Post-Translational Modifications of Retroviral HIV-1 Gag Precursors: An Overview of Their Biological Role

Protein post-translational modifications (PTMs) play key roles in eukaryotes since they finely regulate numerous mechanisms used to diversify the protein functions and to modulate their signaling networks. Besides, these chemical modifications also take part in the viral hijacking of the host, and also contribute to the cellular response to viral infections. All domains of the human immunodeficiency virus type 1 (HIV-1) Gag precursor of 55-kDa (Pr55^Gag), which is the central actor for viral RNA specific recruitment and genome packaging, are post-translationally modified. In this review, we summarize the current knowledge about HIV-1 Pr55^Gag PTMs such as myristoylation, phosphorylation, ubiquitination, sumoylation, methylation, and ISGylation in order to figure out how these modifications affect the precursor functions and viral replication. Indeed, in HIV-1, PTMs regulate the precursor trafficking between cell compartments and its anchoring at the plasma membrane, where viral assembly occurs. Interestingly, PTMs also allow Pr55^Gag to hijack the cell machinery to achieve viral budding as they drive recognition between viral proteins or cellular components such as the ESCRT machinery. Finally, we will describe and compare PTMs of several other retroviral Gag proteins to give a global overview of their role in the retroviral life cycle.     

The Autonomous Glycosylation of Large DNA Viruses

Glycosylation of surface molecules is a key feature of several eukaryotic viruses, which use the host endoplasmic reticulum/Golgi apparatus to add carbohydrates to their nascent glycoproteins. In recent years, a newly discovered group of eukaryotic viruses, belonging to the Nucleo-Cytoplasmic Large DNA Virus (NCLDV) group, was shown to have several features that are typical of cellular organisms, including the presence of components of the glycosylation machinery. Starting from initial observations with the chlorovirus PBCV-1, enzymes for glycan biosynthesis have been later identified in other viruses; in particular in members of the Mimiviridae family. They include both the glycosyltransferases and other carbohydrate-modifying enzymes and the pathways for the biosynthesis of the rare monosaccharides that are found in the viral glycan structures. These findings, together with genome analysis of the newly-identified giant DNA viruses, indicate that the presence of glycogenes is widespread in several NCLDV families. The identification of autonomous viral glycosylation machinery leads to many questions about the origin of these pathways, the mechanisms of glycan production, and eventually their function in the viral replication cycle. The scope of this review is to highlight some of the recent results that have been obtained on the glycosylation systems of the large DNA viruses, with a special focus on the enzymes involved in nucleotide-sugar production.     

Transgenic Chicks Expressing Interferon-Inducible Transmembrane Protein 1 (IFITM1) Restrict Highly Pathogenic H5N1 Influenza Viruses

Mammalian cells utilize a wide spectrum of pathways to antagonize the viral replication. These pathways are typically regulated by antiviral proteins and can be constitutively expressed but also exacerbated by interferon induction. A myriad of interferon-stimulated genes (ISGs) have been identified in mounting broad-spectrum antiviral responses. Members of the interferon-induced transmembrane (IFITM) family of proteins are unique among these ISGs due to their ability to prevent virus entry through the lipid bilayer into the cell. In the current study, we generated transgenic chickens that constitutively and stably expressed chicken IFITM1 (chIFITM1) using the avian sarcoma-leukosis virus (RCAS)-based gene transfer system. The challenged transgenic chicks with clinical dose 10⁴ egg infective dose 50 (EID₅₀) of highly pathogenic avian influenza virus (HPAIV) subtype H5N1 (clade 2.2.1.2) showed 100% protection and significant infection tolerance. Although challenged transgenic chicks displayed 60% protection against challenge with the sub-lethal dose (EID₅₀ 10⁵), the transgenic chicks showed delayed clinical symptoms, reduced virus shedding, and reduced histopathologic alterations compared to non-transgenic challenged control chickens. These finding indicate that the sterile defense against H5N1 HPAIV offered by the stable expression of chIFITM1 is inadequate; however, the clinical outcome can be substantially ameliorated. In conclusion, chIFITM proteins can inhibit influenza virus replication that can infect various host species and could be a crucial barrier against zoonotic infections.     

Role of Virally-Encoded Deubiquitinating Enzymes in Regulation of the Virus Life Cycle

The ubiquitin (Ub) proteasome system (UPS) plays a pivotal role in regulation of numerous cellular processes, including innate and adaptive immune responses that are essential for restriction of the virus life cycle in the infected cells. Deubiquitination by the deubiquitinating enzyme, deubiquitinase (DUB), is a reversible molecular process to remove Ub or Ub chains from the target proteins. Deubiquitination is an integral strategy within the UPS in regulating survival and proliferation of the infecting virus and the virus-invaded cells. Many viruses in the infected cells are reported to encode viral DUB, and these vial DUBs actively disrupt cellular Ub-dependent processes to suppress host antiviral immune response, enhancing virus replication and thus proliferation. This review surveys the types of DUBs encoded by different viruses and their molecular processes for how the infecting viruses take advantage of the DUB system to evade the host immune response and expedite their replication.     

Blaze a New Trail: Plant Virus Xylem Exploitation

Viruses are trailblazers in hijacking host systems for their own needs. Plant viruses have been shown to exploit alternative avenues of translocation within a host, including a challenging route through the xylem, to expand their niche and establish systemic spread, despite apparent host-imposed obstacles. Recent findings indicate that plant viruses from many families could successfully hack xylem cells in a broad range of plant hosts, including herbaceous and perennial woody plants. Similar to virus-related structures present in the phloem, virus particles and membrane-containing viral replication complexes are often observed in the xylem. Except for a few single-stranded DNA viruses in the family Geminiviridae and a negative-sense single-stranded RNA rhabdovirus, Lettuce necrotic yellows virus, the majority of the viruses that were detected in the xylem belong to the group of positive-sense RNA viruses. The diversity of the genome organization and virion morphology of those viruses indicates that xylem exploitation appears to be a widely adapted strategy for plant viruses. This review outlines the examples of the xylem-associated viruses and discusses factors that regulate virus inhabitation of the xylem as well as possible strategies of virus introduction into the xylem. In some cases, plant disease symptoms have been shown to be closely related to virus colonization of the xylem. Inhibiting viral xylem invasion could raise potential attractive approaches to manage virus diseases. Therefore, the identification of the host genes mediating virus interaction with the plant xylem tissue and understanding the underlying mechanisms call for more attention.     

The Roles of Ubiquitination in Pathogenesis of Influenza Virus Infection

The ubiquitin system denotes a potent post-translational modification machinery that is capable of activation or deactivation of target proteins through reversible linkage of a single ubiquitin or ubiquitin chains. Ubiquitination regulates major cellular functions such as protein degradation, trafficking and signaling pathways, innate immune response, antiviral defense, and virus replication. The RNA sensor RIG-I ubiquitination is specifically induced by influenza A virus (IAV) to activate type I IFN production. Influenza virus modulates the activity of major antiviral proteins in the host cell to complete its full life cycle. Its structural and non-structural proteins, matrix proteins and the polymerase complex can regulate host immunity and antiviral response. The polymerase PB1-F2 of mutated 1918 IAV, adapts a novel IFN antagonist function by sending the DDX3 into proteasomal degradation. Ultimately the fate of virus is determined by the outcome of interplay between viral components and host antiviral proteins and ubiquitination has a central role in the encounter of virus and its host cell.     

Intracellular Redox-Modulated Pathways as Targets for Effective Approaches in the Treatment of Viral Infection

Host-directed therapy using drugs that target cellular pathways required for virus lifecycle or its clearance might represent an effective approach for treating infectious diseases. Changes in redox homeostasis, including intracellular glutathione (GSH) depletion, are one of the key events that favor virus replication and contribute to the pathogenesis of virus-induced disease. Redox homeostasis has an important role in maintaining an appropriate Th1/Th2 balance, which is necessary to mount an effective immune response against viral infection and to avoid excessive inflammatory responses. It is known that excessive production of reactive oxygen species (ROS) induced by viral infection activates nuclear factor (NF)-kB, which orchestrates the expression of viral and host genes involved in the viral replication and inflammatory response. Moreover, redox-regulated protein disulfide isomerase (PDI) chaperones have an essential role in catalyzing formation of disulfide bonds in viral proteins. This review aims at describing the role of GSH in modulating redox sensitive pathways, in particular that mediated by NF-kB, and PDI activity. The second part of the review discusses the effectiveness of GSH-boosting molecules as broad-spectrum antivirals acting in a multifaceted way that includes the modulation of immune and inflammatory responses.     

Viral Interactions with Adaptor-Protein Complexes: A Ubiquitous Trait among Viral Species

Numerous viruses hijack cellular protein trafficking pathways to mediate cell entry or to rearrange membrane structures thereby promoting viral replication and antagonizing the immune response. Adaptor protein complexes (AP), which mediate protein sorting in endocytic and secretory transport pathways, are one of the conserved viral targets with many viruses possessing AP-interacting motifs. We present here different mechanisms of viral interference with AP complexes and the functional consequences that allow for efficient viral propagation and evasion of host immune defense. The ubiquity of this phenomenon is evidenced by the fact that there are representatives for AP interference in all major viral families, covered in this review. The best described examples are interactions of human immunodeficiency virus and human herpesviruses with AP complexes. Several other viruses, like Ebola, Nipah, and SARS-CoV-2, are pointed out as high priority disease-causative agents supporting the need for deeper understanding of virus-AP interplay which can be exploited in the design of novel antiviral therapies.     

Genome Features of a New Double-Stranded RNA Helper Virus (LBCbarr) from Wine Torulaspora delbrueckii Killer Strains

The killer phenotype of Torulaspora delbrueckii (Td) and Saccharomyces cerevisiae (Sc) is encoded in the genome of medium-size dsRNA viruses (V-M). Killer strains also contain a helper large size (4.6 kb) dsRNA virus (V-LA) which is required for maintenance and replication of V-M. Another large-size (4.6 kb) dsRNA virus (V-LBC), without known helper activity to date, may join V-LA and V-M in the same yeast. T. delbrueckii Kbarr1 killer strain contains the killer virus Mbarr1 in addition to two L viruses, TdV-LAbarr1 and TdV-LBCbarr1. In contrast, the T. delbrueckii Kbarr2 killer strain contains two M killer viruses (Mbarr1 and M1) and a LBC virus (TdV-LBCbarr2), which has helper capability to maintain both M viruses. The genomes of TdV-LBCbarr1 and TdV-LBCbarr2 were characterized by high-throughput sequencing (HTS). Both RNA genomes share sequence identity and similar organization with their ScV-LBC counterparts. They contain all conserved motifs required for translation, packaging, and replication of viral RNA. Their Gag-Pol amino-acid sequences also contain the features required for cap-snatching and RNA polymerase activity. However, some of these motifs and features are similar to those of LA viruses, which may explain that at least TdV-LBCbarr2 has a helper ability to maintain M killer viruses. Newly sequenced ScV-LBC genomes contained the same motifs and features previously found in LBC viruses, with the same genome location and secondary structure. Sequence comparison showed that LBC viruses belong to two clusters related to each species of yeast. No evidence for associated co-evolution of specific LBC with specific M virus was found. The presence of the same M1 virus in S. cerevisiae and T. delbrueckii raises the possibility of cross-species transmission of M viruses.     

The Genomic 3′ UTR of Flaviviruses Is a Translation Initiation Enhancer

Viruses rely on the cellular machinery of host cells to synthesize their proteins, and have developed different mechanisms enabling them to compete with cellular mRNAs for access to it. The genus Flavivirus is a large group of positive, single-stranded RNA viruses that includes several important human pathogens, such as West Nile, Dengue and Zika virus. The genome of flaviviruses bears a type 1 cap structure at its 5′ end, needed for the main translation initiation mechanism. Several members of the genus also use a cap-independent translation mechanism. The present work provides evidence that the WNV 5′ end also promotes a cap-independent translation initiation mechanism in mammalian and insect cells, reinforcing the hypothesis that this might be a general strategy of flaviviruses. In agreement with previous reports, we show that this mechanism depends on the presence of the viral genomic 3′ UTR. The results also show that the 3′ UTR of the WNV genome enhances translation of the cap-dependent mechanism. Interestingly, WNV 3′ UTR can be replaced by the 3′ UTR of other flaviviruses and the translation enhancing effect is maintained, suggesting a molecular mechanism that does not involve direct RNA-RNA interactions to be at work. In addition, the deletion of specific structural elements of the WNV 3′ UTR leads to increased cap-dependent and cap-independent translation. These findings suggest the 3′ UTR to be involved in a fine-tuned translation regulation mechanism.     

Different RNA Elements Control Viral Protein Synthesis in Polerovirus Isolates Evolved in Separate Geographical Regions

Most plant viruses lack the 5′-cap and 3′-poly(A) structures, which are common in their host mRNAs, and are crucial for translation initiation. Thus, alternative translation initiation mechanisms were identified for viral mRNAs, one of these being controlled by an RNA element in their 3′-ends that is able to enhance mRNA cap-independent translation (3′-CITE). The 3′-CITEs are modular and transferable RNA elements. In the case of poleroviruses, the mechanism of translation initiation of their RNAs in the host cell is still unclear; thus, it was studied for one of its members, cucurbit aphid-borne yellows virus (CABYV). We determined that efficient CABYV RNA translation requires the presence of a 3′-CITE in its 3′-UTR. We showed that this 3′-CITE requires the presence of the 5′-UTR in cis for its eIF4E-independent activity. Efficient virus multiplication depended on 3′-CITE activity. In CABYV isolates belonging to the three phylogenetic groups identified so far, the 3′-CITEs differ, and recombination prediction analyses suggest that these 3′-CITEs have been acquired through recombination with an unknown donor. Since these isolates have evolved in different geographical regions, this may suggest that their respective 3′-CITEs are possibly better adapted to each region. We propose that translation of other polerovirus genomes may also be 3′-CITE-dependent.     

Mass Spectrometry Analysis Coupled with de novo Sequencing Reveals Amino Acid Substitutions in Nucleocapsid Protein from Influenza A Virus

Amino acid substitutions in influenza A virus are the main reasons for both antigenic shift and virulence change, which result from non-synonymous mutations in the viral genome. Nucleocapsid protein (NP), one of the major structural proteins of influenza virus, is responsible for regulation of viral RNA synthesis and replication. In this report we used LC-MS/MS to analyze tryptic digestion of nucleocapsid protein of influenza virus (A/Puerto Rico/8/1934 H1N1), which was isolated and purified by SDS poly-acrylamide gel electrophoresis. Thus, LC-MS/MS analyses, coupled with manual de novo sequencing, allowed the determination of three substituted amino acid residues R452K, T423A and N430T in two tryptic peptides. The obtained results provided experimental evidence that amino acid substitutions resulted from non-synonymous gene mutations could be directly characterized by mass spectrometry in proteins of RNA viruses such as influenza A virus.     

Enzymatic Assays to Explore Viral mRNA Capping Machinery

In eukaryotes, mRNA is modified by the addition of the 7-methylguanosine (m⁷G) 5′ cap to protect mRNA from premature degradation, thereby enhancing translation and enabling differentiation between self (endogenous) and non-self RNAs (e. g., viral ones). Viruses often develop their own mRNA capping pathways to augment the expression of their proteins and escape host innate immune response. Insights into this capping system may provide new ideas for therapeutic interventions and facilitate drug discovery, e. g., against viruses that cause pandemic outbreaks, such as beta-coronaviruses SARS-CoV (2002), MARS-CoV (2012), and the most recent SARS-CoV-2. Thus, proper methods for the screening of large compound libraries are required to identify lead structures that could serve as a basis for rational antiviral drug design. This review summarizes the methods that allow the monitoring of the activity and inhibition of enzymes involved in mRNA capping.     

Computational Analysis of Short Linear Motifs in the Spike Protein of SARS-CoV-2 Variants Provides Possible Clues into the Immune Hijack and Evasion Mechanisms of Omicron Variant

Short linear motifs (SLiMs) are short linear sequences that can mediate protein–protein interaction. Mimicking eukaryotic SLiMs to compete with extra- or intracellular binding partners, or to sequester host proteins is the crucial strategy of viruses to pervert the host system. Evolved proteins in viruses facilitate minimal protein–protein interactions that significantly affect intracellular signaling networks. Unfortunately, very little information about SARS-CoV-2 SLiMs is known, especially across SARS-CoV-2 variants. Through the ELM database-based sequence analysis of spike proteins from all the major SARS-CoV-2 variants, we identified four overriding SLiMs in the SARS-CoV-2 Omicron variant, namely, LIG_TRFH_1, LIG_REV1ctd_RIR_1, LIG_CaM_NSCaTE_8, and MOD_LATS_1. These SLiMs are highly likely to interfere with various immune functions, interact with host intracellular proteins, regulate cellular pathways, and lubricate viral infection and transmission. These cellular interactions possibly serve as potential therapeutic targets for these variants, and this approach can be further exploited to combat emerging SARS-CoV-2 variants.     

Development and Use of a Kinetical and Real-Time Monitoring System to Analyze the Replication of Hepatitis C Virus

In microbiological research, it is important to understand the time course of each step in a pathogen’s lifecycle and changes in the host cell environment induced by infection. This study is the first to develop a real-time monitoring system that kinetically detects luminescence reporter activity over time without sampling cells or culture supernatants for analyzing the virus replication. Subgenomic replicon experiments with hepatitis C virus (HCV) showed that transient translation and genome replication can be detected separately, with the first peak of translation observed at 3–4 h and replication beginning around 20 h after viral RNA introduction into cells. From the bioluminescence data set measured every 30 min (48 measurements per day), the initial rates of translation and replication were calculated, and their capacity levels were expressed as the sums of the measured signals in each process, which correspond to the areas on the kinetics graphs. The comparison of various HuH-7-derived cell lines showed that the bioluminescence profile differs among cell lines, suggesting that both translation and replication capacities potentially influence differences in HCV susceptibility. The effects of RNA mutations within the 5′ UTR of the replicon on viral translation and replication were further analyzed in the system developed, confirming that mutations to the miR-122 binding sites primarily reduce replication activity rather than translation. The newly developed real-time monitoring system should be applied to the studies of various viruses and contribute to the analysis of transitions and progression of each process of their life cycle.     

Virus–Host Interaction Gets Curiouser and Curiouser. PART II: Functional Transcriptomics of the E. coli DksA-Deficient Cell upon Phage P1vir Infection

The virus–host interaction requires a complex interplay between the phage strategy of reprogramming the host machinery to produce and release progeny virions, and the host defense against infection. Using RNA sequencing, we investigated the phage–host interaction to resolve the phenomenon of improved lytic development of P1vir phage in a DksA-deficient E. coli host. Expression of the ant1 and kilA P1vir genes in the wild-type host was the highest among all and most probably leads to phage virulence. Interestingly, in a DksA-deficient host, P1vir genes encoding lysozyme and holin are downregulated, while antiholins are upregulated. Gene expression of RepA, a protein necessary for replication initiating at the phage oriR region, is increased in the dksA mutant; this is also true for phage genes responsible for viral morphogenesis and architecture. Still, it seems that P1vir is taking control of the bacterial protein, sugar, and lipid metabolism in both, the wild type and dksA⁻ hosts. Generally, bacterial hosts are reacting by activating their SOS response or upregulating the heat shock proteins. However, only DksA-deficient cells upregulate their sulfur metabolism and downregulate proteolysis upon P1vir infection. We conclude that P1vir development is enhanced in the dksA mutant due to several improvements, including replication and virion assembly, as well as a less efficient lysis.     

Complex Relationships between HIV-1 Integrase and Its Cellular Partners

RNA viruses, in pursuit of genome miniaturization, tend to employ cellular proteins to facilitate their replication. HIV-1, one of the most well-studied retroviruses, is not an exception. There is numerous evidence that the exploitation of cellular machinery relies on nucleic acid-protein and protein-protein interactions. Apart from Vpr, Vif, and Nef proteins that are known to regulate cellular functioning via interaction with cell components, another viral protein, integrase, appears to be crucial for proper virus-cell dialog at different stages of the viral life cycle. The goal of this review is to summarize and systematize existing data on known cellular partners of HIV-1 integrase and their role in the HIV-1 life cycle.