HIV-1 release is mediated through two motifs in the p6 region of Gag, PTAP and LYPXnL, which recruit cellular proteins Tsg101 and Alix, respectively. The Nucleocapsid region of Gag (NC), which binds the Bro1 domain of Alix, also plays an important role in HIV-1 release, but the underlying mechanism remains unclear. Here we show that the first 202 residues of the Bro1 domain (Broi) are sufficient to bind Gag. Broi interferes with HIV-1 release in an NC–dependent manner and arrests viral budding at the plasma membrane. Similar interrupted budding structures are seen following over-expression of a fragment containing Bro1 with the adjacent V domain (Bro1-V). Although only Bro1-V contains binding determinants for CHMP4, both Broi and Bro1-V inhibited release via both the PTAP/Tsg101 and the LYPXnL/Alix pathways, suggesting that they interfere with a key step in HIV-1 release. Remarkably, we found that over-expression of Bro1 rescued the release of HIV-1 lacking both L domains. This rescue required the N-terminal region of the NC domain in Gag and the CHMP4 binding site in Bro1. Interestingly, release defects due to mutations in NC that prevented Bro1 mediated rescue of virus egress were rescued by providing a link to the ESCRT machinery via Nedd4.2s over-expression. Our data support a model in which NC cooperates with PTAP in the recruitment of cellular proteins necessary for its L domain activity and binds the Bro1–CHMP4 complex required for LYPXnL–mediated budding.

Chronic hepatitis B and D infections are major causes of liver disease and hepatocellular carcinoma worldwide. Efficient therapeutic approaches for cure are absent. Sharing the same envelope proteins, hepatitis B virus and hepatitis delta virus use the sodium/ taurocholate cotransporting polypeptide (a bile acid transporter) as a receptor to enter hepatocytes. However, the detailed mechanisms of the viral entry process are still poorly understood. Here, we established a high-throughput infectious cell culture model enabling functional genomics of hepatitis delta virus entry and infection. Using a targeted RNA interference entry screen, we identified glypican 5 as a common host cell entry factor for hepatitis B and delta viruses. 2 Among these, 5%-10% are likely coinfected with hepatitis delta virus (HDV) and exhibit an increased HCC risk.3 HDV is a small RNA satellite virus of HBV that uses the HBV envelope proteins to assemble into infectious particles and enter its target cell. 4 Nucleos(t)ide analogues and interferon-based treatment can control HBV infection, but virus eradication and cure remain largely unattainable. 5 While HDV can partially respond to interferon-based treatment, 6 long-term response is marginal.6,7

The envelope proteins of hepatitis B virus (HBV) bear an N-linked glycosylation site at N146 within the immunodominant a-determinant in the antigenic loop (AGL) region. This glycosylation site is never fully functional, leading to a nearly 1/1 ratio of glycosylated/nonglycosylated isoforms in the viral envelope. Here we investigated the requirement for a precise positioning of Nlinked glycan at amino acid 146 and the functions associated with the glycosylated and nonglycosylated isoforms. We observed that the removal of the N146 glycosylation site by mutagenesis was permissive to envelope protein synthesis and stability and to secretion of subviral particles (SVPs) and hepatitis delta virus (HDV) virions, but it was detrimental to HBV virion production. Several positions in the AGL could substitute for position 146 as the glycosylation acceptor site. At position 146, neither a glycan chain nor asparagine was absolutely required for infectivity, but there was a preference for a polar residue. Envelope proteins bearing 5 AGL glycosylation sites became hyperglycosylated, leading to an increased capacity for SVP secretion at the expense of HBV and HDV virion secretion. Infectivity-compatible N-glycosylation sites could be inserted at 3 positions (positions 115, 129, and 136), but when all three positions were glycosylated, the hyperglycosylated mutant was substantially attenuated at viral entry, while it acquired resistance to neutralizing antibodies. Taken together, these findings suggest that the nonglycosylated N146 is essential for infectivity, while the glycosylated form, in addition to its importance for HBV virion secretion, is instrumental in shielding the a-determinant from neutralizing antibodies. IMPORTANCEAt the surface of HBV particles, the immunodominant a-determinant is the main target of neutralizing antibodies and an essential determinant of infectivity. It contains an N-glycosylation site at position 146, which is functional on only half of the envelope proteins. Our data suggest that the coexistence of nonglycosylated and glycosylated N146 at the surface of HBV reflects the dual function of this determinant in infectivity and immune escape. Hence, a modification of the HBV glycosylation pattern affects not only virion assembly and infectivity but also immune escape.T he hepatitis B virus (HBV) is an enveloped DNA virus and the prototype of the Hepadnaviridae family. HBV is characterized by a strict tropism for human hepatocytes and the ability to cause acute and chronic infections. It is estimated that worldwide, approximately 240 million individuals are HBV chronic carriers and are at risk of developing liver cirrhosis and hepatocellular carcinoma (1). HBV hepatotropism is determined, for the most part, by the HBV envelope proteins at viral entry. A single open reading frame in the HBV genome encodes three envelope proteins that differ from each other by the size of their N-terminal ectodomain. They bear the HBV surface antigen (HBsAg) in their common S domain and are referred to as the l...

Hepatitis delta virus (HDV) particles are coated with the envelope proteins (large, middle, and small) of the hepatitis B virus (HBV). The large protein bears an infectivity determinant in its pre-S1 domain, whereas a second determinant has been proposed to map to the cysteine-rich antigenic loop (AGL) within the S domain of all three envelope proteins (G. Abou Jaoudé and C. Sureau, J. Virol. 79:10460-10466, 2006). In this study, the AGL cysteines were substituted by serine or alanine, and the mutants were evaluated for their function at viral entry using HDV particles and susceptible HepaRG cells. Mutations of cysteines 121 to 149 were tolerant of the production of HDV virions. The mutations altered the structure and antigenicity of the conserved "a" determinant of the AGL, as measured by conformation-sensitive antibodies, and they created a block to infectivity. Substitution of Cys-90 or Cys-221, located outside of the AGL, had no impact on the "a" determinant or viral entry. Furthermore, infectivity was maintained when the AGL CxxC motif at position 121 to 124 was modified by single-amino-acid deletion or insertion, suggesting that cysteines 121 and 124 are not catalyzers of thiol/disulfide exchange. However, membrane-impermeable inhibitors of thiol/disulfide isomerazation demonstrated a dose-dependent inhibition of infection in an in vitro assay when applied to the virus prior to inoculation or during the virus-cell interaction period. Overall, the results demonstrate the essential role of the AGL cysteines at viral entry, and they establish a correlation between the cysteine disulfide network, the conformation of the "a" determinant, and infectivity.Hepatitis B virus (HBV) is responsible for acute and chronic liver disease that affects more than 400 million individuals worldwide (12). HBV is characterized by a very narrow host range that is likely to reflect a highly specific interaction between the viral envelope proteins and the receptor(s) at the surface of human hepatocytes. The mechanism of HBV entry is still poorly understood-the receptor remains unknownand only recently have determinants of infectivity been mapped to discrete domains within the amino acid sequence of the envelope proteins (2,4,16,24). HBV particles bear three sequence-related envelope proteins: the small protein (SHBsAg) consists of a 226-amino-acid-long transmembrane protein, the middle protein (M-HBsAg) includes the S domain and an additional N-terminal pre-S2 ectodomain that is 55 amino acids in length, and the large protein (L-HBsAg) comprises a N-terminal pre-S1 domain (109 amino acids) in addition to pre-S2 and S domains (20). Synthesis occurs at the endoplasmic reticulum (ER) membrane, but it leads almost exclusively to the secretion of empty subviral particles (SVPs). The assembly of mature HBV virions is a rare event that results from an interaction between the matrix domain of LHBsAg and the HBV nucleocapsid (6).The HBV envelope proteins also have the capacity to interact with the hepatitis delta virus (HDV) ribonucleoprote...