Density Of Virions Research Articles

Repurposing cholesterol lowering drugs in the treatment and management of monkeypox.

Dear Editor, While the likelihood of monkeypox (MPX) becoming a pandemic, very little is known about the virus. The smallpox-like symptoms caused by the MPX virus are less severe. Given the current possibility of a pandemic, it is crucial that any negative impacts of a MPX epidemic on public health be kept to a minimum. At the time of writing this letter, Centre for Disease control and Prevention has reported 77,092 confirmed cases across the globe in more than 89 countries. According to Centre for Disease control and Prevention, there is currently no cure for MPX infections; nevertheless, most patients recover after receiving supportive care, such as rehydration. MPX virus and Smallpox virus share genetic similarity and there is evidence to suggest that smallpox vaccination provides some protection against MPX, leading to a more favorable clinical presentation. To prevent high risk individuals, two vaccines namely JYNNEOS and ACAM2000 are used against MPX1. Among several pharmacological interventions suggested, antiviral drugs Brincidofovir and Tecovirimat show promising therapeutic effects. It inhibits the production of protein required for extracellular virus production2. Clinical trials of these antivirals are currently underway and may take a longtime to decipher its effectiveness and safety. Learning from coronavirus disease-2019 (COVID-19) pandemic, several clinical trials and observational studies have shown that statins can protect against death and reduce the symptoms of respiratory infections3. In addition to this, cholesterol lowering drugs (fenofibrates and PCSK9 inhibitors) also presented beneficial effects in COVID-19 patients with hypercholesterolemia4,5. These drugs exhibit antiviral activity by altering cell membrane cholesterol and non–lipid-related pleiotropic effects such as anti-inflammatory and immunomodulatory effects6. The entry of fusogenic, enveloped viruses into host cells requires lipid envelope, especially cholesterol. HSV and HIV-1 requires cholesterol on the host membrane and on the viral envelope for its entry. In envelope glycoprotein clustering of HIV-1, cholesterol is a major molecular platform regulating this process7. Cellular cholesterol depletion by statins reduced the production of human parainfluenza virus type 1 (hPIV1) particles by interfering virus assembly8. It also affects the stability, density of virions of influenza A virus and respiratory syncytial virus9. Lovastain and gemfibrozil inhibited hP1V1 virus assembly production through disruption of lipid raft integrity. Dengue virus infection induced the expression of proprotein convertase subtilisin/kexin type 9 (PCSK9), which decreases the recycling of low-density lipoprotein receptor to promote cholesterol redistribution into ER repressing ER-resident STING and type I interferon activation10. To combat this, PCSK9 inhibitors significantly improved the secretion of type I interferons. Statins are also repurposed in treating the patients following Ebola and Influenza infections. Overall it is clear that, statins exhibits antiviral effects by blocking/altering cholesterol trafficking, redistribution, virus attachment, virions production, maturation, release, and virions stability. MPX seems to become another worldwide public health crisis, evoking parallels to the ongoing COVID-19 epidemic and its forebears. Repurposing cholesterol lowering drugs could be an effective approach in MPX. However, there is a lacune in understanding the role of cholesterol and its redistribution in MPX. Reducing cholesterol seems to be an effective antiviral approach, it could also dampen the host’s immune system11. One must also keep in mind that since viruses rely on lipid flux, employing statins to reduce cholesterol levels that is needed for viral maintenance rather than global reduction could be an effective approach. Hypercholesterolemic patients infected with MPX should continue taking cholesterol lowering drugs. Administering PCSK9 inhibitors, statins and fenofibrates could be considered as an adjuvant therapy along with standard drugs employed in in MPX treatment. Ethical approval Not applicable. Sources of funding None. Author contribution S.S., S.K.R.S. performed literature search and drafted the letter. Conflicts of interest disclosure The authors S.S. and S.K.R.S. certify that they have no affiliations with or involvement in any organization or entity with any financial interest (such as honoraria; educational grants; participation in speakers’ bureaus; membership, employment, consultancies, stock ownership, or other equity interest; and expert testimony or patent-licensing arrangements), or nonfinancial interest (such as personal or professional relationships, affiliations, knowledge or beliefs) in the subject matter or materials discussed in this manuscript. Research registration unique identifying number (UIN) None. Guarantor None.

Virus Propagation and Plaque Assay for Dengue Virus.

This chapter describes the methods of propagation and titration for DENV-1 to -4, which are required for most of the experiments using live viruses. DENV does not reach titers as high as those of other viruses or as high as desired for their use in biological assays. Although DENV grows in many different cell lines derived from both vertebrate and invertebrate cells, the most common cell lines used for virus isolation and propagation are mosquito cells C6/36 from Aedes albopictus. Amongst several methods for virus quantification, plaque assay stands out as being very practical and inexpensive. This technique is carried out essentially to estimate virion density in a particular material, being extremely important when evaluating the effect of an antiviral treatment or antibody neutralization capacity, for example. In this assay, viral particles are serially diluted and added onto confluent cell monolayers immersed in a semisolid medium, which is responsible for limiting virus spread throughout the culture. Therefore, regarding the medium consistency, once a virion successfully infects a cell, the newly produced particles can only infect neighboring cells, eventually leading them to death. This phenomenon can be observed as round gaps or plaques in the culture after staining live cells with a crystal violet solution. Then, plaques are counted and used to determine plaque-forming units per milliliter. Here, we describe a protocol established by our group for dengue virus (DENV) titration with porcine kidney (PS) cells.

Modeling the viral load expelled in saliva droplets carrying SARS-CoV-2

One of the most likely routes of transmission of COVID-19 is through the saliva droplets that are produced while speaking, coughing or sneezing by infected people. The expelled droplets, measuring between 0.4 and 450 μm, follow trajectories determined, mainly, by the gravitational, and air frictional forces. We solve numerically the equations of motion in which the linear and quadratic velocity terms in the drag force are considered. In order to model the virus load expelled during respiratory events, we assume log-log Gaussian distributions, with peaks around 10 μm, and analyze four size ranges: the aerosol (0.4–5 μm), the small (5.1–10 μm), the middle (10.1–100 μm), and big (100.1–450 μm) droplet size regimes. In the aerosol regime, the frictional forces quickly stop the droplets in their horizontal movement and they fall extremely slowly pulled down by the gravitational force. The residence time, in a calm environment, goes from 3.83 days to 33.3 min. More massive droplets take shorter times and hit the ground meters away from the source. By assuming a constant density of virions per milliliter, we estimate the expelled amount into the environment. The middle and small size droplets contain the highest amount but since the aerosol droplets remain in the air such a long time, they represent also a high risk for infection. We emphasize the importance of face protection to minimize COVID-19, transmission.

Interpretation of SARS-CoV-2 behaviour on different substrates and denaturation of virions using ethanol: an atomic force microscopy study.

Coronavirus (SARS-CoV-2) is a respiratory infection virus that was first detected in Wuhan, China. The virus causes COVID-19 disease and the outbreak was recognised as a pandemic by the World Health Organization (WHO) in March 2020. SARS-CoV-2 virion was first imaged using cryo-electron microscopy by the Chinese Center for Disease Control and Prevention (CDC). Atomic Force Microscopy is a unique technique that can allow imaging of biomolecules under different conditions. In this work, we used Atomic Force Microscopy to characterize SARS-CoV-2 on tissue culture polystyrene (TCPS) and glass coverslip surfaces. We isolated SARS-CoV-2 and drop casted it on coverslip glass and tissue culture polystyrene surfaces. We analyzed height profiles, density, and aggregation behavior of the virion on glass and polystyrene surfaces. We observed the coffee ring effect on the drop casted samples and close packing of virions near the coffee rings on both surfaces with relatively higher virion distribution on the tissue culture polystyrene (TCPS) substrates. We compare virion agglomeration on the two types of surfaces. Finally, we applied ethanol disinfectant to virions on the surface to visualize the effect of ethanol and image the ultrastructure of SARS-CoV-2.

On the fractional-order model of HIV-1 infection of CD4+ T-cells under the influence of antiviral drug treatment

ABSTRACT In this article, the fractional-order model, namely HIV-1 infection of CD4+ T-cells combined with the effect of antiviral drug treatment, is investigated. The model includes three unknown factors, (i) the uninfected CD4+ T-cells, (ii) the infected CD4+ T-cells, and (iii) the density of virions in the plasma. The most effective techniques such as the variation-of-parameters method, the variational iteration method, the homotopy perturbation method and the adomian decomposition method are implemented to tackle the mathematical models of Caputo’s fractional derivative. Graphical illustration and numerical results are presented to show the validity and accuracy of the obtained results. It is found that the fractional-order model can be easily solved by means of analytical approaches with less computational cost and the HPM technique is slightly better than others. It is also perceived that the fractional-order approximate solutions can get closer to classical approximate solutions when .

The Fractional Differential Model of HIV-1 Infection of CD4+ T-Cells with Description of the Effect of Antiviral Drug Treatment.

In this paper, the fractional-order differential model of HIV-1 infection of CD4+ T-cells with the effect of drug therapy has been introduced. There are three components: uninfected CD4+ T-cells, x, infected CD4+ T-cells, y, and density of virions in plasma, z. The aim is to gain numerical solution of this fractional-order HIV-1 model by Laplace Adomian decomposition method (LADM). The solution of the proposed model has been achieved in a series form. Moreover, to illustrate the ability and efficiency of the proposed approach, the solution will be compared with the solutions of some other numerical methods. The Caputo sense has been used for fractional derivatives.

Internal Disequilibria and Phenotypic Diversification during Replication of Hepatitis C Virus in a Noncoevolving Cellular Environment.

Viral quasispecies evolution upon long-term virus replication in a noncoevolving cellular environment raises relevant general issues, such as the attainment of population equilibrium, compliance with the molecular-clock hypothesis, or stability of the phenotypic profile. Here, we evaluate the adaptation, mutant spectrum dynamics, and phenotypic diversification of hepatitis C virus (HCV) in the course of 200 passages in human hepatoma cells in an experimental design that precluded coevolution of the cells with the virus. Adaptation to the cells was evidenced by increase in progeny production. The rate of accumulation of mutations in the genomic consensus sequence deviated slightly from linearity, and mutant spectrum analyses revealed a complex dynamic of mutational waves, which was sustained beyond passage 100. The virus underwent several phenotypic changes, some of which impacted the virus-host relationship, such as enhanced cell killing, a shift toward higher virion density, and increased shutoff of host cell protein synthesis. Fluctuations in progeny production and failure to reach population equilibrium at the genomic level suggest internal instabilities that anticipate an unpredictable HCV evolution in the complex liver environment.IMPORTANCE Long-term virus evolution in an unperturbed cellular environment can reveal features of virus evolution that cannot be explained by comparing natural viral isolates. In the present study, we investigate genetic and phenotypic changes that occur upon prolonged passage of hepatitis C virus (HCV) in human hepatoma cells in an experimental design in which host cell evolutionary change is prevented. Despite replication in a noncoevolving cellular environment, the virus exhibited internal population disequilibria that did not decline with increased adaptation to the host cells. The diversification of phenotypic traits suggests that disequilibria inherent to viral populations may provide a selective advantage to viruses that can be fully exploited in changing environments.

New Insights into the Understanding of Hepatitis C Virus Entry and Cell-to-Cell Transmission by Using the Ionophore Monensin A.

In our study, we characterized the effect of monensin, an ionophore that is known to raise the intracellular pH, on the hepatitis C virus (HCV) life cycle. We showed that monensin inhibits HCV entry in a pangenotypic and dose-dependent manner. Monensin induces an alkalization of intracellular organelles, leading to an inhibition of the fusion step between viral and cellular membranes. Interestingly, we demonstrated that HCV cell-to-cell transmission is dependent on the vesicular pH. Using the selective pressure of monensin, we selected a monensin-resistant virus which has evolved to use a new entry route that is partially pH and clathrin independent. Characterization of this mutant led to the identification of two mutations in envelope proteins, the Y297H mutation in E1 and the I399T mutation in hypervariable region 1 (HVR1) of E2, which confer resistance to monensin and thus allow HCV to use a pH-independent entry route. Interestingly, the I399T mutation introduces an N-glycosylation site within HVR1 and increases the density of virions and their sensitivity to neutralization with anti-apolipoprotein E (anti-ApoE) antibodies, suggesting that this mutation likely induces conformational changes in HVR1 that in turn modulate the association with ApoE. Strikingly, the I399T mutation dramatically reduces HCV cell-to-cell spread. In summary, we identified a mutation in HVR1 that overcomes the vesicular pH dependence, modifies the biophysical properties of particles, and drastically reduces cell-to-cell transmission, indicating that the regulation by HVR1 of particle association with ApoE might control the pH dependence of cell-free and cell-to-cell transmission. Thus, HVR1 and ApoE are critical regulators of HCV propagation. Although several cell surface proteins have been identified as entry factors for hepatitis C virus (HCV), the precise mechanisms regulating its transmission to hepatic cells are still unclear. In our study, we used monensin A, an ionophore that is known to raise the intracellular pH, and demonstrated that cell-free and cell-to-cell transmission pathways are both pH-dependent processes. We generated monensin-resistant viruses that displayed different entry routes and biophysical properties. Thanks to these mutants, we highlighted the importance of hypervariable region 1 (HVR1) of the E2 envelope protein for the association of particles with apolipoprotein E, which in turn might control the pH dependency of cell-free and cell-to-cell transmission.

Role of hypervariable region 1 for the interplay of hepatitis C virus with entry factors and lipoproteins.

Hepatitis C virus (HCV) particles associate with lipoproteins and infect cells by using at least four cell entry factors. These factors include scavenger receptor class B type I (SR-BI), CD81, claudin 1 (CLDN1), and occludin (OCLN). Little is known about specific functions of individual host factors during HCV cell entry and viral domains that mediate interactions with these factors. Hypervariable region 1 (HVR1) within viral envelope protein 2 (E2) is involved in the usage of SR-BI and conceals the viral CD81 binding site. Moreover, deletion of this domain alters the density of virions. We compared lipoprotein interaction, surface attachment, receptor usage, and cell entry between wild-type HCV and a viral mutant lacking this domain. Deletion of HVR1 did not affect CD81, CLDN1, and OCLN usage. However, unlike wild-type HCV, HVR1-deleted viruses were not neutralized by antibodies and small molecules targeting SR-BI. Nevertheless, modulation of SR-BI cell surface expression altered the infection efficiencies of both viruses to similar levels. Analysis of affinity-purified virions revealed comparable levels of apolipoprotein E (ApoE) incorporation into viruses with or without HVR1. However, ApoE incorporated into these viruses was differentially recognized by ApoE-specific antibodies. Thus, SR-BI has at least two functions during cell entry. One of them can be neutralized by SR-BI-targeting molecules, and it is critical only for wild-type HCV. The other one is important for both viruses but apparently is not inactivated by the SR-BI binding antibodies and small molecules evaluated here. In addition, HVR1 modulates the conformation and/or epitope exposure of virus particle-associated ApoE. HCV cell entry is SR-BI dependent irrespective of the presence or absence of HVR1. Moreover, this domain modulates the properties of ApoE on the surface of virus particles. These findings have implications for the development of SR-BI-targeting antivirals. Furthermore, these findings highlight separable functions of SR-BI during HCV cell entry and reveal a novel role of HVR1 for the properties of virus-associated lipoproteins.

Encapsidation of Host-Derived Factors Correlates with Enhanced Infectivity of Sindbis Virus

The genus Alphavirus consists of a group of enveloped, single-stranded RNA viruses, many of which are transmitted by arthropods to a wide range of vertebrate host species. Here we report that Sindbis virus (SINV) produced from a representative mammalian cell line consists of at least two unique particle subpopulations, separable on the basis of virion density. In contrast, mosquito-derived SINV consists of a homogeneous population of particles. Our findings indicate that the denser particle subpopulation, SINV(Heavy), is more infectious on a per-particle basis than SINV(Light). SINV produced in mosquito cell lines (SINV(C6/36)) exhibited particle-to-PFU ratios similar to those observed for SINV(Heavy). In mammalian cells, viral RNA was synthesized and accumulated more rapidly following infection with SINV(Heavy) or SINV(C6/36) than following infection with SINV(Light), due partly to enhanced translation of viral genomic RNA early in infection. Analysis of the individual particle subpopulations indicated that SINV(Heavy) and SINV(C6/36) contain host-derived factors whose presence correlates with the enhanced translation, RNA synthesis, and infectivity observed for these particles.

The Conserved Set of Host Proteins Incorporated into HIV-1 Virions Suggests a Common Egress Pathway in Multiple Cell Types

HIV-1 incorporates a large array of host proteins into virions. Determining the host protein composition in HIV virions has technical difficulties, including copurification of microvesicles. We developed an alternative purification technique using cholesterol that differentially modulates the density of virions and microvesicles (density modification, DM) allowing for high-yield virion purification that is essential for tandem mass spectrometric and quantitative proteomic (iTRAQ) analysis. DM purified virions were analyzed using iTRAQ and validated against Optiprep (60% iodixanol) purified virions. We were able to characterize host protein incorporation in DM-purified HIV particles derived from CD4+ T-cell lines; we compared this data set to a reprocessed data set of monocyte-derived macrophages (MDM) derived HIV-1 using the same bioinformatics pipeline. Seventy-nine clustered proteins were shared between the MDM derived and T-cell derived data set. These clusters included an extensive collection of actin isoforms, HLA proteins, chaperones, and a handful of other proteins, many of which have previously been documented to interact with viral proteins. Other proteins of note were ERM proteins, the dynamin domain containing protein EH4, a phosphodiesterase, and cyclophilin A. As these proteins are incorporated in virions produced in both cell types, we hypothesize that these proteins may have direct interactions with viral proteins or may be important in the viral life cycle. Additionally, identified common set proteins are predicted to interact with >1000 related human proteins. Many of these secondary interacting proteins are reported to be incorporated into virions, including ERM proteins and adhesion molecules. Thus, only a few direct interactions between host and viral proteins may dictate the host protein composition in virions. Ultimately, interaction and expression differences in host proteins between cell types may drive virion phenotypic diversity, despite conserved viral protein-host protein interactions between cell types.

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

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.

Aqueous extracts from peppermint, sage and lemon balm leaves display potent anti-HIV-1 activity by increasing the virion density

BackgroundAqueous extracts from leaves of well known species of the Lamiaceae family were examined for their potency to inhibit infection by human immunodeficiency virus type 1 (HIV-1).ResultsExtracts from lemon balm (Melissa officinalis L.), peppermint (Mentha × piperita L.), and sage (Salvia officinalis L.) exhibited a high and concentration-dependent activity against the infection of HIV-1 in T-cell lines, primary macrophages, and in ex vivo tonsil histocultures with 50% inhibitory concentrations as low as 0.004%. The aqueous Lamiaceae extracts did not or only at very high concentrations interfere with cell viability. Mechanistically, extract exposure of free virions potently and rapidly inhibited infection, while exposure of surface-bound virions or target cells alone had virtually no antiviral effect. In line with this observation, a virion-fusion assay demonstrated that HIV-1 entry was drastically impaired following treatment of particles with Lamiaceae extracts, and the magnitude of this effect at the early stage of infection correlated with the inhibitory potency on HIV-1 replication. Extracts were active against virions carrying diverse envelopes (X4 and R5 HIV-1, vesicular stomatitis virus, ecotropic murine leukemia virus), but not against a non-enveloped adenovirus. Following exposure to Lamiaceae extracts, the stability of virions as well as virion-associated levels of envelope glycoprotein and processed Gag protein were unaffected, while, surprisingly, sucrose-density equilibrium gradient analyses disclosed a marked increase of virion density.ConclusionAqueous extracts from Lamiaceae can drastically and rapidly reduce the infectivity of HIV-1 virions at non-cytotoxic concentrations. An extract-induced enhancement of the virion's density prior to its surface engagement appears to be the most likely mode of action. By harbouring also a strong activity against herpes simplex virus type 2, these extracts may provide a basis for the development of novel virucidal topical microbicides.

Application of spectrophotometry to evaluate the concentration of purified White Spot Syndrome Virus

White Spot Syndrome Virus (WSSV) is a highly virulent pathogen of shrimp. In previous work, a simple and efficient method has been established in our laboratory to purify intact WSSV virions from infected crayfish tissues. To perform studies of WSSV infection mechanism, pathogenesis and gene function by using this purified virion, quantitative assay for the virus becomes increasingly important. In this study, the optical density of the purified virion samples was measured at 600 nm wavelength using spectrophotometer and the corresponding concentration was counted by transmission electron microscopy. The statistical results revealed a high correlation between optical density and concentration of WSSV virions ( r = 0.993; n = 5). Finally, a conversion coefficient “ f” (3.34 × 10 8 virions/μl) was obtained and a formula was established: C (virions/μl) = f OD 600 = 3.34 × 10 8 × OD 600, which can be conveniently used to convert the optical density of purified WSSV preparation into the virion concentration.

An in vitro model of hepatitis C virion production

The hepatitis C virus (HCV) is a major cause of liver disease worldwide. The understanding of the viral life cycle has been hampered by the lack of a satisfactory cell culture system. The development of the HCV replicon system has been a major advance, but the system does not produce virions. In this study, we constructed an infectious HCV genotype 1b cDNA between two ribozymes that are designed to generate the exact 5′ and 3′ ends of HCV. A second construct with a mutation in the active site of the viral RNA-dependent RNA polymerase (RdRp) was generated as a control. The HCV-ribozyme expression construct was transfected into Huh7 cells. Both HCV structural and nonstructural proteins were detected by immunofluorescence and Western blot. RNase protection assays showed positive- and negative-strand HCV RNA. Sequence analysis of the 5′ and 3′ ends provided further evidence of viral replication. Sucrose density gradient centrifugation of the culture medium revealed colocalization of HCV RNA and structural proteins in a fraction with the density of 1.16 g/ml, the putative density of HCV virions. Electron microscopy showed viral particles of ≈50 nm in diameter. The level of HCV RNA in the culture medium was as high as 10 million copies per milliliter. The HCV-ribozyme construct with the inactivating mutation in the RdRp did not show evidence of viral replication, assembly, and release. This system supports the production and secretion of high-level HCV virions and extends the repertoire of tools available for the study of HCV biology.

On the behavior of solutions in viral dynamical models

We consider simple mathematical models for the early population dynamics of the human immunodefficiency type 1 virus (HIV-1). Although these systems of differential equations may be solved by numerical methods, few general theoretical results are available due to nonlinearities. We analyze a model whose components are plasma densities of uninfected CD4+ T-cells and infected cells (assumed in this model to be proportional to virion density). In addition to analyzing the nature of the equilibrium points, we show that there are no periodic or limit-cycle solutions. Depending on the values of the parameters, solutions either tend without oscillation to an equilibrium point with zero virion density or to an equilibrium point in which there are a nonzero number of virions. In the latter case the approach to equilibrium may be through damped oscillations or without oscillation.

Dissociation of genome dimerization from packaging functions and virion maturation of human immunodeficiency virus type 1.

The dimer initiation site/dimer linkage sequence (DIS/DLS) region of the human immunodeficiency virus type 1 (HIV-1) RNA genome is thought to play important roles at various stages of the virus life cycle. Recently we showed that the DIS/DLS region affects RNA-RNA interaction in intact virus particles, by demonstrating that duplication of the region in viral RNA caused the production of virus particles containing partially monomeric RNAs. We have extended this finding and succeeded for the first time in creating mutant particles which contain only monomeric RNAs without modifying any viral proteins. In terms of RNA encapsidation ability, virion density, and protein processing, the mutant particles were comparable to wild-type particles. The level of production of viral DNA by the mutant virus construct in infected cells was also comparable to that of the constructs that produced exclusively dimeric RNA, indicating that monomeric viral RNA could be the template for strand transfer. These results indicated that the RNA dimerization of HIV-1 could be separated from viral RNA packaging and was not absolutely required for RNA packaging, virion maturation, and reverse transcription.

Kilham polyomavirus: activation of gene expression and DNA replication in mouse fibroblast cells by an enhancer substitution.

The Kilham strain of polyomavirus (KV) infects vascular endothelial cells in vivo (J. E. Greenlee, Infect. Immun. 26:705-713, 1979), but no permissive cell type for growth of the virus in vitro has been identified. The failure of KV DNA to replicate in mouse fibroblast cells after transfection suggested that viral gene expression had narrow cell specificity. A KV substitution mutant having a part of the regulatory region of KV DNA replaced with a segment of the polyomavirus transcriptional enhancer was constructed. The substitution mutant was able to replicate in transfected 3T3 cells, and the newly replicated viral DNA associated with protein to form particles with the density of virions in CsCl equilibrium gradients. However, these particles were noninfectious when tested on 3T3 cells, suggesting that absorption or uptake of virus particles was defective for these cells. Analysis of early and late promoter activities by luciferase reporter gene expression showed that the enhancer substitution had a moderate positive effect on early gene expression and a large effect on the expression of the late genes. KV large T antigen inhibited the activities of both the wild-type and the substitution mutant early promoter, whereas only the mutant late promoter was activated under the same conditions. A comparison of the KV and polyomavirus large T antigens showed that they were not interchangeable in the initiation of KV and polyomavirus DNA synthesis. Furthermore, the wild-type KV origin of DNA replication was less active than the mutant structure in the presence of saturating amounts of KV large T antigen. Together, our data demonstrate several differences between the two types of large T antigen in their interactions with cellular proteins.

Human immunodeficiency virus type 1 virion density is not determined by nucleocapsid basic residues.

The human immunodeficiency virus type 1 (HIV-1) Gag polyprotein is sufficient for assembly and release of virion-like particles from the plasma membrane. To promote assembly, the Gag polyprotein must polymerize to form a shell that lines the inner membrane of nascent virions. Several techniques have been used to functionally map the domain required for Gag polymerization (the I domain). Among these methods, isopycnic centrifugation has been used under the assumption that changes in virion density reflect impairment in Gag-Gag interaction. If virion density is determined by efficient Gag-Gag interaction, then mutation of basic residues in the nucleocapsid (NC) domain should disrupt virion density, since these residues constitute the I domain. However, we have previously shown that simultaneous disruption of up to 10 HIV-1 NC basic residues has no obvious effect on virion density. To rule out the possibility that HIV-1 NC basic residues other than those previously mutated might be important for virion density, mutations were introduced at the remaining sites and the ability of these mutations to affect Gag-Gag interaction and virion density was analyzed. Included in our analysis is a mutant in which all NC basic residues are replaced with alanine. Our results show that disruption of HIV-1 NC basic residues has an enormous effect on Gag-Gag interaction but only a minimal effect on the density of those virions that are still produced. Therefore, the determinants of the I domain and of virion density are genetically distinguishable.

First passage time to detection in stochastic population dynamical models for HIV-1

The detection of HIV-1 levels in human hosts is cast as a first exit time problem for a multidimensional diffusion process. We consider a four-component model for early HIV-1 dynamics including uninfected CD4+ T-cells, latently infected cells, actively infected cells, and HIV-1 virions. An analytical framework is presented for the distribution of the time at which a given virion level is attained. A one-dimensional diffusion approximation for a branching process leads to an estimate for the distribution of the virion density and an expression for the mean detection time for any given detection threshold.