BackgroundThe human immunodeficiency virus type 1 (HIV-1) Vpu protein degrades CD4 and counteracts a restriction factor termed tetherin (CD317; Bst-2) to enhance virion release. It has been suggested that both functions can be genetically separated by mutation of a serine residue at position 52. However, recent data suggest that the S52 phosphorylation site is also important for the ability of Vpu to counteract tetherin. To clarify this issue, we performed a comprehensive analysis of HIV-1 with a mutated casein kinase-II phosphorylation site in Vpu in various cell lines, primary blood lymphocytes (PBL), monocyte-derived macrophages (MDM) and ex vivo human lymphoid tissue (HLT).ResultsWe show that mutation of serine 52 to alanine (S52A) entirely disrupts Vpu-mediated degradation of CD4 and strongly impairs its ability to antagonize tetherin. Furthermore, casein-kinase II inhibitors blocked the ability of Vpu to degrade tetherin. Overall, Vpu S52A could only overcome low levels of tetherin, and its activity decreased in a manner dependent on the amount of transiently or endogenously expressed tetherin. As a consequence, the S52A Vpu mutant virus was unable to replicate in macrophages, which express high levels of this restriction factor. In contrast, HIV-1 Vpu S52A caused CD4+ T-cell depletion and spread efficiently in ex vivo human lymphoid tissue and PBL, most likely because these cells express comparably low levels of tetherin.ConclusionOur data explain why the effect of the S52A mutation in Vpu on virus release is cell-type dependent and suggest that a reduced ability of Vpu to counteract tetherin impairs HIV-1 replication in macrophages, but not in tissue CD4+ T cells.

Hepatitis B virus with a G145R mutation in the small surface protein is considered the quintessential immune escape mutant because it frequently is found in vaccinated individuals with breakthrough infections and liver transplant recipients under anti-hepatitis B surface antigen (HBsAg) immunoglobulin prophylaxis. Nowadays the prevalence of the variant progressively increases. However, because spread of a virus depends not only on immune pressure but also on the viral phenotype, we investigated the biologic properties of the G145R variant. The G145R mutation was introduced into wild-type (Wt) virus genome by in vitro mutagenesis. After transfection into human hepatoma cells, the DNA, RNA, and protein synthesis and viral secretion ability of the mutant were studied. Furthermore, cotransfection studies were performed with the G145R variant and a Wt virus S-protein expressing construct and vice versa. Production and stability of viral messenger RNAs (mRNAs), DNA, and proteins were not affected by the G145R mutation. In contrast, secretion of mutant virions was reduced significantly. Only 20% of virions were found in the medium of G145R variant-transfected cells compared with Wt virus. Furthermore, mutant virions were more sensitive to detergent treatment suggesting a diminished stability. In cotransfection studies, Wt virus S-protein rescued secretion of mutant virions, whereas mutant S-protein had a transdominant negative effect on secretion of Wt virus. Both mechanisms may support persistence of the defective mutant in a mixed population with Wt virus. In conclusion, the significant defect of the G145R mutant for secretion of infectious virions and the diminished stability of mutant virions may limit global spread of the mutant. H epatitis B virus (HBV) exhibits a mutation rate more than 4 orders of magnitude higher than that of other DNA viruses. 1 Naturally occurring variants may accumulate over time if they have a survival benefit over wild-type (Wt) virus, such as a higher ability to replicate DNA, an enhanced secretion ability, an increased infectivity or stability of virions, and so forth. In addition, certain variants may be selected under immune pressure or therapy with antiviral drugs.Nowadays, immune-escape variants that emerge under active and/or passive vaccination and are responsible for vaccination-breakthrough infections are of particular clinical relevance. Several such mutants carrying single or multiple amino acid substitutions in the major antigenic region of the virus, the a-determinant of the small surface protein, have been described. [2][3][4] The mutant by far most frequently detected all over the world in vaccinated individuals and liver transplant recipients treated with monoclonal or polyclonal anti-hepatitis B surface antigen (HBsAg) hyperimmunoglobulin carries a glycine to arginine substitution at amino acid position 145 (G145R). 3,[5][6][7][8][9][10][11] There is no doubt that despite selection of immuneescape variants, vaccination programs have so far been highly effective in reduci...

Hepatitis B viruses exhibit a narrow host range specificity that is believed to be mediated by a domain of the large surface protein, designated L. For duck hepatitis B virus, it has been shown that the pre-S domain of L binds to carboxypeptidase D, a cellular receptor present in many species on a wide variety of cell types. Nonetheless, only hepatocytes become infected. It has remained vague which viral features determine host range specificity and organotropicity. By using chymotrypsin to treat duck hepatitis B virus, we addressed the question of whether a putative fusogenic region within the amino-terminal end of the small surface protein may participate in viral entry and possibly constitute one of the determinants of the host range of the virus. Addition of the enzyme to virions resulted in increased infectivity. Remarkably, even remnants of enzyme-treated subviral particles proved to be inhibitory to infection. A noninfectious deletion mutant devoid of the binding region for carboxypeptidase D could be rendered infectious for primary duck hepatocytes by treatment with chymotrypsin. Although because of the protease treatment mutant and wild-type viruses may have become infectious in an unspecific and receptor-independent manner, their host range specificity was not affected, as shown by the inability of the virus to replicate in different hepatoma cell lines, as well as primary chicken hepatocytes. Instead, the organotropicity of the virus could be reduced, which was demonstrated by infection of primary duck kidney cells.The family Hepadnaviridae, with the human hepatitis B virus (HBV) as the prototype member, consists of small, DNAcontaining, enveloped viruses which cause liver diseases in humans, rodents, and birds (22). A characteristic feature of HBV and other hepadnaviruses is a strong species specificity where, e.g., HBV infects humans and chimpanzees (1) and duck HBV (DHBV) infects only certain duck and goose species (49). Moreover, all of the hepadnaviruses show relatively strict organotropicity, infecting mainly hepatocytes.Facilitated by the identification of neutralizing epitopes (12) and the allocation of host range-specific determinants to the pre-S region of L (29), it was possible to demonstrate the binding of DHBV to a putative hepatocellular receptor molecule via a certain domain on pre-S (7,8,30,39,63). This receptor molecule has been identified as gp180, a transmembrane protein of the carboxypeptidase D family that, however, has also been found in nonliver duck tissues that are not susceptible to DHBV infection (16,38,62). Furthermore, it has been shown that LMH cells, a chicken hepatoma cell line which resists infection but is capable of virus production after transfection of full-length DHBV DNA, also express gp180 (38). In addition, it was demonstrated that this molecule is concentrated in the Golgi apparatus, to cycle to and from the plasma membrane, where it colocalizes with pre-S (6, 7). Expression of duck gp180 in HuH7 cells, a human hepatoma cell line, led merely to internalizatio...

The e antigen (eAg) of duck hepatitis B virus (DHBV) is a glycosylated secretory protein with a currently unknown function. We concentrated this antigen from the supernatants of persistently infected primary duck liver cell cultures by ammonium sulphate precipitation, adsorption chromatography over concanavalin A Sepharose, preparative isoelectric focusing and molecular sieve chromatography. The combined treatment of duck liver cells with DHBV eAg (DHBe) concentrate and a-methyl-D-mannopyranoside strongly inhibited DHBV replication at de novo infection. When DHBe was added to non-infected primary duck liver cells, it was found to be associated with liver sinusoidal endothelial cells. This binding could be inhibited by the addition of a-methyl-D-mannopyranoside and other sugar molecules. The inhibitory effect of DHBe on infection could play a role in maintaining viral persistence.