Kaposi’s Sarcoma-Associated Herpesvirus (KSHV) is an oncogenic virus which has adapted unique mechanisms to modulate the cellular microenvironment of its human host. The pathogenesis of KSHV is intimately linked to its manipulation of cellular signaling pathways, including the extracellular signal-regulated kinase (ERK) mitogen-activated protein kinase (MAPK) pathway. We have previously shown that KSHV ORF45 contributes to the sustained activation of both ERK and p90 ribosomal S6 kinase (RSK, a major functional mediator of ERK/MAPK signaling) during KSHV lytic replication. ORF45-activated RSK is required for optimal KSHV lytic gene expression and progeny virion production, though the underlying mechanisms downstream of this activation are still unclear. We hypothesized that the activation of RSK by ORF45 causes differential phosphorylation of cellular and viral substrates, affecting biological processes essential for efficient KSHV lytic replication. Accordingly, we observed widespread and significant differences in protein phosphorylation upon induction of lytic replication. Mass-spectrometry-based phosphoproteomic screening identified putative substrates of ORF45-activated RSK in KSHV-infected cells. Bioinformatic analyses revealed that nuclear proteins, including several transcriptional regulators, were overrepresented among these candidates. We validated the ORF45/RSK-dependent phosphorylation of several putative substrates by employing KSHV BAC mutagenesis, kinase inhibitor treatments, and/or CRISPR-mediated knockout of RSK in KSHV-infected cells. Furthermore, we assessed the consequences of knocking out these substrates on ORF45/RSK-dependent regulation of gene expression and KSHV progeny virion production. Finally, we show data to support that ORF45 regulates the translational efficiency of a subset of viral/cellular genes with complex secondary structure in their 5’ UTR. Altogether, these data shed light on the mechanisms by which KSHV ORF45 manipulates components of the host cell machinery via modulation of RSK activity. Thus, this study has important implications for the pathobiology of KSHV and other diseases in which RSK activity is dysregulated.

Cell-to-cell communication in embryo sacs is thought to regulate the development of female gametes in flowering plants, but the details remain poorly understood. Here, we report a mitochondrial protein, GAMETE CELL DEFECTIVE 1 (GCD1), enriched in gametophytes that is essential for final maturation of female gametes. Using Arabidopsis gcd1 mutants, we found that final maturation of the egg and central cells is not required for double fertilization but is necessary for embryogenesis initiation and endosperm development. Furthermore, nonautonomous effects, observed when GCD1 or AAC2 function is disrupted, suggest that mitochondrial function influences reciprocal signaling between central and egg cells to regulate maturation of the partner (egg or central) cell. Our findings confirm that cell-to-cell communication is important in functional maturation of female gametic cells and suggest that both egg and central cells sense and transmit their mitochondrial metabolic status as an important cue that regulates the coordination of gamete maturation.

We recently showed that the interaction between Kaposi's sarcoma-associated herpesvirus (KSHV) tegument proteins ORF33 and ORF45 is crucial for progeny virion production, but the exact functions of KSHV ORF33 during lytic replication were unknown (J. Gillen, W. Li, Q. Liang, D. Avey, J. Wu, F. Wu, J. Myoung, and F. Zhu, J Virol 89:4918 -4931, 2015, http://dx.doi.org/10 .1128/JVI.02925-14). Therefore, here we investigated the relationship between ORF33 and ORF38, whose counterparts in both alpha-and betaherpesviruses interact with each other. Using specific monoclonal antibodies, we found that both proteins are expressed during the late lytic cycle with similar kinetics and that both are present in mature virions as components of the tegument. Furthermore, we confirmed that ORF33 interacts with ORF38. Interestingly, we observed that ORF33 tightly associates with the capsid, whereas ORF38 associates with the envelope. We generated ORF33-null, ORF38-null, and double-null mutants and found that these mutants apparently have identical phenotypes: the mutations caused no apparent effect on viral gene expression but reduced the yield of progeny virion by about 10-fold. The progeny virions also lack certain virion component proteins, including ORF45. During viral lytic replication, the virions associate with cytoplasmic vesicles. We also observed that ORF38 associates with the membranes of vesicles and colocalizes with the Golgi membrane or early endosome membrane. Further analyses of ORF33/ORF38 mutants revealed the reduced production of virion-containing vesicles, suggesting that ORF33 and ORF38 are involved in the transport of newly assembled viral particles into cytoplasmic vesicles, a process important for viral maturation and egress. IMPORTANCEHerpesvirus assembly is an essential step in virus propagation that leads to the generation of progeny virions. It is a complicated process that depends on the delicate regulation of interactions among virion proteins. We previously revealed an essential role of ORF45-ORF33 binding for virus assembly. Here, we report that ORF33 and its binding partner, ORF38, are required for infectious virus production due to their important role in the tegumentation process. Moreover, we found that both ORF33 and ORF38 are involved in the transportation of virions through vesicles during maturation and egress. Our results provide new insights into the important roles of ORF33 and ORF38 during viral assembly, a process critical for virus propagation that is intimately linked to KSHV pathobiology.

Herpesviruses acquire their envelope by budding into the lumen of cytoplasmic membrane vesicles. This process is initiated by component(s) on viral particles, which recognize the budding site where the viral glycoproteins are present and recruit cellular cargo transport and sorting machinery to the site to complete the budding process. Proteins in the tegument layer, connecting capsid and envelope, are candidates for the recognition of budding sites on vesicle membrane and induction of budding and final envelopment. We examined several outer and matrix tegument proteins of Kaposi’s sarcoma-associated herpesvirus (KSHV) and found that ORF45 associates with lipid rafts (LRs) of cellular membrane. LRs are membrane micro-domains, which have been implicated as relay stations in intracellular signaling and transport including viral entry and virion assembly. The ability of ORF45 to target LR is dependent on the mono-ubiquitylation of ORF45 at Lys297 as the mutation at Lys297 (K297R) abolished LR-association of ORF45. The K297R mutation also impairs ORF45 and viral particle co-localization with trans-Golgi network and endosomes, but facilitates ORF45 and viral particles co-localizing with lysosomes. More importantly, the recombinant KSHV carrying ORF45 K297R mutant (BAC-K297R) was found severely defective in producing mature and infectious virion particles in comparison to wild type KSHV (BAC16). Taken together, our results reveal a new function of KSHV tegument protein ORF45 in targeting LR of host cell membrane, promoting viral particles co-localization with trans-Golgi and endosome vesicles and facilitating the maturation and release of virion particles, suggesting that ORF45 plays a role in bringing KSHV particles to the budding site on cytoplasmic vesicle membrane and triggering the viral budding process for final envelopment and virion maturation.

Human immunodeficiency virus 1 (HIV-1) infection is initiated by binding of the viral envelope glycoprotein (Env) to the cell-surface receptor CD4. Although high resolution structures of Env complexed with soluble domains of CD4 have been determined, the binding process is less understood on native membranes. Here, we apply cryo-electron tomography (cryo-ET) to monitor Env-CD4 interactions at membrane-membrane interfaces formed between HIV-1 and CD4-presenting virus-like particles. Env-CD4 complexes organized into clusters and rings, bringing opposing membranes closer together. Additionally, Env-CD4 clustering was dependent on capsid maturation. Subtomogram averaging and classification revealed that Env bound one, two, and finally three CD4 molecules, upon which Env adopted a partially open state. Our data indicate that asymmetric HIV-1 Env trimers bound to one and two CD4 molecules are detectable intermediates during virus binding to host cell membranes, which likely has consequences for antibody-mediated immune responses and vaccine immunogen design.

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. 2015. Erratum for Fu et al., Activation of p90 ribosomal S6 kinases by ORF45 of Kaposi's sarcoma-associated herpesvirus is critical for optimal production of infectious viruses. J Virol 89:4705.