Principles of Virology. Jane Flint

Human immunodeficiency virus Pol Reverse transcriptase; proviral DNA synthesis type 1 IN Integrase; integration of proviral DNA into the cellular genome PR Protease; production of infectious particles Rhabdovirus Vesicular stomatitis virus L RNA-dependent RNA polymerase; synthesis of viral mRNA and vRNA

^aVaccinia virions contain some 20 enzymes, only a few of which are listed.

Cellular Macromolecules

      Virus particles can also contain cellular macromolecules that play important roles during the infectious cycle, such as the cellular histones that condense and organize polyomaviral and papillomaviral DNAs. Because they are formed by budding, enveloped viruses can readily incorporate cellular proteins and other macromolecules. For example, cellular glycoproteins may not be excluded from the membrane from which the viral envelope is derived. Furthermore, as a bud enlarges and pinches off during virus assembly, internal cellular components may be trapped within it. Enveloped viruses are also generally more difficult to purify than naked viruses. Indeed, analysis by the sensitive proteomic methods provided by mass spectrometry has identified from 50 to 100 cellular proteins in purified, enveloped particles of various herpesviruses, filoviruses, and rhabdoviruses. Consequently, it can be difficult to distinguish cellular components specifically incorporated into enveloped virus particles from those trapped randomly or copurifying with the virus. Nevertheless, in some cases it is clear that cellular molecules are important components of virus particles: these molecules are reproducibly observed at a specific stoichiometry and can be shown to be essential or play pivotal roles in the infectious cycle. The cellular components captured in retrovirus particles have been particularly well characterized.

      The primer for initiation of synthesis of the (−) strand DNA during reverse transcription in retroviral genomes is a specific cellular transfer RNA (tRNA), such as tRNALys3 in the case of human immunodeficiency virus type 1. These RNAs are incorporated into virus particles via association with the reverse transcriptase domains of one type of polyprotein (Gag-Pol) that in turn is assembled into particles via interactions with the Gag polyprotein (Chapter 13). The cognate human lysyl tRNA synthase is also selectively packaged into human immunodeficiency virus type 1 particles and facilitates initiation of reverse transcription (Chapter 10).

      A variety of membrane proteins have been observed in retrovirus particles, but appear to be acquired nonspecifically by virtue of their presence at the sites of particle budding. In contrast, several cytoplasmic proteins of the host cell are packaged specifically. It was reported more than 20 years ago that human immunodeficiency virus type 1 particles contain cellular cyclophilin A (PPIase A). This chaperone is the major cytoplasmic member of a ubiquitous family of peptidyl-prolyl isomerases. It is bound to the N terminus of the capsid (CA) protein and catalyzes isomerization of a single Gly-Pro bond in the protein. Substitutions in CA that impair binding of cyclophilin A reduce the infectivity of virus particles in some cell types. However, the effects of depletion of the cellular chaperone and of its inhibitors in virus-producing cells or in newly infected target cells have established that it is PPIase A present in new target cells, not the protein carried by incoming virus particles, that is important. Its presence in virus particles is simply a secondary consequence of interactions with the CA domain of viral polyproteins.

      Clathrin heavy chain, which mediates formation of endosomes (Chapter 5), is also selectively incorporated into the particles of many retroviruses. In the case of human immunodeficiency virus type 1, clathrin is recruited by a specific interaction with the integrase (IN) domain of the Gag-Pol polyprotein. Substitutions in the IN region that impair association with clathrin reduce production of virus particles, and are thought to lead to premature processing of the polyproteins from which particles assemble (Chapter 13).

      Cellular components present in virus particles may serve to facilitate virus reproduction, a property exemplified by the cellular tRNA primers for retroviral reverse transcription. However, incorporation of cellular components can also provide antiviral defense. As discussed in Volume II, Chapters 3 and 6, packaging of a cellular enzyme that converts cytosine to uracil (APOBEC3) into human immunodeficiency virus type 1 particles at the end of one infectious cycle leads to degradation and hypermutation of viral DNA synthesized early in the next cycle of infection.

Mechanical Properties of Virus Particles

      Investigation of Mechanical Properties of Virus Particles

As illustrated in the preceding sections, studies of purified virus particles by methods such as X-ray crystallography and cryo-EM can yield high-resolution descriptions of the interactions among particle components responsible for the assembly and sturdiness of these nanomachines. Such studies are typically performed under extreme conditions, for example, very low temperature, and the structures described are based on the averaging of very large numbers of particles. Consequently, these approaches provide no information about the dynamic or mechanical properties that underlie the functions of virus particles. Such information can be collected by various biophysical methods, including small-angle X-ray scattering and calorimetry. Atomic force microscopy, which permits both imaging of virus particles and measurement of mechanical properties, has been especially useful.

In this method, a very sharp tip attached to a cantilever scans the surface of a sample immobilized on a solid support (Fig. 4.30A). Deflections of the cantilever to or from the surface during scanning are detected by a laser beam via a position-sensitive detector. In this way, the topography of the surface of a sample such as a virus particle can be imaged, albeit at relatively low resolution (Fig. 4.30B). To assess mechanical properties, the tip is applied (nanoindentation) to a specific point on the surface, such as an axis of icosahedral symmetry, to deform the particle. Measurement of the force applied as a function of distance (degree of indentation) allows measurement of such parameters as elasticity and the force required to break the particle at that point. This approach has led to fascinating insights into how virus particles meet the seemingly paradoxical requirements for high stability to protect viral genomes and efficient disassembly during entry into a host cell.

Figure 4.30 Atomic force microscopy and its application to human adenovirus particles. (A) A schematic illustration of nanoindentation of virus particles. Virus particles are attached to a solid surface, such as a glass coverslip or mica, and the tip of an atomic force microscope (chosen to be smaller than the radius of the particles of interest) brought into contact. The tip is attached to a cantilever that moves to or from the surface of the particle as it is scanned. Deflections of a laser beam focused on the tip during scanning are recorded via a photodiode, allowing construction