prM-reactive antibodies reveal a role for partially mature virions in dengue virus pathogenesis.

Cleavage of the flavivirus premembrane (prM) structural protein during maturation can be inefficient. The contribution of partially mature flavivirus virions that retain uncleaved prM to pathogenesis during primary infection is unknown. To investigate this question, we characterized the functional properties of newly-generated dengue virus (DENV) prM-reactive monoclonal antibodies (mAbs) in vitro and using a mouse model of DENV disease. Anti-prM mAbs neutralized DENV infection in a virion maturation state-dependent manner. Alanine scanning mutagenesis and cryoelectron microscopy of anti-prM mAbs in complex with immature DENV defined two modes of attachment to a single antigenic site. In vivo, passive transfer of intact anti-prM mAbs resulted in an antibody-dependent enhancement of disease. However, protection against DENV-induced lethality was observed when the transferred mAbs were genetically modified to inhibit their ability to interact with Fcγ receptors. These data establish that in addition to mature forms of the virus, partially mature infectious prM+ virions can also contribute to pathogenesis during primary DENV infections.

Assembly and Evolution of Poxviruses.

Poxvirus assembly has been an intriguing area of research for several decades. While advancements in experimental techniques continue to yield fresh insights, many questions are still unresolved. Large genome sizes of up to 380 kbp, asymmetrical structure, an exterior lipid bilayer, and a cytoplasmic life cycle are some notable characteristics of these viruses. Inside the particle are two lateral bodies and a protein wall-bound-biconcave core containing the viral nucleocapsid. The assembly progresses through five major stages-endoplasmic reticulum (ER) membrane alteration and rupture, crescent formation, immature virion formation, genome encapsidation, virion maturation and in a subset of viruses, additional envelopment of the virion prior to its dissemination. Several large dsDNA viruses have been shown to follow a comparable sequence of events. In this chapter, we recapitulate our understanding of the poxvirus morphogenesis process while reviewing the most recent advances in the field. We also briefly discuss how virion assembly aids in our knowledge of the evolutionary links between poxviruses and other Nucleocytoplasmic Large DNA Viruses (NCLDVs).

Evaluating HIV-1 Infectivity and Virion Maturation across Varied Producer Cells with a Novel FRET-Based Detection and Quantification Assay.

The maturation of HIV-1 virions is a crucial process in viral replication. Although T-cells are a primary source of virus production, much of our understanding of virion maturation comes from studies using the HEK293T human embryonic kidney cell line. Notably, there is a lack of comparative analyses between T-cells and HEK293T cells in terms of virion maturation efficiency in existing literature. We previously developed an advanced virion visualization system based on the FRET principle, enabling the effective distinction between immature and mature virions via fluorescence microscopy. In this study, we utilized pseudotyped, single-round infectious viruses tagged with FRET labels (HIV-1 Gag-iFRET∆Env) derived from Jurkat (a human T-lymphocyte cell line) and HEK293T cells to evaluate their virion maturation rates. HEK293T-derived virions demonstrated a maturity rate of 81.79%, consistent with other studies and our previous findings. However, virions originating from Jurkat cells demonstrated a significantly reduced maturation rate of 68.67% (p < 0.0001). Correspondingly, viruses produced from Jurkat cells exhibited significantly reduced infectivity compared to those derived from HEK293T cells, with the relative infectivity measured at 65.3%. This finding is consistent with the observed relative maturation rate of viruses produced by Jurkat cells. These findings suggest that initiation of virion maturation directly correlates with viral infectivity. Our observation highlights the dynamic nature of virus-host interactions and their implications for virion production and infectivity.

Emergence of Compensatory Mutations Reveals the Importance of Electrostatic Interactions between HIV-1 Integrase and Genomic RNA.

HIV-1 integrase (IN) has a noncatalytic function in virion maturation through its binding to the viral RNA genome (gRNA). Class II IN substitutions inhibit IN-gRNA binding and result in the formation of virions with aberrant morphologies marked by mislocalization of the gRNA between the capsid lattice and the lipid envelope. These viruses are noninfectious due to a block at an early reverse transcription stage in target cells. HIV-1 IN utilizes basic residues within its C-terminal domain (CTD) to bind to the gRNA; however, the molecular nature of how these residues mediate gRNA binding and whether other regions of IN are involved remain unknown. To address this, we have isolated compensatory substitutions in the background of a class II IN mutant virus bearing R269A/K273A substitutions within the IN-CTD. We found that the nearby D256N and D270N compensatory substitutions restored the ability of IN to bind gRNA and led to the formation of mature infectious virions. Reinstating the local positive charge of the IN-CTD through individual D256R, D256K, D278R, and D279R substitutions was sufficient to specifically restore IN-gRNA binding and reverse transcription for the IN R269A/K273A as well as the IN R262A/R263A class II mutants. Structural modeling suggested that compensatory substitutions in the D256 residue created an additional interaction interface for gRNA binding, whereas other substitutions acted locally within the unstructured C-terminal tail of IN. Taken together, our findings highlight the essential role of CTD in gRNA binding and reveal the importance of pliable electrostatic interactions between the IN-CTD and the gRNA. IMPORTANCE In addition to its catalytic function, HIV-1 integrase (IN) binds to the viral RNA genome (gRNA) through positively charged residues (i.e., R262, R263, R269, K273) within its C-terminal domain (CTD) and regulates proper virion maturation. Mutation of these residues results in the formation of morphologically aberrant viruses blocked at an early reverse transcription stage in cells. Here we show that compensatory substitutions in nearby negatively charged aspartic acid residues (i.e., D256N, D270N) restore the ability of IN to bind gRNA for these mutant viruses and result in the formation of accurately matured infectious virions. Similarly, individual charge reversal substitutions at D256 as well as other nearby positions (i.e., D278, D279) are all sufficient to enable the respective IN mutants to bind gRNA, and subsequently restore reverse transcription and virion infectivity. Taken together, our findings reveal the importance of highly pliable electrostatic interactions in IN-gRNA binding.

The Hepatitis B Virus Envelope Proteins: Molecular Gymnastics Throughout the Viral Life Cycle.

New hepatitis B virions released from infected hepatocytes are the result of an intricate maturation process that starts with the formation of the nucleocapsid providing a confined space where the viral DNA genome is synthesized via reverse transcription. Virion assembly is finalized by the enclosure of the icosahedral nucleocapsid within a heterogeneous envelope. The latter contains integral membrane proteins of three sizes, collectively known as hepatitis B surface antigen, and adopts multiple conformations in the course of the viral life cycle. The nucleocapsid conformation depends on the reverse transcription status of the genome, which in turn controls nucleocapsid interaction with the envelope proteins for virus exit. In addition, after secretion the virions undergo a distinct maturation step during which a topological switch of the large envelope protein confers infectivity. Here we review molecular determinants for envelopment and models that postulate molecular signals encoded in the capsid scaffold conducive or adverse to the recruitment of envelope proteins.

Role of Host Cell Secretory Machinery in Zika Virus Life Cycle.

The high human cost of Zika virus infections and the rapid establishment of virus circulation in novel areas, including the United States, present an urgent need for countermeasures against this emerging threat. The development of an effective vaccine against Zika virus may be problematic because of the cross reactivity of the antibodies with other flaviviruses leading to antibody-dependent enhancement of infection. Moreover, rapidly replicating positive strand RNA viruses, including Zika virus, generate large spectrum of mutant genomes (quasi species) every replication round, allowing rapid selection of variants resistant to drugs targeting virus-specific proteins. On the other hand, viruses are ultimate cellular parasites and rely on the host metabolism for every step of their life cycle, thus presenting an opportunity to manipulate host processes as an alternative approach to suppress virus replication and spread. Zika and other flaviviruses critically depend on the cellular secretory pathway, which transfers proteins and membranes from the ER through the Golgi to the plasma membrane, for virion assembly, maturation and release. In this review, we summarize the current knowledge of interactions of Zika and similar arthropod-borne flaviviruses with the cellular secretory machinery with a special emphasis on virus-specific changes of the secretory pathway. Identification of the regulatory networks and effector proteins required to accommodate the trafficking of virions, which represent a highly unusual cargo for the secretory pathway, may open an attractive and virtually untapped reservoir of alternative targets for the development of superior anti-viral drugs.

Infection-Induced Changes Within the Endocytic Recycling Compartment Suggest a Roadmap of Human Cytomegalovirus Egress.

Human cytomegalovirus (HCMV) is an important pathogen in developing fetuses, neonates, and individuals with compromised immune systems. Gaps in our understanding of the mechanisms required for virion assembly stand in the way of development of antivirals targeting late stages of viral replication. During infection, HCMV causes a dramatic reorganization of the host endosecretory system, leading to the formation of the cytoplasmic virion assembly complex (cVAC), the site of virion assembly. As part of cVAC biogenesis, the composition and behavior of endosecretory organelles change. To gain more comprehensive understanding of the impact HCMV infection has on components of the cellular endocytic recycling compartment (ERC), we used previously published transcriptional and proteomic datasets to predict changes in the directionality of ERC trafficking. We identified infection-associated changes in gene expression that suggest shifts in the balance between endocytic and exocytic recycling pathways, leading to formation of a secretory trap within the cVAC. Conversely, there was a corresponding shift favoring outbound secretory vesicle trafficking, indicating a potential role in virion egress. These observations are consistent with previous studies describing sequestration of signaling molecules, such as IL-6, and the synaptic vesicle-like properties of mature HCMV virions. Our analysis enabled development of a refined model incorporating old and new information related to the behavior of the ERC during HCMV replication. While limited by the paucity of integrated systems-level data, the model provides an informed basis for development of experimentally testable hypotheses related to mechanisms involved in HCMV virion maturation and egress. Information from such experiments will provide a robust roadmap for rational development of novel antivirals for HCMV and related viruses.

Mutations in HPV18 E1

The most highly expressed protein during the productive phase of the human papillomavirus (HPV) life cycle is E1^E4. Its full role during infection remains to be established. HPV E1^E4 is expressed during both the early and late stages of the virus life cycle and contributes to viral genome amplification. In an attempt to further outline the functions of E1^E4, and determine whether it plays a role in viral capsid assembly and viral infectivity, we examined wild-type E1^E4 as well as four E1^E4 truncation mutants. Our study revealed that HPV18 genomes containing the shortest truncated form of E1^E4, the 17/18 mutant, produced viral titers that were similar to wild-type virus and significantly higher compared to virions containing the three longer E1^E4 mutants. Additionally, the infectivity of virus containing the shortest E1^E4 mutation was equivalent to wild-type and significantly higher than the other three mutants. In contrast, infectivity was completely abrogated for virus containing the longer E1^E4 mutants, regardless of virion maturity. Taken together, our results indicate for the first time that HPV18 E1^E4 impacts capsid assembly and viral infectivity as well as virus maturation.

Enhancing dengue virus maturation using a stable furin over-expressing cell line.

Flaviviruses are positive-stranded RNA viruses that incorporate envelope (E) and premembrane (prM) proteins into the virion. Furin-mediated cleavage of prM defines a required maturation step in the flavivirus lifecycle. Inefficient prM cleavage results in structurally heterogeneous virions with unique antigenic and functional characteristics. Recent studies with dengue virus suggest that viruses produced in tissue culture cells are less mature than those produced in primary cells. In this study, we describe a Vero cell line that ectopically expresses high levels of human furin (Vero-furin) for use in the production of more homogenous mature flavivirus populations. Laboratory-adapted and clinical dengue virus isolates grow efficiently in Vero-furin cells. Biochemical and structural techniques demonstrate efficient prM cleavage in Vero-furin derived virus preparations. These virions also were less sensitive to neutralization by antibodies that bind efficiently to immature virions. This furin-expressing cell line will be of considerable utility for flavivirus neutralization and structural studies.

Specific mutations in the C-terminus domain of HBV surface antigen significantly correlate with low level of serum HBV-DNA in patients with chronic HBV infection.

BACKGROUND: To define HBsAg-mutations correlated with different serum HBV-DNA levels in HBV chronically-infected drug-naive patients. METHODS: This study included 187 patients stratified into the following ranges of serum HBV-DNA:12-2000 IU/ml, 2000-100,000 IU/ml, and >100,000 IU/ml. HBsAg-mutations were associated with HBV-DNA levels by applying a Bayesian-Partitional-Model and Fisher-exact test. Mutant and wild-type HBV genotype-D genomes were expressed in Huh7 cells and HBsAg-production was determined in cell-supernatants at 3 days-post-transfection. RESULTS: Specific HBsAg-mutations (M197T,-S204N-Y206C/H-F220L) were significantly correlated with serum HBV-DNA <2000 IU/ml (posterior-probability>90%, P < 0.05). The presence of Y206C/H and/or F220L was also associated with lower median (IQR) HBsAg-levels and lower median (IQR) transaminases (for HBsAg:250[115-840] IU/ml for Y206C/H and/or F220L versus 4300[640-11,838] IU/ml for wild-type, P = 0.023; for ALT:28[21-40] IU/ml versus 53[34-90] IU/ml, P < 0.001). These mutations were localized in the HBsAg C-terminus, known to be involved in virion and/or HBsAg secretion. The co-occurrence of Y206C + F220L was found significant by cluster-analysis, (P = 0.02). In addition, in an in-vitro model Y206C + F220L determined a 2.8-3.3 fold-reduction of HBsAg-amount released in supernatants compared to single mutants and wt (Y206C + F220L = 5,679 IU/ml; Y206H = 16,305 IU/ml; F220L = 18,368 IU/ml; Y206C = 18,680 IU/ml; wt = 14,280 IU/ml, P < 0.05). CONCLUSIONS: Specific HBsAg-mutations (compartmentalized in the HBsAg C-terminus) correlated with low-serum HBV-DNA and HBsAg-levels. These findings can be important to understand mechanisms underlying low HBV replicative potential including the inactive-carrier state.