MicrobiologyBytes

Lentiviruses are mammalian retroviruses known to infect cattle, cats, horses, sheep, and primates. They are the focus of intense study due to their causative association with AIDS in human. Although our knowledge on the origin and early evolution of HIV has grown exponentially over the past few years, much remains unresolved about the deeper relationships between primate and non-primate lentiviruses, the origin of lentiviruses, and their mode of structural evolution over long periods of evolutionary time. This is because these viruses evolve extremely rapidly, in a conflicting relationship with their hosts, and while their high mutation rate provides a wealth of information documenting their recent history, it also quickly erases evidence of their deeper ancestry. The lifecycle of retroviruses is atypical compared to other viruses in that after appropriate receptor recognition and entry in a specific cell type, their RNA genome is reverse transcribed into double-stranded DNA and integrated into the host genome as a provirus. Occasionally this process can take place in the host germline, and the integrated copy, also called endogenous retrovirus (ERV), may be transmitted vertically from parent to offspring and reach fixation in the host population. As such, ERVs constitute a fossil record of past viral infections that potentially provide an alternative way of gaining insights into the deep evolutionary history of present day exogenous retroviruses.

Although many ERVs have been characterized in mammals (e.g. 8% of the human genome), apparently very few derive from lentiviruses. Two reasons have traditionally been put forward to explain their absence in mammalian genomes: (i) they are of relatively recent evolutionary origin and endogenization has not yet commonly occurred, and/or (ii) they were not able to enter germ cells because of a very specific cell tropism. Recently however, an endogenous lentivirus called RELIK has been identified in the genome of rabbits and hares, whose germline integration was dated at least 12 millions years old. This discovery not only showed that lentiviruses were able to infiltrate mammalian germlines, but also demonstrated that this group of viruses is probably much older than what could previously be inferred based on sequence comparison of extant exogenous lentiviruses. New research now shows that a retrovirus related to HIV became stably integrated into the genomes of lemurs around 4.2 million years ago. The discovery of prosimian immunodeficiency virus (pSIV) offers new insights into the evolution of lentiviruses.

Based on “fossil” sequences collected from different lemur species, the researchers computationally reconstructed an apparently intact and complete DNA sequence for the ancestral prosimian lentivirus. The discovery that two different species of lemurs endemic to Madagascar suffered, independently and quasi-simultaneously, multiple germline infections of pSIV provides evidence that lentiviruses have repeatedly infiltrated the germline of prosimian species. These findings should allow future functional analysis of the extinct virus and advance our understanding of the biology of lentiviruses, including HIV. In addition, the characterization of this ancient lentivirus in lemurs raises the possibility that HIV-like retroviruses are still circulating today in the mammalian fauna of Madagascar.

Parallel Germline Infiltration of a Lentivirus in Two Malagasy Lemurs. 2009 PLoS Genet 5(3): e1000425
Retroviruses normally infect the somatic cells of their host and are transmitted horizontally, i.e., in an exogenous way. Occasionally, however, some retroviruses can also infect and integrate into the genome of germ cells, which may allow for their vertical inheritance and fixation in a given species; a process known as endogenization. Lentiviruses, a group of mammalian retroviruses that includes HIV, are known to infect primates, ruminants, horses, and cats. Unlike many other retroviruses, these viruses have not been demonstrably successful at germline infiltration. Here, we report on the discovery of endogenous lentiviral insertions in seven species of Malagasy lemurs from two different genera – Cheirogaleus and Microcebus. Combining molecular clock analyses and cross-species screening of orthologous insertions, we show that the presence of this endogenous lentivirus in six species of Microcebus is the result of one endogenization event that occurred about 4.2 million years ago. In addition, we demonstrate that this lentivirus independently infiltrated the germline of Cheirogaleus and that the two endogenization events occurred quasi-simultaneously. Using multiple proviral copies, we derive and characterize an apparently full length and intact consensus for this lentivirus. These results provide evidence that lentiviruses have repeatedly infiltrated the germline of prosimian species and that primates have been exposed to lentiviruses for a much longer time than what can be inferred based on sequence comparison of circulating lentiviruses. The study sets the stage for an unprecedented opportunity to reconstruct an ancestral primate lentivirus and thereby advance our knowledge of host–virus interactions.

Related:

• Phoenix from the ashes: The 5 million year old virus
• T Cell Responses to Human Endogenous Retroviruses in HIV-1 Infection
• Human endogenous retroviruses: from ancestral pathogens to bona fide genes
• HIV infection is due to the sins of the fathers
• Rooting the tree

PKR is a sentinel kinase constitutively expressed in all cells as an inactive protein that is subsequently activated by virus RNA produced during an infection. The active kinase perturbs virus replication by phosphorylating protein substrates in the cell. RNA helicase A (RHA) is a novel substrate for PKR. Viruses usurp this helicase to replicate their own genome. Phosphorylation of RHA by PKR perturbs the ability of the helicase to bind virus RNA. Correspondingly, PKR prevents the capacity of RHA to enhance expression of genetic elements encoded by the human immunodeficiency virus (HIV). In addition, HIV virions packaged within cells that also express protein fragments of RHA have enhanced infectivity. These fragments of RHA occur within a protein domain previously established to bind RNA but increasingly recognized to mediate protein–protein interactions. This supports an emerging role for these protein domains to coordinate the cell’s response to pathogen-associated RNA. The findings identify a new cell-signaling pathway important in the response to virus infection.

An Antiviral Response Directed by PKR Phosphorylation of the RNA Helicase A. 2009 PLoS Pathog 5(2): e1000311
The double-stranded RNA-activated protein kinase R (PKR) is a key regulator of the innate immune response. Activation of PKR during viral infection culminates in phosphorylation of the α subunit of the eukaryotic translation initiation factor 2 (eIF2α) to inhibit protein translation. A broad range of regulatory functions has also been attributed to PKR. However, as few additional PKR substrates have been identified, the mechanisms remain unclear. Here, PKR is shown to interact with an essential RNA helicase, RHA. Moreover, RHA is identified as a substrate for PKR, with phosphorylation perturbing the association of the helicase with double-stranded RNA (dsRNA). Through this mechanism, PKR can modulate transcription, as revealed by its ability to prevent the capacity of RHA to catalyze transactivating response (TAR)–mediated type 1 human immunodeficiency virus (HIV-1) gene regulation. Consequently, HIV-1 virions packaged in cells also expressing the decoy RHA peptides subsequently had enhanced infectivity. The data demonstrate interplay between key components of dsRNA metabolism, both connecting RHA to an important component of innate immunity and delineating an unanticipated role for PKR in RNA metabolism.

Related:

• Viruses and Apoptosis

Kaposi’s sarcoma (KS) is a multifocal tumour only found in a few groups of people, including elderly Mediterranean men, individuals in Africa and patients with immune disorders. The tumours arise from the formation of new blood or lymphatic vessels (angiogenesis or lymphangiogenesis) due to the proliferation of endothelial cells. In 1994 Chang and Moore identified a new virus, Kaposi’s sarcoma-associated herpesvirus (KSHV) as the cause of these tumours.

Unlike other herpesviruses, the seroprevalence of KSHV is not ubiquitous, perhaps only 5% in those countries with low KSHV rates such as the USA and Northern Europe). Like all herpesviruses, KSHV infection persists for the life of the host and can enter either of two states: latency or lytic reactivation. In latency, the minimum number of viral genes is expressed to maintain the virus genome in dividing cells, evading immune detection. Lytic reactivation occurs when the virus re-enters productive replication to generate new progeny, lysing the host cell in the process. And like all herpesviruses KSHV likes to mess with the immune system of its host. The KSHV genome contains 86 genes, almost a quarter of which encode proteins with immunoregulatory activities such as T- and B-cell function, complement activation, the innate antiviral interferon response and natural killer cell activity. Many of these gene are homologues of cellular proteins.

• The KSHV proteins MIR1 and MIR2 ubiquitinate the cytoplasmic tail of MHC-I which triggers endocytosis and proteasomal degradation. This protects KSHV-infected cells from NK-mediated lysis. MIR2 can also down-regulate other components of the immune synapse, ICAM (CD54) and PECAM (CD31) by the same mechanism.
• The KSHV vOX2 protein causes the cellular CD200 receptor to deliver an inhibitory signal to granulocytes, although the mechanism by which this acts is not yet well defined.
• The KCP protein is present on the surface of KSHV virions and infected cells and protects them from complement attack by accelerating the decay of the classical pathway C3 convertase enzyme complex.
• The K15 protein activates MAP kinases and this affects immune function.
• KSHV encodes a family of 12 miRNAs. These regulate both B- and T-cell function.
• The K1 protein reduces the presence of B cell receptors on the surface of B cells and interferes with the production of cytokines, and inhibits apoptosis.
• The MIR2 protein down-regulates tetherin, which is involved in normal B-cell differentiation.
• Three KSHV chemokine homologues (vCCL1–3) have affinity for chemokine receptors (CCRs) and this affects T-cell responses.
• KSHV proteins inhibit interferon pathways.

Since KSHV infection results in lifelong persistence of the virus, these immunomodulation activities are clearly successful in preventing its elimination by the immune system. Many questions about KSHV infection remain unanswered, but we have learned valuable lessons about the normal function of the immune system through studying this virus.

Modulation of the immune system by Kaposi’s sarcoma-associated herpesvirus. Trends Microbiol. Feb 18 2009

Subscribe to podcasts (free):
[iTunes] Enhanced podcasts & videos
[RSS] mp3 podcasts (audio only)
Play this episode: Enhanced version
Audio only

Related:

• MicrobiologyBytes: Human Herpesvirus 8 (HHV-8/KSHV)
• Cellular Genes Targeted by KSHV MicroRNAs
• Herpesviruses – not all bad?

HIV is evolving rapidly to escape the human immune system. Researchers have shown HIV is able to adapt rapidly to counter human genes controlling immune system molecules that can target it for destruction. Progression to AIDS is tied to genes which control production of key immune system molecules called human leucocyte antigens (HLAs). Humans differ in the HLA genes they have, and even small differences can have a big impact on how quickly AIDS develops. Researchers found mutations that enabled HIV effectively to neutralise the effect of a particular HLA gene were more frequent in populations with a high prevalence of that specific gene.

BBC News

Adaptation of HIV-1 to human leukocyte antigen class I. Nature, 25 February 2009
The rapid and extensive spread of the human immunodeficiency virus (HIV) epidemic provides a rare opportunity to witness host–pathogen co-evolution involving humans. A focal point is the interaction between genes encoding human leukocyte antigen (HLA) and those encoding HIV proteins. HLA molecules present fragments (epitopes) of HIV proteins on the surface of infected cells to enable immune recognition and killing by CD8+ T cells; particular HLA molecules, such as HLA-B*57, HLA-B*27 and HLA-B*51, are more likely to mediate successful control of HIV infection1. Mutation within these epitopes can allow viral escape from CD8+ T-cell recognition. Here we analysed viral sequences and HLA alleles from >2,800 subjects, drawn from 9 distinct study cohorts spanning 5 continents. Initial analysis of the HLA-B*51-restricted epitope, TAFTIPSI (reverse transcriptase residues 128–135), showed a strong correlation between the frequency of the escape mutation I135X and HLA-B*51 prevalence in the 9 study cohorts (P = 0.0001). Extending these analyses to incorporate other well-defined CD8+ T-cell epitopes, including those restricted by HLA-B*57 and HLA-B*27, showed that the frequency of these epitope variants (n = 14) was consistently correlated with the prevalence of the restricting HLA allele in the different cohorts (together, P < 0.0001), demonstrating strong evidence of HIV adaptation to HLA at a population level. This process of viral adaptation may dismantle the well-established HLA associations with control of HIV infection that are linked to the availability of key epitopes, and highlights the challenge for a vaccine to keep pace with the changing immunological landscape presented by HIV.

Related:

• Why is HIV a pathogen?
• How does HIV cause AIDS?
• Immune exhaustion in HIV infection
• HIV-2 – a kinder AIDS virus?

The pathogenesis of HIV begins with a profound depletion of CD4+ T cells in the gut followed by a long period of clinically silent but dynamic virus replication and diversification with high host cell turnover before the onset of AIDS. The AIDS-defining opportunistic infections and tumors mark the end-point of a long balancing act between virus and host that occurs when CD4+ T cell numbers fall below a level that can sustain immunity. Comparative studies of lentivirus infections in other species show that AIDS is not an inevitable outcome of infection because simian immunodeficiency virus in natural hosts seldom causes disease. What distinguishes pathogenic from ‘passenger’ infection is a systemic activation of immune responses followed by destruction of the integrity of lymphoid follicles. Macrophage and dendritic cell infection also contribute to pathogenesis. Maedi-Visna virus infection in sheep, which targets these cells but not T lymphocytes, also leads to progressive disease and death that resembles the wasting and brain diseases of HIV without the T cell immunodeficiency. Thus, lessons from pathogenic and nonpathogenic lentivirus infections provide insight into the complex syndrome called AIDS.

Why is HIV a pathogen? Trends Microbiol. 2008 16(12):555-60

Related:

• How does HIV cause AIDS?
• Immune exhaustion in HIV infection
• HIV “can never be cured”
• HIV-2 – a kinder AIDS virus?

One of the first attempts to use gene therapy to treat HIV has produced promising results in clinical trials. When the therapy was tested on 74 patients, it was shown to be safe and appeared to reduce the effect of the virus on the immune system. In theory, one treatment should be enough to replace the need for a lifetime of antiretroviral therapy. The latest therapy involves giving patients blood stem cells modified to carry a molecule called OZ1, which is designed to stop HIV reproducing itself by targeting two key proteins. The patients in the trial either received the therapy, or a dummy treatment. After 48 weeks the researchers found there was no statistically significant difference in the amount of HIV circulating in the blood of the two groups of patients. However, after 100 weeks the patients who received the gene therapy had higher levels of CD4+ cells – the key cells of the immune system which are specifically destroyed by HIV.

BBC News

Phase 2 gene therapy trial of an anti-HIV ribozyme in autologous CD34+ cells. Nature Medicine, 15 February 2009
Gene transfer has potential as a once-only treatment that reduces viral load, preserves the immune system and avoids lifetime highly active antiretroviral therapy. This study, which is to our knowledge the first randomized, double-blind, placebo-controlled, phase 2 cell-delivered gene transfer clinical trial, was conducted in 74 HIV-1–infected adults who received a tat-vpr–specific anti-HIV ribozyme (OZ1) or placebo delivered in autologous CD34+ hematopoietic progenitor cells. There were no OZ1-related adverse events. There was no statistically significant difference in viral load between the OZ1 and placebo group at the primary end point (average at weeks 47 and 48), but time-weighted areas under the curve from weeks 40–48 and 40–100 were significantly lower in the OZ1 group. Throughout the 100 weeks, CD4+ lymphocyte counts were higher in the OZ1 group. This study indicates that cell-delivered gene transfer is safe and biologically active in individuals with HIV and can be developed as a conventional therapeutic product.

Related:

• HIV “can never be cured”
• Retroviruses
• HIV-2 – a kinder AIDS virus?
• HIV mutation and the immune response
• Immune exhaustion in HIV infection
• How does HIV cause AIDS?

GlaxoSmithKline, the world’s second biggest pharmaceutical company, is to provide cheap drugs to millions of people in the developing world. Andrew Witty, the new head of the company, has said he will cut prices on all medicines, including HIV treatments, in the 50 poorest countries to no more than 25 per cent of the levels in Britain and the US. The company will also give back 20 per cent of profits to be spent on hospitals and clinics and share knowledge about potential drugs currently protected by patents. Drug companies have been repeatedly criticised for failing to drop prices for HIV drugs as millions have died in Africa and Asia.

Source

A retrovirus has a lipoprotein envelope lined with a layer of matrix protein (MA), surrounding a nucleoprotein core. In the core, the diploid RNA genome in complex with nucleocapsid protein (NC) and the replication enzymes is enclosed within the capsid – a shell of CA protein. MA, CA and NC are derived from a common precursor, the Gag polyprotein, which assembles into a thick-walled spherical shell in the immature virus. After it buds off from the host cell, the viral protease is activated, releasing CA subunits that assemble into capsids. Capsids of a given retrovirus vary in structure, and the predominant types vary among retroviruses; for instance, those of HIV are conical, those of Rous sarcoma virus (RSV) are irregular polyhedra and those of murine leukemia virus are round. Some virions contain more than one capsid, and nested (multilayer) capsids are also observed.

For a retrovirus such as HIV to be infectious, a properly formed capsid is needed; however, unusually among viruses, retrovirus capsids are highly variable in structure. According to the fullerene conjecture, they are composed of hexamers and pentamers of capsid protein (CA), with the shape of a capsid varying according to how the twelve pentamers are distributed and its size depending on the number of hexamers. Hexamers have been studied in planar and tubular arrays, but the predicted pentamers have not been observed. Here we report cryo-electron microscopic analyses of two in-vitro-assembled capsids of Rous sarcoma virus. Both are icosahedrally symmetric: one is composed of 12 pentamers, and the other of 12 pentamers and 20 hexamers. Fitting of atomic models of the two CA domains into the reconstructions shows three distinct inter-subunit interactions. These observations substantiate the fullerene conjecture, show how pentamers are accommodated at vertices, support the inference that nucleation is a crucial morphologic determinant, and imply that electrostatic interactions govern the differential assembly of pentamers and hexamers.

Visualization of a missing link in retrovirus capsid assembly. Nature 457: 694-698 (5 February 2009)

Related:

• Retroviruses
• Retrovirus infection of resting cells

Retroviruses have been studied for over 100 years since the discovery by Ellerman and Bang in 1908 that cell-free tissue filtrates could transmit leukaemia in chickens. The first pathogenic human retrovirus (HTLV) was discovered in 1981 and HIV, the causative agent of AIDS was discovered in 1983, but this presentation concentrates on the basic biology of retroviruses.

Retrovirus particles consist of a core, which contains the RNA genome of the virus plus the nucleocapsid (NC) protein and reverse transcriptase (RT), integrase (IN) and protease (PR) enzymes. The core lies inside an icosahedral capsid (CA protein) which is surrounded by the matrix (MA) which links the capsid to the lipid envelope. The transmembrane protein (TM) and surface glycoprotein (SU) are associated with the envelope.

All retrovirus genomes consist of two molecules of RNA, which are equivalent to mRNA. These range in size from ~7-11kb. Retrovirus genomes have four unique features:

1. They are the only viruses which are fully diploid.
2. They are the only RNA viruses whose genome is produced by cellular transcriptional machinery (without any participation by a virus-encoded polymerase).
3. They are the only viruses whose genome requires a specific cellular RNA (tRNA) for replication.
4. They are the only plus-sense RNA viruses whose genome does not serve directly as mRNA immediately after infection.

The gene order in all retroviruses is the same:

5′ – gag – pol – env – 3′

Some retroviruses have additional genes, such as the tax and rex genes in HTLV and tat and rev in HIV.

To initiate infection, the SU envelope glycoprotein binds to a specific receptor on the surface of the host target cell. The specificity of this interaction does much to determine the cell-tropism of different retroviruses, or even different isolates of the same virus (e.g. in HIV). Receptor binding results in conformational changes in the glycoprotein spike, revealing the (previously masked) fusion domain in the TM protein and resulting in fusion of the virus envelope with the cell membrane. Penetration and uncoating are poorly understood, but it is now known that uncoating is only partial, resulting eventually in a core (nucleocapsid) particle within the cytoplasm. Reverse transcription occurs inside the ordered structure of this core particle. During reverse transcription, the two single-stranded genome RNA molecules are converted into one double-stranded DNA version of the virus genome, which has the addition of long terminal repeats (LTRs).

The double-stranded DNA form of the virus genome is integrated into the chromatin of the host cell by the integrase (IN) enzyme, where it is known as a provirus. Promoter sequences in the upstream LTR direct expression of virus genes using host cell RNA polymerase. Alternative splicing is used to express virus genes gag, pol and env. In addition to encoding the gag proteins, the full length mRNA transcript also forms new genomes which are packaged into virus particles.

The genetics of retroviruses are complex:

• High mutation rate – reverse transcription is an error-prone process.
• Recombination – occurs during reverse transcription, promoted by the combination of two strands of RNA into one double-stranded DNA provirus.
• Interactions with the host cell – insertional mutagenesis, transduction.

In addition to infectious viruses, retrotransposons are endogenous retrovirus-like genetic elements which make up much of the human genome.

Subscribe to podcasts (free):
[iTunes] Enhanced podcasts & videos
[RSS] mp3 podcasts (audio only)
Play this episode: Enhanced version
Audio only

So what have retroviruses ever done for us?
Apart from forming much of our genome: Syncytin is a captive retroviral envelope protein involved in human placental morphogenesis. 2000 Nature 403: 785-9.

Retroviruses are responsible for a wide range of diseases:

• Paralysis
• Wasting
• Ataxia
• Arthritis
• Dementia
• Neuropathy
• Transformation
• Immunodeficiency

Retroviruses have been studied intensively for over 100 years as causes of disease and more recently as gene vectors.

Related:

• Latest News: Retroviruses