Hassan Javanbakht

Retrovirus Restriction by TRIM5α Variants from Old World and New World Primates

ABSTRACT The TRIM5α proteins of humans and some Old World monkeys have been shown to block infection of particular retroviruses following virus entry into the host cell. Infection of most New World monkey cells by the simian immunodeficiency virus of macaques (SIVmac) is restricted at a similar point. Here we examine the antiretroviral activity of TRIM5α orthologs from humans, apes, Old World monkeys, and New World monkeys. Chimpanzee and orangutan TRIM5α proteins functionally resembled human TRIM5α, potently restricting infection by N-tropic murine leukemia virus (N-MLV) and moderately restricting human immunodeficiency virus type 1 (HIV-1) infection. Notably, TRIM5α proteins from several New World monkey species restricted infection by SIVmac and the SIV of African green monkeys, SIVagm. Spider monkey TRIM5α, which has an expanded B30.2 domain v3 region due to a tandem triplication, potently blocked infection by a range of retroviruses, including SIVmac, SIVagm, HIV-1, and N-MLV. Tandem duplications in the TRIM5α B30.2 domain v1 region of African green monkeys are also associated with broader antiretroviral activity. Thus, variation in TRIM5α proteins among primate species accounts for the observed patterns of postentry restrictions in cells from these animals. The TRIM5α proteins of some monkey species exhibit dramatic lengthening of particular B30.2 variable regions and an expanded range of susceptible retroviruses.

The B30.2(SPRY) Domain of the Retroviral Restriction Factor TRIM5α Exhibits Lineage-Specific Length and Sequence Variation in Primates

ABSTRACT Tripartite motif (TRIM) proteins are composed of RING, B-box 2, and coiled coil domains. Some TRIM proteins, such as TRIM5α, also possess a carboxy-terminal B30.2(SPRY) domain and localize to cytoplasmic bodies. TRIM5α has recently been shown to mediate innate intracellular resistance to retroviruses, an activity dependent on the integrity of the B30.2 domain, in particular primate species. An examination of the sequences of several TRIM proteins related to TRIM5 revealed the existence of four variable regions (v1, v2, v3, and v4) in the B30.2 domain. Species-specific variation in TRIM5α was analyzed by amplifying, cloning, and sequencing nonhuman primate TRIM5 orthologs. Lineage-specific expansion and sequential duplication occurred in the TRIM5α B30.2 v1 region in Old World primates and in v3 in New World monkeys. We observed substitution patterns indicative of selection bordering these particular B30.2 domain variable elements. These results suggest that occasional, complex changes were incorporated into the TRIM5α B30.2 domain at discrete time points during the evolution of primates. Some of these time points correspond to periods during which primates were exposed to retroviral infections, based on the appearance of particular endogenous retroviruses in primate genomes. The results are consistent with a role for TRIM5α in innate immunity against retroviruses.

Retroviral Restriction Factor TRIM5α Is a Trimer

ABSTRACT The retrovirus restriction factor TRIM5α targets the viral capsid soon after entry. Here we show that the TRIM5α protein oligomerizes into trimers. The TRIM5α coiled-coil and B30.2(SPRY) domains make important contributions to the formation and/or stability of the trimers. A functionally defective TRIM5α mutant with the RING and B-box 2 domains deleted can form heterotrimers with wild-type TRIM5α, accounting for the observed dominant-negative activity of the mutant protein. Trimerization potentially allows TRIM5α to interact with threefold pseudosymmetrical structures on retroviral capsids.

Suppression of hepatitis B virus DNA accumulation in chronically infected cells using a bacterial CRISPR/Cas RNA-guided DNA endonuclease

Hepatitis B virus (HBV) remains a major human pathogen, with over 240 million individuals suffering from chronic HBV infections. These can persist for decades due to the lack of therapies that can effectively target the stable viral covalently closed circular (ccc) DNA molecules present in infected hepatocytes. Using lentiviral transduction of a bacterial Cas9 gene and single guide RNAs (sgRNAs) specific for HBV, we observed effective inhibition of HBV DNA production in in vitro models of both chronic and de novo HBV infection. Cas9/sgRNA combinations specific for HBV reduced total viral DNA levels by up to ~1000-fold and HBV cccDNA levels by up to ~10-fold and also mutationally inactivated the majority of the residual viral DNA. Together, these data provide proof of principle for the hypothesis that CRISPR/Cas systems have the potential to serve as effective tools for the depletion of the cccDNA pool in chronically HBV infected individuals.

The Human TRIM5α Restriction Factor Mediates Accelerated Uncoating of the N-Tropic Murine Leukemia Virus Capsid

ABSTRACT The host cell factors TRIM5αhu and Fv-1 restrict N-tropic murine leukemia virus (N-MLV) infection at an early postentry step before or after reverse transcription, respectively. Interestingly, the identity of residue 110 of the MLV capsid determines susceptibility to both TRIM5αhu and Fv-1. In this study, we investigate the fate of the MLV capsid in cells expressing either the TRIM5αhu or Fv-1 restriction factor. The expression of TRIM5αhu, but not Fv-1, specifically promoted the premature conversion of particulate N-MLV capsids within infected cells to soluble capsid proteins. The TRIM5αhu-mediated disassembly of particulate N-MLV capsids was dependent upon residue 110 of the viral capsid. Furthermore, the deletion or disruption of TRIM5αhu domains necessary for potent N-MLV restriction completely abrogated the disappearance of particulate N-MLV capsids observed with wild-type TRIM5αhu. These results suggest that premature disassembly of the viral capsid contributes to the restriction of N-MLV infection by TRIM5αhu, but not by Fv-1.

Incorporation of Lysyl-tRNA Synthetase into Human Immunodeficiency Virus Type 1

ABSTRACT During human immunodeficiency virus type 1 (HIV-1) assembly, tRNALys isoacceptors are selectively incorporated into virions and tRNA 3 Lys is used as the primer for reverse transcription. We show herein that the tRNALys-binding protein, lysyl-tRNA synthetase (LysRS), is also selectively packaged into HIV-1. The viral precursor protein Pr55 gag alone will package LysRS into Pr55 gag particles, independently of tRNALys. With the additional presence of the viral precursor protein Pr160 gag-pol , tRNALys and LysRS are both packaged into the particle. While the predominant cytoplasmic LysRS has an apparent M r of 70,000, viral LysRS associated with tRNALys packaging is shorter, with an apparent M r of 63,000. The truncation occurs independently of viral protease and might be required to facilitate interactions involved in the selective packaging and genomic placement of primer tRNA 3 Lys .

A Randomized, Double-Blind Placebo Controlled Trial of Balapiravir, a Polymerase Inhibitor, in Adult Dengue Patients

Background. Dengue is the most common arboviral infection of humans. There are currently no specific treatments for dengue. Balapiravir is a prodrug of a nucleoside analogue (called R1479) and an inhibitor of hepatitis C virus replication in vivo. Methods. We conducted in vitro experiments to determine the potency of balapiravir against dengue viruses and then an exploratory, dose-escalating, randomized placebo-controlled trial in adult male patients with dengue with <48 hours of fever. Results. The clinical and laboratory adverse event profile in patients receiving balapiravir at doses of 1500 mg (n = 10) or 3000 mg (n = 22) orally for 5 days was similar to that of patients receiving placebo (n = 32), indicating balapiravir was well tolerated. However, twice daily assessment of viremia and daily assessment of NS1 antigenemia indicated balapiravir did not measurably alter the kinetics of these virological markers, nor did it reduce the fever clearance time. The kinetics of plasma cytokine concentrations and the whole blood transcriptional profile were also not attenuated by balapiravir treatment. Conclusions. Although this trial, the first of its kind in dengue, does not support balapiravir as a candidate drug, it does establish a framework for antiviral treatment trials in dengue and provides the field with a clinically evaluated benchmark molecule. Clinical Trials Registration. NCT01096576.

Retrovirus-Specific Packaging of Aminoacyl-tRNA Synthetases with Cognate Primer tRNAs

ABSTRACT The tRNAs used to prime reverse transcription in human immunodeficiency virus type 1 (HIV-1), Rous sarcoma virus (RSV), and Moloney murine leukemia virus (Mo-MuLV) are \batchmode \documentclass[fleqn,10pt,legalpaper]{article} \usepackage{amssymb} \usepackage{amsfonts} \usepackage{amsmath} \pagestyle{empty} \begin{document} \(tRNA_{3}^{Lys}\) \end{document} , tRNATrp, and tRNAPro, respectively. Using antibodies to the three cognate human aminoacyl-tRNA synthetases, we found that only lysyl-tRNA synthetase (LysRS) is present in HIV-1, only tryptophanyl-tRNA synthetase (TrpRS) is present in RSV, and neither these two synthetases nor prolyl-tRNA synthetase (ProRS) is present in Mo-MuLV. LysRS and TrpRS are present in HIV-1 and RSV at approximately 25 and 12 molecules/virion, respectively. These results support the hypothesis that, in HIV-1 and RSV, the cognate aminoacyl-tRNA synthetase may be used as the signal for targeting the selective packaging of primer tRNAs into retroviruses. The absence of ProRS in Mo-MuLV is consistent with reports that selective packaging of tRNAPro in this virus is less important for achieving optimum annealing of the primer to Mo-MuLV genomic RNA.

Cellular Distribution of Lysyl-tRNA Synthetase and Its Interaction with Gag during Human Immunodeficiency Virus Type 1 Assembly

ABSTRACT Lysyl-tRNA synthetase (LysRS) is packaged into human immunodeficiency virus type 1 (HIV-1) via its interaction with Gag, and this enzyme facilitates the selective packaging of tRNA3Lys, the primer for initiating reverse transcription, into HIV-1. The Gag/LysRS interaction is detected at detergent-resistant membrane but not in membrane-free cell compartments that contain Gag and LysRS. LysRS is found (i) in the nucleus, (ii) in a cytoplasmic high-molecular-weight aminoacyl-tRNA synthetase complex (HMW aaRS complex), (iii) in mitochondria, and (iv) associated with plasma membrane. The cytoplasmic form of LysRS lacking the mitochondrial import signal was previously shown to be efficiently packaged into virions, and in this report we also show that LysRS compartments in nuclei, in the HMW aaRS complex, and at the membrane are also not required as a primary source for viral LysRS. Exogenous mutant LysRS species unable to either enter the nucleus or bind to the cell membrane are still incorporated into virions. Many HMW aaRS components are not packaged into the virion along with LysRS, and the interaction of LysRS with p38, a protein that binds tightly to LysRS in the HMW aaRS complex, is not required for the incorporation of LysRS into virions. These data indicate that newly synthesized LysRS may interact rapidly with Gag before the enzyme has the opportunity to move to the above-mentioned cellular compartments. In confirmation of this idea, we found that newly synthesized LysRS is associated with Gag after a 10-min pulse with [35S]cysteine/methionine. This observation is also supported by previous work indicating that the incorporation of LysRS into HIV-1 is very sensitive to the inhibition of new synthesis of LysRS.

Mutational Inactivation of Herpes Simplex Virus 1 MicroRNAs Identifies Viral mRNA Targets and Reveals Phenotypic Effects in Culture

ABSTRACT Herpes simplex virus 1 (HSV-1), a ubiquitous human pathogen, expresses several viral microRNAs (miRNAs). These, along with the latency-associated transcript, represent the only viral RNAs detectable in latently infected neuronal cells. Here, for the first time, we analyze which HSV-1 miRNAs are loaded into the RNA-induced silencing complex (RISC), the key effector of miRNA function. Only 9 of the 17 reported HSV-1 miRNAs, i.e., miR-H1 to miR-H8 plus miR-H11, were found to actually load into the RISC. Surprisingly, this analysis also revealed that HSV-1 miRNAs loaded into the RISC with efficiencies that differed widely; <1% of the miR-H1-3p miRNA detectable in HSV-1-infected cells was loaded into the RISC. Analysis of HSV-1 mutants individually lacking the viral miR-H2, miR-H3, or miR-H4 miRNA revealed that loss of these miRNAs affected the rate of replication of HSV-1 in neuronal cells but not in fibroblasts. Analysis of mRNA and protein expression, as well as assays mapping viral miRNA binding sites in infected cells, showed that endogenous HSV-1 miR-H2 binds to viral ICP0 mRNA and inhibits its expression, while endogenous miR-H4 inhibits the expression of the viral ICP34.5 gene. In contrast, no viral mRNA target for miR-H3 could be detected, even though miR-H3, like miR-H4, is perfectly complementary to ICP34.5 mRNA. Together, these data demonstrate that endogenous HSV-1 miRNA expression can significantly alter viral replication in culture, and they also identify two viral mRNA targets for miR-H2 and miR-H4 that can partially explain this phenotype.