Proteomic and mechanistic dissection of the poxvirus-customized ribosome.

Ribosomes are often viewed as protein synthesis machines that lack intrinsic regulatory capacity. However, studies have established that ribosomes can functionally diversify through changes in the composition of, or post-translational modifications to ribosomal subunit proteins (RPs). We recently found that poxviruses phosphorylate unique sites in the RP, receptor for activated C kinase 1 (RACK1) to enhance viral protein synthesis. Here, we developed approaches for large-scale proteomic analysis of ribosomes isolated from cells infected with different viruses. Beyond RACK1, we identified additional phosphorylation events within RPS2 and RPS28 that arise during poxvirus infection, but not other viruses tested. The modified sites lie within unstructured loop domains that position around the mRNA entry and exit channel, respectively, and site-substitution mutants revealed that each modified residue contributed differently to poxvirus replication. Our findings reveal the broader extent to which poxviruses customize host ribosomes and provide new insights into how ribosomes can functionally diversify.

Human Cytomegalovirus Exploits TACC3 To Control Microtubule Dynamics and Late Stages of Infection.

While it is well established that microtubules (MTs) facilitate various stages of virus replication, how viruses actively control MT dynamics and functions remains less well understood. Recent work has begun to reveal how several viruses exploit End-Binding (EB) proteins and their associated microtubule plus-end tracking proteins (+TIPs), in particular to enable loading of viral particles onto MTs for retrograde transport during early stages of infection. Distinct from other viruses studied to date, at mid- to late stages of its unusually protracted replication cycle, human cytomegalovirus (HCMV) increases the expression of all three EB family members. This occurs coincident with the formation of a unique structure, termed the assembly compartment (AC), which serves as a Golgi-derived MT organizing center. Together, the AC and distinct EB proteins enable HCMV to increase the formation of dynamic and acetylated microtubule subsets to regulate distinct aspects of the viral replication cycle. Here, we reveal that HCMV also exploits EB-independent +TIP pathways by specifically increasing the expression of transforming acidic coiled coil protein 3 (TACC3) to recruit the MT polymerase, chTOG, from initial sites of MT nucleation in the AC out into the cytosol, thereby increasing dynamic MT growth. Preventing TACC3 increases or depleting chTOG impaired MT polymerization, resulting in defects in early versus late endosome organization in and around the AC as well as defects in viral trafficking and spread. Our findings provide the first example of a virus that actively exploits EB-independent +TIP pathways to regulate MT dynamics and control late stages of virus replication. IMPORTANCE Diverse viruses rely on host cell microtubule networks to transport viral particles within the dense cytoplasmic environment and to control the broader architecture of the cell to facilitate their replication. However, precisely how viruses regulate the dynamic behavior and function of microtubule filaments remains poorly defined. We recently showed that the assembly compartment (AC) formed by human cytomegalovirus (HCMV) acts as a Golgi-derived microtubule organizing center. Here, we show that at mid- to late stages of infection, HCMV increases the expression of transforming acidic coiled coil protein 3 (TACC3) to control the localization of the microtubule polymerase, chTOG. This, in turn, enables HCMV to generate dynamic microtubule subsets that organize endocytic vesicles in and around the AC and facilitate the transport of new viral particles released into the cytosol. Our findings reveal the first instance of viral targeting of TACC3 to control microtubule dynamics and virus spread.

Negative charge in the RACK1 loop broadens the translational capacity of the human ribosome.

Although the roles of initiation factors, RNA binding proteins, and RNA elements in regulating translation are well defined, how the ribosome functionally diversifies remains poorly understood. In their human hosts, poxviruses phosphorylate serine 278 (S278) at the tip of a loop domain in the small subunit ribosomal protein RACK1, thereby mimicking negatively charged residues in the RACK1 loops of dicot plants and protists to stimulate translation of transcripts with 5' poly(A) leaders. However, how a negatively charged RACK1 loop affects ribosome structure and its broader translational output is not known. Here, we show that although ribotoxin-induced stress signaling and stalling on poly(A) sequences are unaffected, negative charge in the RACK1 loop alters the swivel motion of the 40S head domain in a manner similar to several internal ribosome entry sites (IRESs), confers resistance to various protein synthesis inhibitors, and broadly supports noncanonical modes of translation.