A Cooperative Interaction between Nontranslated RNA Sequences and NS5A Protein Promotes In Vivo Fitness of a Chimeric Hepatitis C/GB Virus B

GB virus B (GBV-B) is closely related to hepatitis C virus (HCV), infects small non-human primates, and is thus a valuable surrogate for studying HCV. Despite significant differences, the 5′ nontranslated RNAs (NTRs) of these viruses fold into four similar structured domains (I-IV), with domains II-III-IV comprising the viral internal ribosomal entry site (IRES). We previously reported the in vivo rescue of a chimeric GBV-B (vGB/IIIHC) containing HCV sequence in domain III, an essential segment of the IRES. We show here that three mutations identified within the vGB/IIIHC genome (within the 3′NTR, upstream of the poly(U) tract, and NS5A coding sequence) are necessary and sufficient for production of this chimeric virus following intrahepatic inoculation of synthetic RNA in tamarins, and thus apparently compensate for the presence of HCV sequence in domain III. To assess the mechanism(s) underlying these compensatory mutations, and to determine whether 5′NTR subdomains participating in genome replication do so in a virus-specific fashion, we constructed and evaluated a series of chimeric subgenomic GBV-B replicons in which various 5′NTR subdomains were substituted with their HCV homologs. Domains I and II of the GBV-B 5′NTR could not be replaced with HCV sequence, indicating that they contain essential, virus-specific RNA replication elements. In contrast, domain III could be swapped with minimal loss of genome replication capacity in cell culture. The 3′NTR and NS5A mutations required for rescue of the related chimeric virus in vivo had no effect on replication of the subgenomic GBneoD/IIIHC RNA in vitro. The data suggest that in vivo fitness of the domain III chimeric virus is dependent on a cooperative interaction between the 5′NTR, 3′NTR and NS5A at a step in the viral life cycle subsequent to genome replication, most likely during particle assembly. Such a mechanism may be common to all hepaciviruses

Morphogenesis of the hepatitis B virus

The human hepatitis B virus (HBV) is a small hepatotropic enveloped virus associated with chronic infection that can lead to cirrhosis and hepatocellular carcinoma. The HBV genome is a DNA molecule contained in an icosahedral capsid. Although HBV is not a retrovirus, the replication of its genome involves reverse transcription. Another distinctive feature of HBV is the production, in great excess over virions, of non-infectious subviral particles (SVP) consisting of membrane phospholipids and the three envelope proteins (small [S], medium [M] and large [L]). These empty non-infectious particles are highly immunogenic, and their in vitro production is at the basis of the current vaccine against hepatitis B. Despite numerous studies that lead to a better understanding of the HBV replication, little is known about the morphogenesis of the virion and its associated SVP. Recent approaches suggest that the mechanisms responsable for assembly of the virions and the SVP could be distinct

Reassessment of the capacity of the HIV-1 Env cytoplasmic domain to trigger NF-κB activation

The cytoplasmic domain of lentiviral Envelopes (EnvCD) ensures Env incorporation into nascent virions and regulatesEnv trafficking to and from the plasma membrane. It has also been reported to promote transcription from the viralLTR both directly and indirectly. Noticeably, the HIV-1 and SIVmac239EnvCDs were described to trigger nucleartranslocation of NF-κB (Postler, Cell Host Microbes 2012). Given the paramount importance of identifying viral and host factors regulating HIV transcription, cellular signaling pathways and latency, and given that viral replicationcapacity is dependent on Env, we asked whether HIV EnvCDs from different HIV-1 subtypes differently modulatedNF-κB. To that aim, we evaluated the ability of primaryHIV-1 Envs from subtypes B and C to activate the NF-κB pathway.Primary subtype B and C Envs all failed to activate the NF-κB pathway. In contrast, when the EnvCD of HIV-1 Envs wasfused to the the CD8-αchain, it induced ~ 10-fold increase in NF-κB induction, and this increase was much stronger witha truncated form of the HIV EnvCD lacking the 76 C-terminal residues and containing the proposed TAK-1 bindingdomain. Our results indicate that the HIV-1 EnvCD is unlikely to trigger the NF-κB pathway in its native trimeric form

The Envelope Cytoplasmic Tail of HIV-1 Subtype C Contributes to Poor Replication Capacity through Low Viral Infectivity and Cell-to-Cell Transmission.

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