Regulation of HIV-1 Gag-Pol Expression by Shiftless, an Inhibitor of Programmed -1 Ribosomal Frameshifting.

Programmed -1 ribosomal frameshifting (-1PRF) is a widely used translation recoding mechanism. HIV-1 expresses Gag-Pol protein from the Gag-coding mRNA through -1PRF, and the ratio of Gag to Gag-Pol is strictly maintained for efficient viral replication. Here, we report that the interferon-stimulated gene product C19orf66 (herein named Shiftless) is a host factor that inhibits the -1PRF of HIV-1. Shiftless (SFL) also inhibited the -1PRF of a variety of mRNAs from both viruses and cellular genes. SFL interacted with the -1PRF signal of target mRNA and translating ribosomes and caused premature translation termination at the frameshifting site. Downregulation of translation release factor eRF3 or eRF1 reduced SFL-mediated premature translation termination. We propose that SFL binding to target mRNA and the translating ribosome interferes with the frameshifting process. These findings identify SFL as a broad-spectrum inhibitor of -1PRF and help to further elucidate the mechanisms of -1PRF.

HIV-1 Exploits the Host Factor RuvB-like 2 to Balance Viral Protein Expression.

The correct ratio of the HIV-1 structural protein Gag to the envelope protein (Env) is important for maximal virion infectivity. How the virus ensures the production of Gag and Env proteins in an appropriate ratio remains unknown. We report that HIV-1 exploits the host factor RuvB-like 2 (RVB2) to balance relative expression of Gag and Env for efficient production of infectious virions. RVB2 inhibits Gag expression by interacting with both the encoded Matrix (MA) domain of Gag protein and 5' UTR of the translating mRNA and promoting mRNA degradation in a translation-dependent manner. This inhibitory activity of RVB2 is antagonized by Env through competitive interaction with MA, allowing Gag synthesis to proceed when Env levels are adequate for virion assembly. In HIV-1-positive patients, RVB2 levels positively correlate with viral loads and disease progression status. These findings reveal a mechanism by which HIV-1 regulates its protein expression.

Bat origins of MERS-CoV supported by bat coronavirus HKU4 usage of human receptor CD26.

The recently reported Middle East respiratory syndrome coronavirus (MERS-CoV) is phylogenetically closely related to the bat coronaviruses (BatCoVs) HKU4 and HKU5. However, the evolutionary pathway of MERS-CoV is still unclear. A receptor binding domain (RBD) in the MERS-CoV envelope-embedded spike protein specifically engages human CD26 (hCD26) to initiate viral entry. The high sequence identity in the viral spike protein prompted us to investigate if HKU4 and HKU5 can recognize hCD26 for cell entry. We found that HKU4-RBD, but not HKU5-RBD, binds to hCD26, and pseudotyped viruses embedding HKU4 spike can infect cells via hCD26 recognition. The structure of the HKU4-RBD/hCD26 complex revealed a hCD26-binding mode similar overall to that observed for MERS-RBD. HKU4-RBD, however, is less adapted to hCD26 than MERS-RBD, explaining its lower affinity for receptor binding. Our findings support a bat origin for MERS-CoV and indicate the need for surveillance of HKU4-related viruses in bats.

PILRα and PILRß have a siglec fold and provide the basis of binding to sialic acid.

Paired immunoglobulin-like type 2 receptor α (PILRα) and ß (PILRß) belong to the PILR family and are related to innate immune regulation in various species. Despite their high sequence identity, PILRα and PILRß are shown to have variant sialic acid (SA) binding avidities. To explore the molecular basis of this interaction, we solved the crystal structures of PILRα and PILRß at resolutions of 1.6 Å and 2.2 Å, respectively. Both molecules adopt a typical siglec fold but use a hydrophobic bond to substitute the siglec-specific disulfide linkage for protein stabilization. We further used HSV-1 glycoprotein B (gB) as a representative molecule to study the PILR-SA interaction. Deploying site-directed mutagenesis, we demonstrated that three residues (Y2, R95, and W108) presented on the surface of PILRα form the SA binding site equivalent to those in siglecs but are arranged in a unique linear mode. PILRß differs from PILRα in one of these three residues (L108), explaining its inability to engage gB. Mutation of L108 to tryptophan in PILRß restored the gB-binding capacity. We further solved the structure of this PILRß mutant complexed with SA, which reveals the atomic details mediating PILR/SA recognition. In comparison with the free PILR structures, amino acid Y2 oriented variantly in the complex structure, thereby disrupting the linear arrangement of PILR residues Y2, R95, and W108. In conclusion, our study provides significant implications for the PILR-SA interaction and paves the way for understanding PILR-related ligand binding.

ZAP inhibits murine gammaherpesvirus 68 ORF64 expression and is antagonized by RTA.

Zinc finger antiviral protein (ZAP) is an interferon-inducible host antiviral factor that specifically inhibits the replication of certain viruses, including HIV-1 and Ebola virus. ZAP functions as a dimer formed through intermolecular interactions of its N-terminal tails. ZAP binds directly to specific viral mRNAs and inhibits their expression by repressing translation and/or promoting degradation of the target mRNA. ZAP is not a universal antiviral factor, since some viruses grow normally in ZAP-expressing cells. It is not fully understood what determines whether a virus is susceptible to ZAP. We explored the interaction between ZAP and murine gammaherpesvirus 68 (MHV-68), whose life cycle has latent and lytic phases. We previously reported that ZAP inhibits the expression of M2, which is expressed mainly in the latent phase, and regulates MHV-68 latency in cultured cells. Here, we report that ZAP inhibits the expression of ORF64, a tegument protein that is expressed in the lytic phase and is essential for lytic replication. MHV-68 infection induced ZAP expression. However, ZAP did not inhibit lytic replication of MHV-68. We provide evidence showing that the antiviral activity of ZAP is antagonized by MHV-68 RTA, a critical viral transactivator expressed in the lytic phase. We further show that RTA inhibits the antiviral activity of ZAP by disrupting the N-terminal intermolecular interaction of ZAP. Our results provide an example of how a virus can escape ZAP-mediated immunity.

Zinc finger antiviral protein inhibits murine gammaherpesvirus 68 M2 expression and regulates viral latency in cultured cells.

Zinc finger antiviral protein (ZAP) is a host factor that specifically inhibits the replication of certain viruses by binding to specific viral mRNAs and repressing mRNA expression. Here we report that ZAP inhibits expression of murine gammaherpesvirus 68 (MHV-68) M2, which plays important roles in establishment and maintenance of viral latency. Downregulation of endogenous ZAP in cells harboring latent MHV-68 promoted lytic replication of the virus. These results suggest that ZAP inhibits M2 expression and regulates the maintenance of MHV-68 latency.