieved undetectable HBV DNA levels on ETV therapy (7/10). No other emerging substitutions identified decreased ETV susceptibility. In conclusion, ETVr emergence in ETV-treated nucleoside naïve patients over a 2-year period is rare, occurring in two patients with LVDr variants. These findings suggest that the rapid, sustained suppression of HBV replication, combined with a requirement for multiple substitutions, creates a high genetic barrier to ETVr in nucleoside naïve patients.

Entecavir (ETV) is a deoxyguanosine analog approved for use for the treatment of chronic infection with wild-type and lamivudine-resistant (LVDr) hepatitis B virus (HBV).In LVD-refractory patients, 1.0 mg ETV suppressed HBV DNA levels to below the level of detection by PCR (<300 copies/ml) in 21% and 34% of patients by Weeks 48 and 96, respectively. Prior studies showed that virologic rebound due to ETV resistance (ETVr) required preexisting LVDr HBV reverse transcriptase substitutions M204V and L180M plus additional changes at T184, S202, or M250. To monitor for resistance, available isolates from 192 ETV-treated patients were sequenced, with phenotyping performed for all isolates with all emerging substitutions, in addition to isolates from all patients experiencing virologic rebounds. The T184, S202, or M250 substitution was found in LVDr HBV at baseline in 6% of patients and emerged in isolates from another 11/187 (6%) and 12/151 (8%) ETV-treated patients by Weeks 48 and 96, respectively. However, use of a more sensitive PCR assay detected many of the emerging changes at baseline, suggesting that they originated during LVD therapy. Only a subset of the changes in ETVr isolates altered their susceptibilities, and virtually all isolates were significantly replication impaired in vitro. Consequently, only 2/187 (1%) patients experienced ETVr rebounds in year 1, with an additional 14/151 (9%) patients experiencing ETVr rebounds in year 2. Isolates from all 16 patients with rebounds were LVDr and harbored the T184 and/or S202 change. Seventeen other novel substitutions emerged during ETV therapy, but none reduced the susceptibility to ETV or resulted in a rebound. In summary, ETV was effective in LVD-refractory patients, with resistant sequences arising from a subset of patients harboring preexisting LVDr/ETVr variants and with approximately half of the patients experiencing a virologic rebound.More than 350 million people worldwide are chronically infected with hepatitis B virus (HBV) (32); and many will ultimately develop severe liver disease, including cirrhosis, hepatocellular carcinoma, and liver failure. Significant improvements in patient outcomes have been realized since the use of antiviral therapy for HBV. Due to the poor efficacies of these therapies and the emergence of viral resistance, however, additional therapies are needed (16). Prior to 2005, HBV therapies included parenteral regimens containing interferon alfa and the oral nucleoside/nucleotide analogs lamivudine (LVD) and adefovir dipivoxil (ADV). However, interferon alfa shows poor response rates and poor sustained efficacy (ϳ30 to 40% [reviewed in reference 18]), has low tolerability, and is contraindicated in patients with decompensated liver disease. LVD and ADV are associated with the development of viral resistance. LVD resistance (LVDr) is reported to occur in 24% of patients treated for 1 year, and this rate increases to 70% after 4 years (19). The rate of ADV resistance (ADVr) in nucleoside-naïve HBeAg-negative HBV patients has been r...