Kuo-Jung Huang

HIV-1 matrix protein repositioning in nucleocapsid region fails to confer virus-like particle assembly

Abstract The HIV-1 matrix (MA) protein is similar to nucleocapsid (NC) proteins in its propensity for self-interaction and association with RNA. Here we report on our finding that replacing MA with NC results in the production of wild type (wt)-level RNA and virus-like particles (VLPs). In contrast, constructs containing MA as a substitute for NC are markedly defective in VLP production and form virions with lower densities than wt, even though their RNA content is over 50% that of wt level. We also noted that a ΔMN mutant lacking both MA and NC produces a relatively higher amount of VLPs than those in which MA was substituted for NC. Although ΔMN contains approximately 30% the RNA of wt, it still exhibits virion densities equal (or very similar) to those of wt. The data suggest that neither NC nor RNA are major virion density determinants. Furthermore, we noted that NC(ZIP)—a NC replacement with a leucine zipper dimerization motif—produces VLPs as efficiently as wt. However, the markedly reduced assembly efficiency of NC(ZIP) is associated with the formation of VLPs with densities slightly lower than those of wt following MA removal, suggesting that (a) MA is required to help the inserted leucine zipper motif perform efficient Gag multimerization, and (b) MA plays a role in the virus assembly process.

Self-assembly of Severe Acute Respiratory Syndrome Coronavirus Membrane Protein

Coronavirus membrane (M) protein can form virus-like particles (VLPs) when coexpressed with nucleocapsid (N) or envelope (E) proteins, suggesting a pivotal role for M in virion assembly. Here we demonstrate the self-assembly and release of severe acute respiratory syndrome coronavirus (SARS-CoV) M protein in medium in the form of membrane-enveloped vesicles with densities lower than those of VLPs formed by M plus N. Although efficient N-N interactions require the presence of RNA, we found that M-M interactions were RNA-independent. SARS-CoV M was observed in both the Golgi area and plasma membranes of a variety of cells. Blocking M glycosylation does not appear to significantly affect M plasma membrane labeling intensity, M-containing vesicle release, or VLP formation. Results from a genetic analysis indicate involvement of the third transmembrane domain of M in plasma membrane-targeting signal. Fusion proteins containing M amino-terminal 50 residues encompassing the first transmembrane domain were found to be sufficient for membrane binding, multimerization, and Golgi retention. Surprisingly, we found that fusion proteins lacking all three transmembrane domains were still capable of membrane binding, Golgi retention, and interacting with M. The data suggest that multiple SARS-CoV M regions are involved in M self-assembly and subcellular localization.

Mutations at human immunodeficiency virus type 1 reverse transcriptase tryptophan repeat motif attenuate the inhibitory effect of efavirenz on virus production.

HIV-1 virus particle processing is mediated by protease (PR), with enzymatic activation triggered by Gag-Pol/Gag-Pol interaction. We previously reported that truncation mutations at the reverse transcriptase (RT) connection subdomain markedly impair virus particle processing, suggesting an important role for the RT subdomain in PR-mediated virus processing. A highly conserved tryptophan (Trp) repeat motif of the HIV-1 RT connection subdomain is involved in RT dimerization. Our goal in this study was to determine whether mutations at the Trp repeat motif have any effect on PR-mediated virus processing. Our results indicate that even though alanine substitutions at W401 (W401A) or at both W401 and W402 (W401A/W402A) have no major effect on steady-state virus processing, the combined W401A/W402A mutations partially negate and the W401A mutation almost completely negates an efavirenz (EFV)-imposed barrier to virus production. The combination of RT instability and poor enzymatic activity reflects a RT dimerization defect incurred by the mutations. We also found that an artificial p66RT carrying the W401A or W401A/W402A mutations was packaged into virions more efficiently than wild-type p66RT, and that the viral incorporation of p66RT is significantly reduced by EFV, implying a novel effect of EFV on RT-Gag interaction. Our results suggest that the Trp repeat motif may play a role in the Gag-Pol/Gag-Pol interaction that contributes to subsequent PR activation.

A Single Amino Acid Substitution in HIV-1 Reverse Transcriptase Significantly Reduces Virion Release

ABSTRACT HIV-1 protease (PR) mediates the proteolytic processing of virus particles during or after virus budding. PR activation is thought to be triggered by appropriate Gag-Pol/Gag-Pol interaction; factors affecting this interaction either enhance or reduce PR-mediated cleavage efficiency, resulting in markedly reduced virion production or the release of inadequately processed virions. We previously showed that a Gag-Pol deletion mutation involving the reverse transcriptase tryptophan (Trp) repeat motif markedly impairs PR-mediated virus maturation and that an alanine substitution at W401 (W401A) or at both W401 and W402 (W401A/W402A) partially or almost completely negates the enhancement effect of efavirenz (a nonnucleoside reverse transcriptase inhibitor) on PR-mediated virus processing efficiency. These data suggest that the Trp repeat motif may contribute to the PR activation process. Here we demonstrate that due to enhanced Gag cleavage efficiency, W402 alanine or leucine substitution significantly reduces virus production. However, W402 replacement with phenylalanine does not significantly affect virus particle assembly or processing, but it does markedly impair viral infectivity in a single-cycle infection assay. Our results demonstrate that a single amino acid substitution at HIV-1 RT can radically affect virus assembly by enhancing Gag cleavage efficiency, suggesting that in addition to contributing to RT biological function during the early stages of virus replication, the HIV-1 RT tryptophan repeat motif in a Gag-Pol context may play an important role in suppressing the premature activation of PR during late-stage virus replication.

Incorporation of Human Immunodeficiency Virus Type 1 Reverse Transcriptase into Virus-Like Particles

ABSTRACT We demonstrate that a genetically engineered human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) composed mainly of p66 or p51 subunits can be incorporated into virus-like particles (VLPs) when coexpressed with HIV-1 Pr55gag. VLP-associated RT exhibited a detergent-resistant association with immature cores during sucrose gradient equilibrium centrifugation, suggesting that RT is incorporated into VLPs. However, RT that retains downstream integrase (IN) is severely inhibited in terms of incorporation into VLPs. Results from immunofluorescence tests reveal that RT-IN is primarily localized at the perinuclear area and exhibits poor colocalization with Gag. IN removal leads to a redistribution of RT throughout the cytoplasm and improved RT incorporation into VLPs. Similar results were observed for RT-IN in which alanine was substituted for 186-Lys-Arg-Lys-188 residues of the IN putative nuclear localization signal, suggesting that IN karyophilic properties may partly account for the inhibitory effect of IN on RT incorporation. Although the membrane-binding capacity of RT was markedly reduced compared to that of wild-type Gag or Gag-Pol, the correlation of membrane-binding ability with particle incorporation efficiency was incomplete. Furthermore, we observed that membrane-binding-defective myristylation-minus RT can be packaged into VLPs at the same level as its normal myristylated counterpart. This suggests that the incorporation of RT into VLPs is independent of membrane affinity but very dependent on RT-Gag interaction. Results from a genetic analysis suggest that the Gag-interacting regions of RT mainly reside in the thumb subdomain and that the RT-binding domains of Gag are located in the matrix (MA) and p6 regions.

Placement of Leucine Zipper Motifs at the Carboxyl Terminus of HIV-1 Protease Significantly Reduces Virion Production

Natural HIV-1 protease (PR) is homodimeric. Some researchers believe that interactions between HIV-1 Gag-Pol molecules trigger the activation of embedded PR (which mediates Gag and Gag-Pol cleavage), and that Gag-Pol assembly domains outside of PR may contribute to PR activation by influencing PR dimer interaction in a Gag-Pol context. To determine if the enhancement of PR dimer interaction facilitates PR activation, we placed single or tandem repeat leucine zippers (LZ) at the PR C-terminus, and looked for a correlation between enhanced Gag processing efficiency and increased Gag-PR-LZ multimerization capacity. We found significant reductions in virus-like particles (VLPs) produced by HIV-1 mutants, with LZ fused to the end of PR as a result of enhanced Gag cleavage efficiency. Since VLP production can be restored to wt levels following PR activity inhibition, this assembly defect is considered PR activity-dependent. We also found a correlation between the LZ enhancement effect on Gag cleavage and enhanced Gag-PR multimerization. The results suggest that PR dimer interactions facilitated by forced Gag-PR multimerization lead to premature Gag cleavage, likely a result of premature PR activation. Our conclusion is that placement of a heterologous dimerization domain downstream of PR enhances PR-mediated Gag cleavage efficiency, implying that structural conformation, rather than the primary sequence outside of PR, is a major determinant of HIV-1 PR activation.

BST2/CD317 counteracts human coronavirus 229E productive infection by tethering virions at the cell surface

Abstract Bone marrow stromal antigen 2 (BST2), an interferon-inducible antiviral factor, has been shown to block the release of various enveloped viruses from cells. It has also been identified as an innate immune system component. Most enveloped viruses subject to BST2 restriction bud at the plasma membrane. Here we report our findings that (a) the production of human coronavirus 229E (HCoV-229E) progeny viruses, whose budding occurs at the ER-Golgi intermediate compartment (ERGIC), markedly decreases in the presence of BST2; and (b) BST2 knockdown expression results in enhanced HCoV-229E virion production. Electron microscopy analyses indicate that HCoV-229E virions are tethered to cell surfaces or intracellular membranes by BST2. Our results suggest that BST2 exerts a broad blocking effect against enveloped virus release, regardless of whether budding occurs at the plasma membrane or intracellular compartments.

Gag-Pol Transframe Domain p6* Is Essential for HIV-1 Protease-Mediated Virus Maturation

HIV-1 protease (PR) is encoded by pol, which is initially translated as a Pr160gag-pol polyprotein by a ribosomal frameshift event. Within Gag-Pol, truncated p6gag is replaced by a transframe domain (referred to as p6* or p6pol) located directly upstream of PR. p6* has been proposed as playing a role in modulating PR activation. Overlapping reading frames between p6* and p6gag present a challenge to researchers using genetic approaches to studying p6* biological functions. To determine the role of p6* in PR activation without affecting the gag reading frame, we constructed a series of Gag/Gag-Pol expression vectors by duplicating PR with or without p6* between PR pairs, and observed that PR duplication eliminated virus production due to significant Gag cleavage enhancement. This effect was mitigated when p6* was placed between the two PRs. Further, Gag cleavage enhancement was markedly reduced when either one of the two PRs was mutationally inactivated. Additional reduction in Gag cleavage efficiency was noted following the removal of p6* from between the two PRs. The insertion of a NC domain (wild-type or mutant) directly upstream of PR or p6*PR did not significantly improve Gag processing efficiency. With the exception of those containing p6* directly upstream of an active PR, all constructs were either noninfectious or weakly infectious. Our results suggest that (a) p6* is essential for triggering PR activation, (b) p6* has a role in preventing premature virus processing, and (c) the NC domain within Gag-Pol is not a major determinant of PR activation.

Mutations in the HIV-1 reverse transcriptase tryptophan repeat motif affect virion maturation and Gag-Pol packaging.

Our goal was to determine the contribution of HIV-1 reverse transcriptase tryptophan repeat motif residues to virion maturation. With the exception of W402A, we found none of the single substitution mutations exerted major impacts on virus assembly or processing. However, all mutants except for W410A exhibited significant decreases in virus-associated RT, presumably a result of unstable RT mutant degradation. Mutations W398A, W401A and W406A decreased the enhancement effect of efavirenz on PR-mediated Gag processing efficiency, which is in agreement with their destabilizing RT effects. Furthermore, combined double or triple W398, W401 and W406 mutations significantly affected virus processing and Gag-Pol packaging. Further analyses suggest that inefficient PR-mediated Gag cleavage partly accounts for the virion processing defect. Our results support the idea that in addition to playing a role in RT heterodimer stabilization, the RT Trp repeat motif in the Gag-Pol context is also involved in PR activation via Gag-Pol/Gag-Pol interaction.

Identifying SARS-CoV membrane protein amino acid residues linked to virus-like particle assembly.

Severe acute respiratory syndrome coronavirus (SARS-CoV) membrane (M) proteins are capable of self-assembly and release in the form of membrane-enveloped vesicles, and of forming virus-like particles (VLPs) when coexpressed with SARS-CoV nucleocapsid (N) protein. According to previous deletion analyses, M self-assembly involves multiple M sequence regions. To identify important M amino acid residues for VLP assembly, we coexpressed N with multiple M mutants containing substitution mutations at the amino-terminal ectodomain, carboxyl-terminal endodomain, or transmembrane segments. Our results indicate that a dileucine motif in the endodomain tail (218LL219) is required for efficient N packaging into VLPs. Results from cross-linking VLP analyses suggest that the cysteine residues 63, 85 and 158 are not in close proximity to the M dimer interface. We noted a significant reduction in M secretion due to serine replacement for C158, but not for C63 or C85. Further analysis suggests that C158 is involved in M-N interaction. In addition to mutations of the highly conserved 107-SWWSFNPE-114 motif, substitutions at codons W19, W57, P58, W91, Y94 or F95 all resulted in significantly reduced VLP yields, largely due to defective M secretion. VLP production was not significantly affected by a tryptophan replacement of Y94 or F95 or a phenylalanine replacement of W19, W57 or W91. Combined, these results indicate the involvement of specific M amino acids during SARS-CoV virus assembly, and suggest that aromatic residue retention at specific positions is critical for M function in terms of directing virus assembly.