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In Memory of Stephen Oroszlan

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A special issue of Viruses (ISSN 1999-4915). This special issue belongs to the section "Animal Viruses".

Deadline for manuscript submissions: closed (30 April 2021) | Viewed by 40567

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Dr. Alan Rein

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HIV Dynamics and Replication Program, National Cancer Institute-Frederick, National Institutes of Health, USA
Interests: molecular mechanisms of retrovirus replication; mechanisms of action of antiretroviral host restriction factors
Special Issues, Collections and Topics in MDPI journals

Special Issue Information

Dear Colleagues,

Stephen Oroszlan was a pioneer in the analysis of retroviral proteins. His groundbreaking contributions to the field paved the way for the development of inhibitors of retroviral proteases, now among the most potent weapons in our arsenal of anti-HIV-1 therapies.

Sadly, Steve passed away in May 2020. Viruses is planning a Special Issue in honor of his work and scientific legacy. We invite submissions on subjects related to Steve’s work, such as proteolysis in viral maturation, post-translational modifications of viral proteins, recoding mechanisms used by viruses, etc., particularly those reporting recent progress in these fields. Original research reports, reviews, and brief commentaries on Steve’s influence on the field would all be welcome.

Dr. Alan R. Rein
Guest Editor

Manuscript Submission Information

Manuscripts should be submitted online at www.mdpi.com by registering and logging in to this website. Once you are registered, click here to go to the submission form. Manuscripts can be submitted until the deadline. All submissions that pass pre-check are peer-reviewed. Accepted papers will be published continuously in the journal (as soon as accepted) and will be listed together on the special issue website. Research articles, review articles as well as short communications are invited. For planned papers, a title and short abstract (about 100 words) can be sent to the Editorial Office for announcement on this website.

Submitted manuscripts should not have been published previously, nor be under consideration for publication elsewhere (except conference proceedings papers). All manuscripts are thoroughly refereed through a single-blind peer-review process. A guide for authors and other relevant information for submission of manuscripts is available on the Instructions for Authors page. Viruses is an international peer-reviewed open access monthly journal published by MDPI.

Please visit the Instructions for Authors page before submitting a manuscript. The Article Processing Charge (APC) for publication in this open access journal is 2600 CHF (Swiss Francs). Submitted papers should be well formatted and use good English. Authors may use MDPI's English editing service prior to publication or during author revisions.

Keywords

HIV-1
retroviruses
viral proteins
viral proteases
fatty acid modification
translational suppression

Published Papers (12 papers)

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Editorial

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3 pages, 743 KiB

Open AccessEditorial

Stephen Oroszlan and Retroviral Proteins

by Alan Rein

Viruses 2022, 14(2), 290; https://0-doi-org.brum.beds.ac.uk/10.3390/v14020290 - 29 Jan 2022

Viewed by 1732

Abstract

Stephen Oroszlan received his early education in Hungary, graduating in 1950 from the Technical University in Budapest with a degree in chemical engineering [...] Full article

(This article belongs to the Special Issue In Memory of Stephen Oroszlan)

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Research

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14 pages, 3947 KiB

Open AccessArticle

Modulation of Viral Programmed Ribosomal Frameshifting and Stop Codon Readthrough by the Host Restriction Factor Shiftless

by Sawsan Napthine, Chris H. Hill, Holly C. M. Nugent and Ian Brierley

Viruses 2021, 13(7), 1230; https://0-doi-org.brum.beds.ac.uk/10.3390/v13071230 - 25 Jun 2021

Cited by 21 | Viewed by 4031

Abstract

The product of the interferon-stimulated gene C19orf66, Shiftless (SHFL), restricts human immunodeficiency virus replication through downregulation of the efficiency of the viral gag/pol frameshifting signal. In this study, we demonstrate that bacterially expressed, purified SHFL can decrease the efficiency of programmed ribosomal frameshifting in vitro at a variety of sites, including the RNA pseudoknot-dependent signals of the coronaviruses IBV, SARS-CoV and SARS-CoV-2, and the protein-dependent stimulators of the cardioviruses EMCV and TMEV. SHFL also reduced the efficiency of stop-codon readthrough at the murine leukemia virus gag/pol signal. Using size-exclusion chromatography, we confirm the binding of the purified protein to mammalian ribosomes in vitro. Finally, through electrophoretic mobility shift assays and mutational analysis, we show that expressed SHFL has strong RNA binding activity that is necessary for full activity in the inhibition of frameshifting, but shows no clear specificity for stimulatory RNA structures. Full article

(This article belongs to the Special Issue In Memory of Stephen Oroszlan)

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20 pages, 2699 KiB

Open AccessArticle

Development of a Bio-Layer Interferometry-Based Protease Assay Using HIV-1 Protease as a Model

by Márió Miczi, Ádám Diós, Beáta Bozóki, József Tőzsér and János András Mótyán

Viruses 2021, 13(6), 1183; https://0-doi-org.brum.beds.ac.uk/10.3390/v13061183 - 21 Jun 2021

Cited by 6 | Viewed by 3013

Abstract

Proteolytic enzymes have great significance in medicine and the pharmaceutical industry and are applied in multiple fields of life sciences. Therefore, cost-efficient, reliable and sensitive real-time monitoring methods are highly desirable to measure protease activity. In this paper, we describe the development of a new experimental approach for investigation of proteolytic enzymes. The method was designed by the combination of recombinant fusion protein substrates and bio-layer interferometry (BLI). The protease (PR) of human immunodeficiency virus type 1 (HIV-1) was applied as model enzyme to set up and test the method. The principle of the assay is that the recombinant protein substrates immobilized to the surface of biosensor are specifically cleaved by the PR, and the substrate processing can be followed by measuring change in the layer thickness by optical measurement. We successfully used this method to detect the HIV-1 PR activity in real time, and the initial rate of the signal decrease was found to be proportional to the enzyme activity. Substrates representing wild-type and modified cleavage sites were designed to study HIV-1 PR’s specificity, and the BLI-based measurements showed differential cleavage efficiency of the substrates, which was proven by enzyme kinetic measurements. We applied this BLI-based assay to experimentally confirm the existence of extended binding sites at the surface of HIV-1 PR. We found the measurements may be performed using lysates of cells expressing the fusion protein, without primary purification of the substrate. The designed BLI-based protease assay is high-throughput-compatible and enables real-time and small-volume measurements, thus providing a new and versatile approach to study proteolytic enzymes. Full article

(This article belongs to the Special Issue In Memory of Stephen Oroszlan)

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14 pages, 2191 KiB

Open AccessArticle

Specificity of the HIV-1 Protease on Substrates Representing the Cleavage Site in the Proximal Zinc-Finger of HIV-1 Nucleocapsid Protein

by János András Mótyán, Márió Miczi, Stephen Oroszlan and József Tőzsér

Viruses 2021, 13(6), 1092; https://0-doi-org.brum.beds.ac.uk/10.3390/v13061092 - 08 Jun 2021

Cited by 1 | Viewed by 2645

Abstract

To explore the sequence context-dependent nature of the human immunodeficiency virus type 1 (HIV-1) protease’s specificity and to provide a rationale for viral mutagenesis to study the potential role of the nucleocapsid (NC) processing in HIV-1 replication, synthetic oligopeptide substrates representing the wild-type and modified versions of the proximal cleavage site of HIV-1 NC were assayed as substrates of the HIV-1 protease (PR). The S1′ substrate binding site of HIV-1 PR was studied by an in vitro assay using KIVKCF↓NCGK decapeptides having amino acid substitutions of N17 residue of the cleavage site of the first zinc-finger domain, and in silico calculations were also performed to investigate amino acid preferences of S1′ site. Second site substitutions have also been designed to produce “revertant” substrates and convert a non-hydrolysable sequence (having glycine in place of N17) to a substrate. The specificity constants obtained for peptides containing non-charged P1′ substitutions correlated well with the residue volume, while the correlation with the calculated interaction energies showed the importance of hydrophobicity: interaction energies with polar residues were related to substantially lower specificity constants. Cleavable “revertants” showed one residue shift of cleavage position due to an alternative productive binding mode, and surprisingly, a double cleavage of a substrate was also observed. The results revealed the importance of alternative binding possibilities of substrates into the HIV-1 PR. The introduction of the “revertant” mutations into infectious virus clones may provide further insights into the potential role of NC processing in the early phase of the viral life-cycle. Full article

(This article belongs to the Special Issue In Memory of Stephen Oroszlan)

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9 pages, 1785 KiB

Open AccessArticle

Crystal Structure of SARS-CoV-2 Main Protease in Complex with the Non-Covalent Inhibitor ML188

by Gordon J. Lockbaum, Archie C. Reyes, Jeong Min Lee, Ronak Tilvawala, Ellen A. Nalivaika, Akbar Ali, Nese Kurt Yilmaz, Paul R. Thompson and Celia A. Schiffer

Viruses 2021, 13(2), 174; https://0-doi-org.brum.beds.ac.uk/10.3390/v13020174 - 25 Jan 2021

Cited by 78 | Viewed by 6683

Abstract

Viral proteases are critical enzymes for the maturation of many human pathogenic viruses and thus are key targets for direct acting antivirals (DAAs). The current viral pandemic caused by SARS-CoV-2 is in dire need of DAAs. The Main protease (M^pro) is the focus of extensive structure-based drug design efforts which are mostly covalent inhibitors targeting the catalytic cysteine. ML188 is a non-covalent inhibitor designed to target SARS-CoV-1 M^pro, and provides an initial scaffold for the creation of effective pan-coronavirus inhibitors. In the current study, we found that ML188 inhibits SARS-CoV-2 M^pro at 2.5 µM, which is more potent than against SAR-CoV-1 M^pro. We determined the crystal structure of ML188 in complex with SARS-CoV-2 M^pro to 2.39 Å resolution. Sharing 96% sequence identity, structural comparison of the two complexes only shows subtle differences. Non-covalent protease inhibitors complement the design of covalent inhibitors against SARS-CoV-2 main protease and are critical initial steps in the design of DAAs to treat CoVID 19. Full article

(This article belongs to the Special Issue In Memory of Stephen Oroszlan)

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17 pages, 4306 KiB

Open AccessArticle

Analysis and Molecular Determinants of HIV RNase H Cleavage Specificity at the PPT/U3 Junction

by Mar Álvarez, Enrique Sapena-Ventura, Joanna Luczkowiak, Samara Martín-Alonso and Luis Menéndez-Arias

Viruses 2021, 13(1), 131; https://0-doi-org.brum.beds.ac.uk/10.3390/v13010131 - 18 Jan 2021

Cited by 2 | Viewed by 2995

Abstract

HIV reverse transcriptases (RTs) convert viral genomic RNA into double-stranded DNA. During reverse transcription, polypurine tracts (PPTs) resilient to RNase H cleavage are used as primers for plus-strand DNA synthesis. Nonnucleoside RT inhibitors (NNRTIs) can interfere with the initiation of plus-strand DNA synthesis by enhancing PPT removal, while HIV RT connection subdomain mutations N348I and N348I/T369I mitigate this effect by altering RNase H cleavage specificity. Now, we demonstrate that among approved nonnucleoside RT inhibitors (NNRTIs), nevirapine and doravirine show the largest effects. The combination N348I/T369I in HIV-1_BH10 RT has a dominant effect on the RNase H cleavage specificity at the PPT/U3 site. Biochemical studies showed that wild-type HIV-1 and HIV-2 RTs were able to process efficiently and accurately all tested HIV PPT sequences. However, the cleavage accuracy at the PPT/U3 junction shown by the HIV-2_EHO RT was further improved after substituting the sequence YQEPFKNLKT of HIV-1_BH10 RT (positions 342–351) for the equivalent residues of the HIV-2 enzyme (HQGDKILKV). Our results highlight the role of β-sheets 17 and 18 and their connecting loop (residues 342–350) in the connection subdomain of the large subunit, in determining the RNase H cleavage window of HIV RTs. Full article

(This article belongs to the Special Issue In Memory of Stephen Oroszlan)

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Review

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15 pages, 2512 KiB

Open AccessReview

Stephan Oroszlan and the Proteolytic Processing of Retroviral Proteins: Following A Pro

by Ronald Swanstrom and Wesley I. Sundquist

Viruses 2021, 13(11), 2218; https://0-doi-org.brum.beds.ac.uk/10.3390/v13112218 - 04 Nov 2021

Cited by 2 | Viewed by 1624

Abstract

Steve Oroszlan determined the sequences at the ends of virion proteins for a number of different retroviruses. This work led to the insight that the amino-terminal amino acid of the mature viral CA protein is always proline. In this remembrance, we review Steve’s work that led to this insight and show how that insight was a necessary precursor to the work we have done in the subsequent years exploring the cleavage rate determinants of viral protease processing sites and the multiple roles the amino-terminal proline of CA plays after protease cleavage liberates it from its position in a protease processing site. Full article

(This article belongs to the Special Issue In Memory of Stephen Oroszlan)

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29 pages, 3132 KiB

Open AccessReview

From Recoding to Peptides for MHC Class I Immune Display: Enriching Viral Expression, Virus Vulnerability and Virus Evasion

by John F. Atkins, Kate M. O’Connor, Pramod R. Bhatt and Gary Loughran

Viruses 2021, 13(7), 1251; https://0-doi-org.brum.beds.ac.uk/10.3390/v13071251 - 27 Jun 2021

Cited by 4 | Viewed by 3139

Abstract

Many viruses, especially RNA viruses, utilize programmed ribosomal frameshifting and/or stop codon readthrough in their expression, and in the decoding of a few a UGA is dynamically redefined to specify selenocysteine. This recoding can effectively increase viral coding capacity and generate a set ratio of products with the same N-terminal domain(s) but different C-terminal domains. Recoding can also be regulatory or generate a product with the non-universal 21st directly encoded amino acid. Selection for translation speed in the expression of many viruses at the expense of fidelity creates host immune defensive opportunities. In contrast to host opportunism, certain viruses, including some persistent viruses, utilize recoding or adventitious frameshifting as part of their strategy to evade an immune response or specific drugs. Several instances of recoding in small intensively studied viruses escaped detection for many years and their identification resolved dilemmas. The fundamental importance of ribosome ratcheting is consistent with the initial strong view of invariant triplet decoding which however did not foresee the possibility of transitory anticodon:codon dissociation. Deep level dynamics and structural understanding of recoding is underway, and a high level structure relevant to the frameshifting required for expression of the SARS CoV-2 genome has just been determined. Full article

(This article belongs to the Special Issue In Memory of Stephen Oroszlan)

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9 pages, 228 KiB

Open AccessReview

Viruses and Bacteria Associated with Cancer: An Overview

by Davide Zella and Robert C. Gallo

Viruses 2021, 13(6), 1039; https://0-doi-org.brum.beds.ac.uk/10.3390/v13061039 - 31 May 2021

Cited by 25 | Viewed by 4102

Abstract

There are several human viruses and bacteria currently known to be associated with cancer. A common theme indicates that these microorganisms have evolved mechanisms to hamper the pathways dedicated to maintaining the integrity of genetic information, preventing apoptosis of the damaged cells and causing unwanted cellular proliferation. This eventually reduces the ability of their hosts to repair the damage(s) and eventually results in cellular transformation, cancer progression and reduced response to therapy. Our data suggest that mycoplasmas, and perhaps certain other bacteria with closely related DnaKs, may also contribute to cellular transformation and hamper certain drugs that rely on functional p53 for their anti-cancer activity. Understanding the precise molecular mechanisms is important for cancer prevention and for the development of both new anti-cancer drugs and for improving the efficacy of existing therapies. Full article

(This article belongs to the Special Issue In Memory of Stephen Oroszlan)

12 pages, 2366 KiB

Open AccessReview

HIV Protease: Historical Perspective and Current Research

by Irene T. Weber, Yuan-Fang Wang and Robert W. Harrison

Viruses 2021, 13(5), 839; https://0-doi-org.brum.beds.ac.uk/10.3390/v13050839 - 06 May 2021

Cited by 45 | Viewed by 5621

Abstract

The retroviral protease of human immunodeficiency virus (HIV) is an excellent target for antiviral inhibitors for treating HIV/AIDS. Despite the efficacy of therapy, current efforts to control the disease are undermined by the growing threat posed by drug resistance. This review covers the historical background of studies on the structure and function of HIV protease, the subsequent development of antiviral inhibitors, and recent studies on drug-resistant protease variants. We highlight the important contributions of Dr. Stephen Oroszlan to fundamental knowledge about the function of the HIV protease and other retroviral proteases. These studies, along with those of his colleagues, laid the foundations for the design of clinical inhibitors of HIV protease. The drug-resistant protease variants also provide an excellent model for investigating the molecular mechanisms and evolution of resistance. Full article

(This article belongs to the Special Issue In Memory of Stephen Oroszlan)

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Other

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5 pages, 171 KiB

Open AccessPerspective

Steve Oroszlan: A Personal Perspective

by Raymond Gilden

Viruses 2021, 13(4), 622; https://0-doi-org.brum.beds.ac.uk/10.3390/v13040622 - 05 Apr 2021

Cited by 2 | Viewed by 1621

Abstract

My memories of Steve go back over 50 years. While precise dates are no longer in my memory bank, circumstances and emotions remain alive and easy to recall. These memories tell the story of a remarkable human being, a true practitioner of his craft always, faithful to the basic principles of scientific pursuit, with integrity, honesty, and enthusiasm well beyond the norm. We had a professional symbiotic relationship that lasted over 20 years, resulting in over 50 publications in scientific journals and meeting abstracts. During that time, our fortunes rose in tandem, and when it was time to go our separate ways, he was more than ready to flourish on his own. Our personal friendship remained constant, and we enjoyed sharing meals and stories with family and friends over the years. In retrospect, I take pride in having played a role in a portion of his remarkable scientific journey. A few key anecdotes will illustrate some aspects of this summary. By way of a disclaimer, this is not a comprehensive review of the vast field of viral oncology and the selection of references is intentionally narrow. No slight is intended to the many outstanding investigators that were our contemporaries and at times collaborators during the period from the early 70s to the mid-80s. Full article

(This article belongs to the Special Issue In Memory of Stephen Oroszlan)

16 pages, 970 KiB

Open AccessPerspective

Across the Hall from Pioneers

by Alan Rein

Viruses 2021, 13(3), 491; https://0-doi-org.brum.beds.ac.uk/10.3390/v13030491 - 16 Mar 2021

Cited by 4 | Viewed by 1555

Abstract

I was fortunate to be associated with the lab of Stephen Oroszlan at the US National Cancer Institute from ~1982 until his conversion to Emeritus status in 1995. His lab made groundbreaking discoveries on retroviral proteins during that time, including many features that could not have been inferred or anticipated from straightforward sequence information. Building on the Oroszlan lab results, my colleagues and I demonstrated that the zinc fingers in nucleocapsid proteins play a crucial role in genomic RNA encapsidation; that the N-terminal myristylation of the Gag proteins of many retroviruses is important for their association with the plasma membrane before particle assembly is completed; and that gammaretroviruses initially synthesize their Env protein as an inactive precursor and then truncate the cytoplasmic tail of the transmembrane protein, activating Env fusogenicity, during virus maturation. We also elucidated several aspects of the mechanism of translational suppression in pol gene expression in gammaretroviruses; amazingly, this is a fundamentally different mechanism of suppression from that in most other retroviral genera. Full article

(This article belongs to the Special Issue In Memory of Stephen Oroszlan)

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