Title: Retroviral Replication and Restriction
Description: Retroviruses are obligate intracellular parasites that carry the information necessary for replication within their genomes. The three polyproteins, Gag, Pol, and Env, encoded by all retroviruses, function to generate progeny virions inside the host cell. Formation of new viral particles requires detailed instructions contained within the Gag polyprotein. Herein we describe our investigation into assembly of the Mason-Pfizer monkey virus (M-PMV). During retrovirus assembly, the transition from immature to a fully infectious mature particle is associated with the operation of molecular switches that trigger dramatic conformational changes of the Gag proteins. A dominant maturation switch that stabilizes the immature capsid lattice is located in the C-terminus of the capsid (CA) protein in many retroviral Gags. The HIV-1 Gag contains a stretch of five amino acid residues termed the 'clasp motif', important for the organization of the hexameric subunits that provide stability to the overall immature HIV-1 shell.

Sequence alignment of the CA C-terminal domains (CTDs) of the HIV-1 and M-PMV highlighted a spacer-like domain in M-PMV that may provide comparable function. In the present study we report an examination of the role of the clasp motif in the M-PMV life cycle. Our results demonstrate that claps motif mutants display major defects in virion assembly and release, and even larger defects in infectivity. Our data identifies the clasp motif as a fundamental contributor to CA-CTD interactions necessary for efficient viral infection. The retroviral life cycle, unlike that of any other viral family, leads to the obligate integration of a proviral DNA into the host genome of somatic cells and in some cases even into the germ line. This remarkable feature of the Retroviridae family of viruses accounts for their extraordinary persistence through time and widespread abundance among vertebrate hosts. Because retroviral infection can have serious consequences to the host, there is great selective pressure to evolve strong networks that act to control incoming viruses.

In the second study presented here, we report a novel cofactor of an antiviral system, Riplet, which operates to augment HIV-1 restriction by ZAP. The zinc-finger antiviral protein (ZAP) is an interferon-stimulated gene (ISG) with potent intrinsic antiviral activity. ZAP inhibits replication of retroviruses including MLV and HIV-1, as well as alphaviruses, filoviruses, hepatitis B virus, etc. ZAP operates at the post-transcriptional stage, reducing the number of viral transcripts available for translation in the cytoplasm, although additional pathways might be at play. The exact mechanisms by which ZAP restricts viral replication are not fully understood. ZAP lacks enzymatic activity and utilizes other cellular proteins to suppress viral replication. TRIM25 and the nuclease KHNYN have been identified as ZAP cofactors, but its activity may well involve other cellular proteins. Here we identify Riplet, a protein known to play a central role in the activation of the retinoic acid-inducible gene I (RIG-I), as a novel ZAP cofactor that acts to augment ZAP’s antiviral activity.

Our data demonstrates that Riplet significantly augments ZAP-mediated restriction of HIV-1. Additionally, we show that Riplet interacts with ZAP via its PRY/SPRY domain and that the ubiquitin ligase activity of Riplet is not required to stimulate ZAP-mediated inhibition. Moreover, we show that Riplet interacts with TRIM25 suggesting that both Riplet and TRIM25 may operate synergistically to augment ZAP-mediated inhibition of HIV-1. The intracellular tropism of viruses is determined by a diverse combination of host proteins that allow infection to proceed efficiently. To achieve successful infection the virus needs the contribution of numerous cellular factors that assist at various steps of the life cycle. Conversely, replication requires resistance to species-specific restriction factors that act to suppress virus infection. The replication of M-PMV has been found to be highly restricted in mouse cell lines. The mechanism underlying the restriction of M-PMV replication in mouse cells has not been characterized.

In the third study presented here, we examined this potent post-entry block and performed an unbiased genome-wide CRISPR-Cas9 screen, selecting for knock-out of host factors that relieved the block. Our data identified several candidate genes that encode proteins involved in virus trafficking and innate immune activation.