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Summary Stable microtubule (MT) subsets form distinct networks from dynamic MTs and acquire distinguishing posttranslational modifications, notably detyrosination and acetylation. Acting as specialized tracks for vesicle and macromolecular transport, their formation is regulated by the end-binding protein, EB1, which recruits proteins that stabilize MTs. We show that HIV-1 induces the formation of acetylated and detyrosinated stable MTs early in infection. Although the MT depolymerizing agent nocodazole affected dynamic MTs, HIV-1 particles localized to nocodazole-resistant stable MTs and infection was minimally affected. EB1 depletion or expression of an EB1 carboxy-terminal fragment that acts as a dominant negative inhibitor of MT stabilization, prevented HIV-1-induced stable MT formation and suppressed early viral infection. Furthermore, we show that the HIV-1 matrix protein targets the EB1-binding protein, Kif4 to induce MT stabilization. Our findings illustrate how specialized MT-binding proteins mediate MT stabilization by HIV-1 and the importance of stable MT subsets in viral infection.

Interferons (IFNs) induce anti-viral programs, regulate immune responses, and exert anti-proliferative effects. To escape anti-tumorigenic effects of IFNs, malignant cells attenuate JAK/STAT signaling and expression of IFN stimulated genes (ISGs). Such attenuation may enhance the susceptibility of tumor cells to oncolytic virotherapy. Here we studied genetic and epigenetic mechanisms of interference with JAK/STAT signaling and their contribution to susceptibility of prostate cancer cells to viral infection. Bioinformatics analysis of gene-expression in cohorts of prostate cancer patients revealed genetic and epigenetic interference with the IFN program. To correlate lack of IFN signaling and susceptibility to viral infection and oncolysis; we employed LNCaP prostate cancer cells as cellular model, and the human metapneumovirus and the epizootic hemorrhagic disease virus as infectious agents. In LNCaP cells, JAK1 is silenced by bi-allelic inactivating mutations and epigenetic silencing, which also silences ISGs. Chemical inhibition of epigenetic silencing partially restored IFN-sensitivity, induced low levels of expression of selected ISGs and attenuated, but failed to block, viral infection and oncolysis. Since viral infection was not blocked by epigenetic modifiers, and these compounds may independently-induce anti-tumor effects, we propose that epigenetic modifiers and virotherapy are compatible in treatment of prostate tumors defective in JAK1 expression and IFN signaling.

While beta-amyloid (Aβ), a classic hallmark of Alzheimer’s disease (AD) and dementia, has long been known to be elevated in the human immunodeficiency virus type 1 (HIV-1)-infected brain, why and how Aβ is produced, along with its contribution to HIV-associated neurocognitive disorder (HAND) remains ill-defined. Here, we reveal that the membrane-associated amyloid precursor protein (APP) is highly expressed in macrophages and microglia, and acts as an innate restriction against HIV-1. APP binds the HIV-1 Gag polyprotein, retains it in lipid rafts and blocks HIV-1 virion production and spread. To escape this restriction, Gag promotes secretase-dependent cleavage of APP, resulting in the overproduction of toxic Aβ isoforms. This Gag-mediated Aβ production results in increased degeneration of primary cortical neurons, and can be prevented by γ-secretase inhibitor treatment. Interfering with HIV-1’s evasion of APP-mediated restriction also suppresses HIV-1 spread, offering a potential strategy to both treat infection and prevent HAND.

Nirmatrelvir, an oral antiviral targeting the 3CL protease of SARS-CoV-2, has been demonstrated to be clinically useful in reducing hospitalization or death due to COVID-191,2. However, as SARS-CoV-2 has evolved to become resistant to other therapeutic modalities3-9, there is a concern that the same could occur for nirmatrelvir. Here, we have examined this possibility by in vitro passaging of SARS-CoV-2 in increasing concentrations of nirmatrelvir using two independent approaches, including one on a large scale in 480 wells. Indeed, highly resistant viruses emerged from both, and their sequences revealed a multitude of 3CL protease mutations. In the experiment done at scale, 53 independent viral lineages were selected with mutations observed at 23 different residues of the enzyme. Yet, several common mutational pathways to nirmatrelvir resistance were preferred, with a majority of the viruses descending from T21I, P252L, or T304I as precursor mutations. Construction and analysis of 13 recombinant SARS-CoV-2 clones, each containing a unique mutation or a combination of mutations showed that the above precursor mutations only mediated low-level resistance, whereas greater resistance required accumulation of additional mutations. E166V mutation conferred the strongest resistance (~300-fold), but this mutation resulted in a loss of viral replicative fitness that was restored by compensatory changes such as L50F and T21I. Structural explanations are discussed for some of the mutations that are proximal to the drug-binding site, as well as cross-resistance or lack thereof to ensitrelvir, another clinically important 3CL protease inhibitor. Our findings indicate that SARS-CoV-2 resistance to nirmatrelvir does readily arise via multiple pathways in vitro, and the specific mutations observed herein form a strong foundation from which to study the mechanism of resistance in detail and to shed light on the design of next generation protease inhibitors.

Summary N-myristoylation is the covalent attachment of myristic acid to the N-terminus of proteins in eukaryotic cells. The Matrix domain (MA) of HIV-1 Gag protein is N-myristoylated and plays an important role in virus budding. In screening for host factors that interact with HIV-1 MA, we found that heme oxygenase 2 (HO-2) specifically binds the myristate moiety of Gag. HO-2 was also found to bind TRAM, an adaptor protein for Toll-like receptor 4 (TLR4), and thereby impact both virus replication and cellular inflammatory responses. A crystal structure revealed that HO-2 binds myristate via a hydrophobic channel adjacent to the heme-binding pocket. Inhibiting HO-2 expression, or blocking myristate binding with a heme analogue, led to marked increases in virus production. HO-2 deficiency caused hyperresponsive TRAM-dependent TLR4 signaling, and hypersensitivity to the TLR4 ligand lipopolysaccharide. Thus, HO-2 is a cellular myristate-binding protein that negatively regulates both virus replication and host inflammatory responses.

BackgroundSynonymous or silent mutations are usually thought to evolve neutrally. However, accumulating recent evidence has demonstrated that silent mutations may destabilize RNA structures or disrupt cis regulatory motifs superimposed on coding sequences. Such observations suggest the existence of stretches of codon sites that are evolutionary conserved at both DNA-RNA and protein levels. Such stretches may point to functionally important regions within protein coding sequences not necessarily reflecting functional constraints on the amino-acid sequence. The HIV-1 genome is highly compact, and often harbors overlapping functional elements at the protein, RNA, and DNA levels. This superimposition of functions leads to complex selective forces acting on all levels of the genome and proteome. Considering the constraints on HIV-1 to maintain such a highly compact genome, we hypothesized that stretches of synonymous conservation would be common within its genome.ResultsWe used a combined computational-experimental approach to detect and characterize regions exhibiting strong purifying selection against synonymous substitutions along the HIV-1 genome. Our methodology is based on advanced probabilistic evolutionary models that explicitly account for synonymous rate variation among sites and rate dependencies among adjacent sites. These models are combined with a randomization procedure to automatically identify the most statistically significant regions of conserved synonymous sites along the genome. Using this procedure we identified 21 conserved regions. Twelve of these are mapped to regions within overlapping genes, seven correlate with known functional elements, while the functions of the remaining four are yet unknown. Among these four regions, we chose the one that deviates most from synonymous rate homogeneity for in-depth computational and experimental characterization. In our assays aiming to quantify viral fitness in both early and late stages of the replication cycle, no differences were observed between the mutated and the wild type virus following the introduction of synonymous mutations.ConclusionsThe contradiction between the inferred purifying selective forces and the lack of effect of these mutations on viral replication may be explained by the fact that the phenotype was measured in single-cycle infection assays in cell culture. Such a system does not account for the complexity of HIV-1 infections in vivo, which involves multiple infection cycles and interaction with the host immune system.

YXXL motifs in cellular and viral proteins have a variety of functions. The matrix (M) protein of the respiratory pathogen human metapneumovirus (hMPV) contains two such conserved motifs-YSKL and YAGL.We mutated these sequences to analyze their contributions to hMPV infectivity. The mutant clones were capable of intracellular replication; however, the YAGL but not YSKL mutants were defective at spreading in infected cultures. We improved the reverse genetics system for hMPV and generated cell lines that stably expressed selectable, replicating full-length genomes for both the wild type and the mutant clones, allowing microscopic and biochemical analyses of these viruses. YAGL mutants produced normal cellular levels of M protein but failed to release virions, while ectopic coexpression of wild-type M generated particles that were restricted to a single cycle of infection. The YAGL motif did not act as a late (L) domain, however, since hMPV budding was independent of the cellular endosomal sorting complex required for transport (ESCRT) machinery and because replacement of the YAGL motif with classical L domains generated defective viruses. Instead, the YAGL mutants had defective M assemblies lacking a normal filamentous appearance and showed poor extractability from the cell compared to the wild-type protein. The mutant proteins were not grossly misfolded, however, as they interacted with cellular membranes and coassembled with wild-type M proteins. Thus, the YAGL motif is an important determinant of hMPV assembly. Furthermore, the selectable hMPV genomes described here should extend the use of reverse genetics systems in the analysis of spreading-defective viruses.Human metapneumovirus (hMPV) is a respiratory pathogen which causes upper and lower respiratory tract infections worldwide in both healthy and immunocompromised individuals across all age groups, resulting in mild to severe respiratory diseases (reviewed in references 18, 35, and 50). It was first identified in 2001 (84) and is classified, together with avian metapneumovirus (aMPV), as a member of the Metapneumovirus genus within the Pneumovirinae subfamily of the Paramyxoviridae family. The nonsegmented, negative-strand RNA genomes of the MPVs are approximately 13.3 kb long and contain eight genes that are separated by gene start (GS) and gene end (GE) sequences and encode nine putative proteins: nucleoprotein (N), phosphoprotein (P), matrix (M) protein, fusion (F) protein, small hydrophobic surface protein (SH), major attachment glycoprotein (G), major polymerase subunit (L), transcription elongation factor (M2.1), and RNA synthesis regulatory factor (M2.2). The last two proteins (collectively named M2 here) are thought to be expressed from overlapping open reading frames in the M2 gene (16, 83). The overall order of the genes in the hMPV genome is 3Ј-N-P-M-F-M2-SH-G-L-5Ј. hMPV is closely related to the important respiratory pathogen human respiratory syncytial virus (hRSV), which is also classified in the Pneumovirinae subfamily, but the order of the...